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WO2025217607A1 - Systèmes et procédés de culture cellulaire - Google Patents

Systèmes et procédés de culture cellulaire

Info

Publication number
WO2025217607A1
WO2025217607A1 PCT/US2025/024411 US2025024411W WO2025217607A1 WO 2025217607 A1 WO2025217607 A1 WO 2025217607A1 US 2025024411 W US2025024411 W US 2025024411W WO 2025217607 A1 WO2025217607 A1 WO 2025217607A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
chamber
liquid
culture chamber
culture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/024411
Other languages
English (en)
Inventor
Taehong Kwon
Samin AKBARI
Trevor RUGGIERO
Jared RUEHR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sartorius Bioanalytical Instruments Inc
Original Assignee
Sartorius Bioanalytical Instruments Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sartorius Bioanalytical Instruments Inc filed Critical Sartorius Bioanalytical Instruments Inc
Publication of WO2025217607A1 publication Critical patent/WO2025217607A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/50—Mixing liquids with solids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0666—Solenoid valves

Definitions

  • the present disclosure generally describes systems and methods.
  • the subject matter described herein involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • a system for performing cell culture comprises a valve, a cell-culture chamber, and a sensor.
  • the valve is in fluidic communication with a plurality of ports.
  • the cell-culture chamber comprises a cell filter. At least a portion of a surface of the cell-culture chamber is transparent to at least one wavelength of light.
  • the cell-culture chamber has a volume of less than or equal to 500 microliters.
  • the system further comprises a mixing chamber, a mixing element, and/or a flow path configured to promote mixing.
  • a method of performing cell culture comprises flowing a liquid through a cell-culture chamber, and, while the liquid is flowing through the cell-culture chamber, retaining the cells in the cell-culture chamber.
  • the cell-culture chamber contains cells.
  • the cell-culture chamber has a volume of less than or equal to 500 microliters.
  • the cell-culture chamber is present in a system for performing cell culture.
  • the system further comprises a pump, a valve, and a sensor.
  • the system further comprises a mixing chamber, a mixing element, and/or a flow path configured to promote mixing.
  • the valve is in fluidic communication with a plurality of ports. At least a portion of a surface of the cell-culture chamber is transparent to at least one wavelength of light.
  • a system for performing cell culture comprises a pump, a valve, a cell-culture chamber, a sensor, an optical detector, and a gas-permeable membrane.
  • the valve is in fluidic communication with a plurality of ports.
  • the cell-culture chamber comprises a cell filter.
  • the cell-culture chamber has a volume of less than or equal to 500 microliters.
  • the system further comprises a mixing chamber, a mixing element, and/or a flow path configured to promote mixing.
  • Paragraph 4 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system is positioned on a substrate.
  • Paragraph 5 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the substrate is a chip.
  • Paragraph 6 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is a micro fluidic chip.
  • Paragraph 7 In some embodiments, a system or method as in any preceding paragraph is provided, wherein a surface of the chip is transparent to at least one wavelength of light.
  • Paragraph 8 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is transparent to at least one wavelength of light.
  • Paragraph 9 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is formed from a material that is biocompatible.
  • Paragraph 10 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is formed from a material that is sterilizable.
  • Paragraph 11 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is formed from a plastic.
  • Paragraph 12 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the plastic comprises polystyrene, PMMA, a cyclic-olefin copolymer, polycarbonate, polysulfone, polyetherimide, polyurethane, and/or a 3D-printed resin.
  • Paragraph 13 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is formed from PDMS.
  • Paragraph 14 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is formed from glass.
  • Paragraph 15 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the chip is disposable.
  • Paragraph 16 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises an incubator.
  • Paragraph 17 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber is positioned inside the incubator.
  • Paragraph 18 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a pump.
  • Paragraph 19 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the pump is a rotary membrane pump, a peristaltic pump, a rotary pump, a piston pump, or a diaphragm pump.
  • the pump is a rotary membrane pump, a peristaltic pump, a rotary pump, a piston pump, or a diaphragm pump.
  • Paragraph 20 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the pump is a rotary membrane pump.
  • Paragraph 22 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising employing the pump to cause the liquid flow through the cell-culture chamber.
  • Paragraph 23 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the valve is a pneumatic valve, a turning valve, a rotary membrane valve, a shape-memory alloy valve, and/or a solenoid valve.
  • Paragraph 24 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the valve is a rotary membrane valve.
  • Paragraph 25 In some embodiments, a system or method as in any preceding paragraph is provided, wherein at least one port is in fluidic communication with a source of a reagent.
  • Paragraph 26 In some embodiments, a system or method as in any preceding paragraph is provided, wherein at least one port is in fluidic communication with a waste chamber.
  • Paragraph 27 In some embodiments, a system or method as in any preceding paragraph is provided, wherein flowing the liquid through the cell-culture chamber comprises flowing the liquid from the source of the reagent, through a port, and then into the cell-culture chamber.
  • Paragraph 28 In some embodiments, a system or method as in any preceding paragraph is provided, wherein flowing the liquid through the cell-culture chamber comprises flowing the liquid out of the chamber, through a port, and then into the waste chamber.
  • Paragraph 29 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising flowing a fluid through a port, through the valve, and into the cell-culture chamber.
  • Paragraph 30 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising flowing a fluid out of the cell-culture chamber, through the valve, and through a port.
  • Paragraph 31 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the fluid comprises cell-culture media.
  • Paragraph 32 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the valve is capable of placing two or more ports in fluidic communication with the first mixing chamber at the same time.
  • Paragraph 33 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the valve is capable of placing two or more ports in fluidic communication with the second mixing chamber at the same time.
  • Paragraph 34 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising pumping a fluid from a port through the valve and into the first mixing chamber.
  • Paragraph 35 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising simultaneously pumping a first fluid from a first port through the valve and into the first mixing chamber, and pumping a second fluid from a second port through the valve and into the first mixing chamber.
  • Paragraph 36 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising pumping a fluid from a port through the valve and into the second mixing chamber.
  • Paragraph 37 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber contains cells.
  • Paragraph 38 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cells comprise adherent cells.
  • Paragraph 39 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the adherent cells comprise HeLa cells, HEK293 cells, iPSC cells, and/or hMSC cells.
  • Paragraph 40 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cells comprise suspended cells.
  • Paragraph 41 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the suspended cells comprise CHO cells, iPSC aggregates, T cells, HEK 293S cells, and/or Jurkat cells.
  • Paragraph 42 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber consists essentially of a channel.
  • Paragraph 43 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the channel is a microfluidic channel.
  • Paragraph 44 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber has a volume of greater than or equal to 30 microliters and less than or equal to 500 microliters.
  • Paragraph 45 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the surface of the cell-culture chamber is transparent to at least one wavelength of light.
  • Paragraph 46 In some embodiments, a system or method as in any preceding paragraph is provided, wherein at least a portion of the cell-culture chamber is transparent to at least one wavelength of light.
  • Paragraph 47 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber is transparent to at least one wavelength of light.
  • Paragraph 48 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the wavelength of light is greater than or equal to 400 nm and less than or equal to 700 nm.
  • Paragraph 49 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the degree of transparency is greater than or equal to 70%.
  • Paragraph 50 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber is formed from a material that is biocompatible.
  • Paragraph 51 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber is formed from a material that is sterilizable.
  • Paragraph 52 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber is formed from a plastic.
  • Paragraph 53 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the plastic comprises polystyrene and/or PMMA.
  • Paragraph 54 In some embodiments, a system or method as in any preceding paragraph is provided, wherein fluidic dead zones occupy less than or equal to 20 vol% of the cell-culture chamber.
  • Paragraph 55 In some embodiments, a system or method as in any preceding paragraph is provided, wherein fluidic dead zones occupy less than or equal to 5 vol% of the cell-culture chamber.
  • Paragraph 56 In some embodiments, a system or method as in any preceding paragraph is provided, wherein system comprises a cell filter that is a filter, is spiral inertial separator, is a hollow-fiber membrane, retains cells by sedimentation, retains cells via a centrifugal force, retains cells by dielectrophoresis, retains cells by acoustic wave separation, and/or retains cells by deterministic lateral displacement.
  • a cell filter that is a filter, is spiral inertial separator, is a hollow-fiber membrane, retains cells by sedimentation, retains cells via a centrifugal force, retains cells by dielectrophoresis, retains cells by acoustic wave separation, and/or retains cells by deterministic lateral displacement.
  • Paragraph 57 In some embodiments, a system or method as in any preceding paragraph is provided, wherein system comprises a cell filter that is a filter.
  • Paragraph 58 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the filter is a microfiltration membrane or a hollow fiber membrane.
  • Paragraph 59 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the filter is a microfiltration membrane.
  • Paragraph 60 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the filter is flat.
  • Paragraph 61 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the filter is positioned on the valve.
  • Paragraph 62 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cells are retained with an efficiency of greater than or equal to 70%.
  • Paragraph 63 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises the mixing chamber.
  • Paragraph 64 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing chamber is in fluidic communication with the cellculture chamber.
  • Paragraph 65 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing chamber is in fluidic communication with the valve.
  • Paragraph 66 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing element comprises a pump.
  • Paragraph 67 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing element comprises a mechanical mixer.
  • Paragraph 68 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mechanical mixer is a magnetically driven stir bar or a micro-impeller.
  • Paragraph 69 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing element comprises a static mixer.
  • Paragraph 70 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing element is positioned in the cell-culture chamber.
  • Paragraph 71 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising mixing the liquid upstream of the cell-culture chamber.
  • Paragraph 72 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing is performed in the mixing chamber.
  • Paragraph 73 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing is performed in the cell-culture chamber.
  • Paragraph 74 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing comprises performing shuttle flow through a filter that retains cells.
  • Paragraph 75 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing comprises performing looping flow through a peristaltic pump.
  • Paragraph 76 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing comprises performing alternating flow through the mixing chamber and/or through the cell-culture chamber.
  • Paragraph 77 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the mixing comprises performing mechanical mixing.
  • Paragraph 78 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the flow path comprises the cell-culture chamber.
  • Paragraph 79 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the flow path comprises a channel in fluidic communication with the cell-culture chamber.
  • Paragraph 80 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises two mechanically stable orientations, and wherein the mixing comprises flow that occurs when the system is transferred from one of the mechanically stable orientations to the other.
  • Paragraph 81 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the gas-permeable membrane forms a wall of the cell-culture chamber.
  • Paragraph 82 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising flowing a gas through a gas-permeable membrane that forms a wall of the cell-culture chamber.
  • Paragraph 83 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the cell-culture chamber is enclosed in a material that is gas- permeable.
  • Paragraph 84 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the gas-permeable membrane comprises a silicone membrane.
  • Paragraph 85 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the gas-permeable membrane comprises open, gas-permeable pores.
  • Paragraph 86 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the gas comprises a reagent.
  • Paragraph 87 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the gas comprises oxygen.
  • Paragraph 88 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the optical detector comprises a microscope.
  • Paragraph 89 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the optical detector comprises an optical sensor.
  • Paragraph 90 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system further comprises a light source.
  • Paragraph 91 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the light source is a source of fluorescent light.
  • Paragraph 92 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising performing an optical measurement.
  • Paragraph 93 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the optical measurement is performed on the cell-culture chamber.
  • Paragraph 94 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the optical measurement comprises measuring an optical density of the liquid.
  • Paragraph 95 In some embodiments, a system or method as in any preceding paragraph is provided, further determining an amount of cells present in the liquid based on the optical measurement.
  • Paragraph 96 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises two mechanically stable orientations, one of which is suitable for performing cell culture in the cell-culture chamber and the other of which is suitable for performing an optical measurement on the cell-culture chamber.
  • Paragraph 97 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the plurality of ports comprises a port through which cell seeding may be performed.
  • Paragraph 98 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the port is sealed by a septum.
  • Paragraph 99 In some embodiments, a system or method as in any preceding paragraph is provided, wherein cell seeding may be performed by piercing the septum with a needle and delivering cells therethrough.
  • Paragraph 100 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising piercing the septum with a needle and delivering cells therethrough.
  • Paragraph 101 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the port comprises a lid.
  • Paragraph 102 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a reservoir through which cell seeding may be performed.
  • Paragraph 103 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the reservoir comprises a lid.
  • Paragraph 104 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the lid is removed during cell seeding.
  • Paragraph 105 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising pumping the cells from the port or reservoir into the cell-culture chamber.
  • Paragraph 106 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the plurality of ports comprises a port through which cell recovery may be performed.
  • Paragraph 107 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the port is sealed by a septum.
  • Paragraph 108 In some embodiments, a system or method as in any preceding paragraph is provided, wherein cell recovery may be performed by piercing the septum with a needle and withdrawing cells therethrough.
  • Paragraph 109 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising piercing the septum with a needle and withdrawing cells therethrough.
  • Paragraph 110 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the sensor is a dissolved-oxygen sensor.
  • Paragraph 111 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising measuring a dissolved-oxygen content of a liquid present in the system.
  • Paragraph 112 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising delivering an amount of oxygen to the liquid based on the measurement of the dissolved-oxygen content.
  • Paragraph 113 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising delivering a cell-culture medium having a dissolved-oxygen content within a range determined based on the measurement of the dissolved-oxygen content.
  • Paragraph 114 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the sensor is a pH sensor.
  • Paragraph 115 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising measuring a pH of a liquid present in the system.
  • Paragraph 116 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising delivering an amount of an acid and/or a base determined based on the measurement of the pH.
  • Paragraph 117 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising delivering a cell-culture medium having a pH content within a range determined based on the measurement of the pH.
  • Paragraph 118 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises two or more sensors.
  • Paragraph 119 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a sensor for a biological product.
  • Paragraph 120 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the biological product is IgG.
  • Paragraph 121 In some embodiments, a system or method as in any preceding paragraph is provided, further comprising measuring an amount of a biological product in a liquid present in the system.
  • Paragraph 122 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a sensor for carbon dioxide.
  • Paragraph 123 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system further comprises a sensor for carbon dioxide, cell concentration, cell viability, nutrients, metabolites, temperature, pressure, humidity, and/or viscosity.
  • Paragraph 124 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the metabolites comprise lactate and/or glucose.
  • Paragraph 125 In some embodiments, a system or method as in any preceding paragraph is provided, -wherein the system comprises an optical sensor, an electrochemical sensor, and/or an acoustic sensor.
  • Paragraph 126 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the liquid is present in the cell-culture chamber and/or was obtained from the cell-culture chamber.
  • Paragraph 127 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the sensor comprises a sensor plug.
  • Paragraph 128 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the sensor comprises a sensor patch.
  • Paragraph 129 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the liquid is flowed intermittently.
  • Paragraph 130 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the liquid is flowed continuously.
  • Paragraph 131 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the liquid is flowed as part of a batch process.
  • Paragraph 132 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the liquid is flowed as part of a fed-batch process.
  • Paragraph 133 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the liquid is flowed as part of a perfusion process.
  • Paragraph 134 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system is positioned on a chip, and wherein the chip further comprises one or more additional systems.
  • Paragraph 135 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the additional system or systems comprises the same components as the system.
  • Paragraph 136 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a chip comprising two or more modules, and wherein each module comprises an identical set of components.
  • Paragraph 137 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the components comprise the pump, the valve, the cell culture chamber, and/or the sensor.
  • Paragraph 138 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a first chamber and a second chamber, and wherein the first and second chambers are positioned in series between a port through which a sample can be introduced and/or removed and the cell-culture chamber.
  • Paragraph 139 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the first chamber acts as a membrane valve.
  • Paragraph 140 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the second chamber acts as a pump that is configured to pump a known volume of fluid therethrough.
  • Paragraph 141 In some embodiments, a system or method as in any preceding paragraph is provided, wherein a membrane passes through the first chamber and the second chamber.
  • Paragraph 142 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the membrane may be raised and lowered to affect the pressure in the first chamber and/or the second chamber.
  • Paragraph 143 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the membrane may be raised to place the first and second chamber in fluidic communication with each other and lowered to remove the first and second chamber from fluidic communication with each other.
  • Paragraph 144 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a component configured to deliver a gas to the cell-culture chamber.
  • Paragraph 145 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the component configured to deliver a gas to the cell-culture chamber is in fluidic communication with the cell-culture chamber via one or more ports.
  • Paragraph 146 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the component configured to deliver a gas to the cell-culture chamber is configured to deliver the gas in a manner that causes the contents of the cellculture chamber to mix.
  • Paragraph 147 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises a translatable sensor.
  • Paragraph 148 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system comprises two translatable sensors.
  • Paragraph 149 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the two sensors are translatable together.
  • Paragraph 150 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the translatable sensor(s) comprise a pH sensor and/or a dissolved oxygen sensor.
  • Paragraph 151 In some embodiments, a system or method as in any preceding paragraph is provided, wherein the system or method comprises measuring the amount of RNA in a fluid present in the cell-culture chamber and adjusting the fluid composition based on such a measurement.
  • FIGs. 1-9 depict exemplary systems, in accordance with some embodiments.
  • FIGs. 10-53 depict various ways in which liquids can be mixed and associated structures, in accordance with some embodiments.
  • FIGs. 54-58 depict exemplary methods, in accordance with some embodiments;
  • FIG. 59 depicts a method by which cells may be introduced into a cell-culture chamber, in accordance with some embodiments;
  • FIG. 60 depicts a valve, in accordance with some embodiments.
  • FIGs. 61-125 depict exemplary systems and components thereof, in accordance with some embodiments.
  • FIG. 126 depicts photographs that show details associated with experiments performed as described in the Examples, in accordance with some embodiments.
  • FIGs. 127-136 are optical micrographs showing experimental data, in accordance with some embodiments.
  • FIG. 137 is a chart showing confluency as a function of elapsed time, in accordance with some embodiments.
  • FIGs. 138 and 139 are optical micrographs showing experimental data, in accordance with some embodiments.
  • FIG. 140 is a chart showing confluency as a function of elapsed time, in accordance with some embodiments.
  • FIG. 141 is a chart showing oxygen as a function of elapsed time, in accordance with some embodiments.
  • FIG. 142 depicts optical micrographs showing experimental data, in accordance with some embodiments.
  • FIG. 143 is a chart showing confluency as a function of time elapsed post activation, in accordance with some embodiments.
  • FIG. 144 is a chart showing live cell count per image as a function of elapsed time, in accordance with some embodiments.
  • FIG. 145 is a chart showing phase object count per image as a function of elapsed time, in accordance with some embodiments.
  • Systems for performing cell culture and methods of performing cell culture comprise one or more features that facilitates the performance of cell culture in a manner that is relatively advantageous.
  • such systems may comprise one or more features that facilitate the performance of cell culture in conjunction with a component provided as part of the system or external thereto, such as an incubator, an imaging system, and/or external sources of reagents.
  • a component provided as part of the system or external thereto such as an incubator, an imaging system, and/or external sources of reagents.
  • such systems may comprise one or more features that facilitate the use of the system in a flow-through manner, facilitate real-time sensing of one or more properties of the liquid in which the cells are being cultured, and/or allow for the system (and/or one or more components thereof) to have a relatively small volume.
  • a system has a combination of some or all of the above-described advantages that facilitates performing multiplex experiments to assess the impact of different cell-culture conditions on cell growth. Some such experiments may desirably be performed without requiring appreciable reagents and/or cells, and/or may desirably be performed in a single incubator. Some methods described herein may relate to performing cell culture in one or more systems described herein.
  • a system described herein may have one or more advantages. Such advantages may include high throughput (which may facilitate performing reliable statistical analysis and/or the rapid testing of different cell-culture conditions), the ability to perform real-time imaging, the ability to perform uniform cell seeding, the ability to perform mixing and/or cell suspension, the ability to measure one or more properties of a liquid therein (e.g., dissolved oxygen content, pH, IgG content), the ability to control one or more properties of a liquid therein (e.g., dissolved oxygen content, pH), the ability to deliver oxygen to a liquid therein, the ability to deliver reagents to a liquid therein in an automated manner, the ability to perform perfusion, the ability to retain cells, the ability to sample liquid therein (e.g., in a cell-free manner), and/or the ability to recover cells present therein.
  • high throughput which may facilitate performing reliable statistical analysis and/or the rapid testing of different cell-culture conditions
  • the ability to perform real-time imaging the ability to perform uniform cell seeding, the ability to perform mixing
  • Cell culture may comprise growing cells in a system described herein.
  • cells may be present in a cell-culture medium in a system described herein and one or more properties of the system may be selected to promote cell growth.
  • the cells being cultured may be suspended (e.g., in a cell-culture medium) and/or adherent (e.g., to a component of a system described herein).
  • FIG. 1 shows one non-limiting example of a system for performing cell culture described herein.
  • FIG. 1 shows a system 100 comprising a cell-culture chamber 102.
  • the cell-culture chamber 102 shown in FIG. 1 may be configured to allow liquid to flow therethrough.
  • a cell-culture chamber allows liquid to flow therethrough via two ports (e.g., the ports 204A and 204B in FIG. 2).
  • liquid may be capable of flowing into the cell-culture chamber through one of the ports, through some or all of the cell-culture chamber, and out the other port.
  • Each port may comprise an opening on a surface of the cell-culture chamber, and/or may have a different design (e.g., a port may comprise a fitting).
  • a cell-culture chamber may allow liquid to be introduced thereto and/or removed therefrom through a component other than a port.
  • a cell-culture chamber may make up part of a channel through which liquid can flow.
  • liquid can flow into and out of a cell-culture chamber through a variety of surfaces thereof.
  • liquid can flow into and out of a cell-culture chamber through opposing surfaces that are perpendicular to the base of the cell-culture chamber.
  • liquid can flow into and/or out of a cell-culture chamber through different locations (e.g., different ports) positioned on a common surface.
  • FIG. 3 One non-limiting example of a cell-culture chamber having such a design (and further comprising two sensors) is shown in FIG. 3.
  • liquid may flow into, out of, and/or through a cell-culture chamber horizontally, upwards, and/or downwards.
  • a cell-culture chamber comprises one or more sides that are hexagonal. It is also possible for a cell-culture chamber to comprise a side having a shape that is substantially hexagonal (e.g., that is close to a hexagon, but for which one or more vertices have been replaced with short sides, such as sides having a length that is at most 10% of the length of one or more other sides present in the shape).
  • the hexagonal side(s) may be opposing sides, may include a top side and/or a bottom side, and/or may include one or more sides that include the direction of liquid flow through the cell-culture chamber. Without wishing to be bound by any particular theory, it is believed that cell-culture chambers comprising one or more hexagonal sides may exhibit less dead volume than cell-culture chambers having different geometries.
  • a cell-culture chamber comprises a cell filter.
  • the cell-culture chamber 202 may comprise a cell filter. This is shown schematically by the cell filter 206 depicted therein.
  • the cell filter may allow for cells to be retained therein even while liquid is flowing therethrough and/or being exchanged. This may be accomplished in a variety of suitable manners. In some embodiments, this is accomplished by the use of a component proximate to a location from which the liquid exits the cell-culture chamber that prevents flow of the cells out of the cell-culture chamber.
  • a membrane or other component that allows liquid to flow therethrough but that prevents cell flow therethrough may be positioned in such a location
  • a membrane or other component may be positioned on a cell-culture chamber and/or on a valve (e.g., a valve proximate to a cell-culture chamber). This is depicted schematically by the cell filter 206 that is positioned in the cell-culture chamber 202 in FIG. 2.
  • Suitable membranes include microfiltration membranes (e.g., flat microfiltration membranes) and hollow-fiber membranes.
  • a membrane may comprise polyethersulfone (e.g., it may be a polyethersulfone membrane comprising 0.2-micron pores or 5-micron pores).
  • the above-described components may be flat or may comprise one or more bends and/or curves.
  • a cell filter comprises a flow path and/or is positioned in a flow path.
  • Such flow paths may comprise the cellculture chamber and allow for the retention of the cells in the flow path (and, by extension, the cell-culture chamber) while allowing liquid to flow in and out of the flow path.
  • a cell-culture chamber may comprise a cell filter that is located external thereto (e.g., a cell filter that takes the form of a flow path that passes through the cell-culture chamber, a cell filter positioned in a flow path that passes through the cell-culture chamber).
  • a cell filter is a system component that retains cells by sedimentation, via a centrifugal force, by dielectrophoresis, by acoustic wave separation, and/or by deterministic lateral displacement.
  • a cell-culture chamber comprises two or more cell filters.
  • a system described herein comprises a cell-culture chamber and further comprises one or more components that are capable of delivering and/or configured to deliver one or more liquids and/or reagents to the cell-culture chamber.
  • a system described herein may comprise a cell-culture chamber and further comprise one or more components that are capable of removing and/or configured to remove one or more liquids and/or reagents from the cell-culture chamber.
  • Such component(s) may take the form of a pump and/or a valve.
  • FIG. 4 shows one non-limiting embodiment of a system 400 comprising a cell-culture chamber 402 and a pump 408.
  • the pump may be in fluidic communication with the cell-culture chamber (e.g., via a conduit, such as the conduit 410 in FIG. 4).
  • the pump may pump liquid into the cell-culture chamber, such as by supplying a positive pressure to a liquid upstream from the cell-culture chamber. This may cause that liquid to flow downstream into the cell-culture chamber.
  • Liquid flowing into the cell-culture chamber may apply pressure to liquid present in the cell-culture chamber, causing it to flow out of the cell-culture chamber.
  • a pump to pump liquid out of the cellculture chamber by applying a negative pressure to a liquid downstream from the cell-culture chamber and/or to a component through which liquid flows out of the cell-culture chamber.
  • the liquid may be pulled out of the cell-culture chamber by the negative pressure, which may cause liquid upstream from the cell-culture chamber to flow into the cell-culture chamber.
  • a pump may be capable of pumping and/or configured to pump in both directions (e.g., to both apply a positive pressure to a liquid to cause flow in one direction and to apply a negative pressure to cause flow in the opposite direction, to apply positive pressure to a liquid on one side thereof to cause flow in one direction and to apply positive pressure to a liquid on the opposite side thereof to cause flow in the opposite direction). It is also possible for a pump to only be capable of pumping and/or configured to pump in a single direction.
  • Suitable pumps may be employed in the systems described herein.
  • suitable pumps include rotary membrane pumps, peristaltic pumps (e.g., tubing pumps), rotary pumps, piston pumps, diaphragm pumps, motor pumps (e.g., syringe pump-like motor pumps), and roller pumps.
  • a system comprises two or more pumps.
  • the two or more pumps may comprise two of the same pumps and/or two pumps that differ from each other in one or more ways.
  • FIG. 5 shows one example of a diaphragm pump and a manner in which it can be operated.
  • a diaphragm pump may comprise a plurality of pump-valves.
  • the pump-valves may be actuated in a sequence that promotes peristaltic flow through the diaphragm pump. For instance, in order to actuate flow from left to right, the sequence of steps shown in FIG. 5 may be performed. First, the leftmost pump-valve may be opened while the other two pump valves are closed. Then, the middle pump-valve can be opened to allow liquid to flow thereinto. Subsequent to this liquid flow, the leftmost pump-valve can be closed. Then, the middle pump-valve can be closed and the rightmost pump-valve can be opened. Together, this sequence of steps can cause liquid to flow from the leftmost pumpvalve to and through the right-most pump-valve.
  • a system comprising a cell-culture chamber further comprises a valve.
  • FIG. 6 shows one non-limiting embodiment of a system 600 comprising a cell-culture chamber 602, a pump 608, a conduit 610, and a valve 612.
  • the valve may be in fluidic communication with the pump and/or the cellculture chamber. In FIG. 6, this is accomplished via the conduit 614 which places the valve 612 in fluidic communication with the pump 608.
  • the valve may be positioned fluidically between the pump and the cell-culture chamber, or the cell-culture chamber may be positioned fluidically between the pump and the valve.
  • a valve is in fluidic communication with a plurality of ports. With respect to the embodiment in FIG. 6, the valve 612 is in fluidic communication with the ports 616 and 618.
  • a valve is capable of placing and/or configured to place one or more such ports in fluidic communication with a pump and/or a cell-culture chamber.
  • a valve may be capable of placing and/or configured to have two or more positions, each of which places a unique subset of the ports in fluidic communication with the pump and/or the cell-culture chamber. In some embodiments, each position of the valve places exactly one port or zero ports in fluidic communication with the pump and/or the cell-culture chamber.
  • a pump comprises one or more positions that place two or more ports (e.g., each in fluidic communication with a source of a reagent) in fluidic communication with a cell-culture chamber, a pump, and/or other system component (e.g., a mixing chamber) at the same time.
  • a position of a valve may comprise the positioning of one or more components thereof.
  • a valve may comprise a pathway that can be rotated among two or more configurations that place different combinations of ports in fluidic communication with each other through the valve.
  • a valve may comprise multiple sub-valves that may be independently opened or closed, and the position of a valve may comprise the positions of each of the sub-valves.
  • Valves may allow for the composition of a liquid flowing into a cell-culture chamber (e.g., under the action of a pump) to be modified by modifying the position of the valve. For instance, a valve may be switched between positions in which it places different combinations of ports in fluidic communication with each other through the valve. A first such port may be in fluidic communication with a pump and/or a cell-culture chamber.
  • ports may be in fluidic communication with sources of liquids having different compositions (e.g., comprising different reagents, comprising a sample), with other ports (e.g., a port through which cell seeding and/or recovery may be performed), and/or with other portions of the system to which liquid from the cell-culture chamber can be directed (e.g., a mixing chamber, a waste chamber).
  • switching the valve position such that different ports are in fluidic communication the first port may switch the composition of the liquid that can be pumped by a pump into a cell-culture chamber.
  • a system described herein comprises a cell-culture chamber and further comprises one or more mixing components.
  • Such components may be capable of mixing liquid flowing into, liquid flowing through, and/or liquid present in the cell-culture chamber and/or may be configured to mix such liquid. The mixing may assist with cell suspension and/or the mixing of any reagents present in such liquid.
  • Such components may also be provided in combination with pumps, valves, and/or other system components described herein.
  • a system comprises a mixing component that is a mixing chamber.
  • FIG. 7 shows one nonlimiting embodiment of a system 700 comprising a mixing chamber 720.
  • the mixing chamber may be in fluidic communication with the cell-culture chamber. This may allow a liquid to be mixed in the mixing chamber and then, once mixed, transported to the cell-culture chamber. As shown in FIG. 7, fluidic communication between a mixing chamber and a cell-culture chamber may be achieved directly (i.e., with no intervening components) via a conduit (e.g., via the conduit 722).
  • a valve is positioned between a mixing chamber and a cell-culture chamber, and the mixing chamber is in fluidic communication with one of the ports with which the valve is in fluidic communication.
  • FIG. 8 the system comprises a mixing chamber and a cell-culture chamber (labeled therein as “Growth Chamber”) between which a valve and a pump are positioned.
  • the valve and pump shown in FIG. 8 are a rotary membrane valve and rotary membrane pump, respectively.
  • FIG. 8 includes one port that is in fluidic communication with the mixing chamber, one port that is in fluidic communication with the pump and the cell-culture chamber, one port that is in fluidic communication with a waste chamber, and a plurality of further ports that are in fluidic communication with different liquids.
  • FIG. 8 also shows a cell filter (e.g., a membrane) that is configured to retain suspended cells in the cell-culture chamber, a gas-permeable membrane positioned between the cell-culture chamber and the mixing chamber, and further fluidics on opposing sides of the cell-culture chamber and the growth chamber. Possible options for such further fluidics are described in further detail elsewhere herein, and should be understood to be present in some, but not all, embodiments described herein. Similarly, it should be understood that the design shown in FIG. 8 is contemplated for use with other types of pumps, valves, components to retain suspended cells, ports, etc. and/or that systems may comprise some but not all of the components shown in FIG. 8.
  • a cell filter e.g., a
  • FIG. 8 shows an open path that is present during use of the mixing chamber depicted therein, a process that is described in further detail in the text that follows and with reference to FIG. 9.
  • FIG. 9 shows a system having the same design FIG. 8, but depicted flipped vertically.
  • mixing may be accomplished by pumping the liquid to be mixed into the mixing chamber (top), performing mixing therein (not shown), and then pumping the mixed liquid into the cell-culture chamber (bottom).
  • a position for the valve may be selected such that the port in fluidic communication with the pump and the cell-culture chamber is placed in fluidic communication through the valve with the port in fluidic communication with the mixing chamber.
  • the pump may be employed to pump liquid from the cell-culture chamber, through the pump, through the valve, and into the mixing chamber.
  • the liquid may be mixed upon entering the mixing chamber.
  • the pump may be employed to pump the liquid back from the mixing chamber, through the valve, through the pump, and into the cell-culture chamber.
  • a system comprises a mixing component that is a mixing element.
  • Systems described herein may comprise static mixing elements and/or active mixing elements. Additionally, mixing elements may be positioned in a variety of suitable locations in the systems described herein. As two examples, mixing elements may be positioned in and/or associated with mixing chambers (e.g., a system may comprise a mixing chamber comprising a mixing element and/or may comprise a mixing chamber with which a mixing element is associated) and/or mixing elements may be positioned in and/or associated with cell-culture chambers (e.g., a system may comprise a cell-culture chamber comprising a mixing element and/or may comprise a cell-culture chamber with which a mixing element is associated).
  • Mixing elements may mix liquid in a variety of suitable manners.
  • a liquid is mixed, possibly in a chamber (e.g., a mixing chamber, a cell-culture chamber), by being pumped and/or subject to flow.
  • a mixing element may comprise a pump.
  • types of flow that a liquid may be subject to include oscillating flow, shuttle flow, looping flow, and alternating flow.
  • one or more such types of low may comprise flow of the liquid through a cell filter taking the form of a filter that retains cells (e.g., shuttle flow through such a filter), through a pump (e.g., looping flow through a peristaltic pump), and/or through one or more chambers present in the system (e.g., through a mixing chamber and/or a cell-culture chamber, such as alternating flow through either or both such chambers).
  • a cell filter taking the form of a filter that retains cells (e.g., shuttle flow through such a filter), through a pump (e.g., looping flow through a peristaltic pump), and/or through one or more chambers present in the system (e.g., through a mixing chamber and/or a cell-culture chamber, such as alternating flow through either or both such chambers).
  • a mixing chamber may be subject to oscillating flow inside a chamber and/or into and/or out of the chamber.
  • Mixing via oscillating flow is shown schematically in FIG. 10.
  • a liquid being mixed may flow backwards and forwards within a chamber (the central box) and/or into and/or out of the chamber.
  • the liquid flowing into and out of the chamber flows along tortuous conduits (e.g., tortuous channels) prior to entering and after exiting the chamber during the oscillating flow. Flow through such conduits may further assist with mixing.
  • Oscillating flow into, out of, and/or within a chamber may be affected by the use of a pump, such as a peristaltic pump, a piston pump, and/or a diaphragm pump.
  • a pump such as a peristaltic pump, a piston pump, and/or a diaphragm pump.
  • oscillating flow that comprises flow into and out of a chamber may be performed in addition to flow in and/or out of the chamber in a different direction.
  • a chamber may comprise two or more pairs of ports (and/or other components) through which liquid may flow into and/or out of the chamber.
  • One such pair may allow for and/or be associated with one or more components that facilitate oscillating flow and another such pair may allow for and/or be associated with one or more components that facilitate flow into and/or out of the chamber along a different fluidic pathway (e.g., one that comprises a pump and/or a valve).
  • the chamber shown therein allows for oscillating flow into and out of the chamber via one pair of ports and perfusion through the chamber through a different pair of ports.
  • chambers in which oscillating flow is capable of being performed and/or is configured to be performed may further comprise one or more cell filters (e.g., one or more filters and/or membranes).
  • cell filters e.g., one or more filters and/or membranes.
  • Such membranes may include a membrane described elsewhere herein that is suitable for retaining suspended cells, a membrane that is capable of performing and/or configured to perform cross-filtration, a membrane that is capable of performing and/or configured to perform through-filtration, and/or a membrane that is capable of reducing and/or is configured to reduce fouling and/or clogging.
  • suitable membrane is a microfiltration membrane.
  • Membranes that are capable of performing and/or configured to perform crossfiltration may be positioned on a surface through which liquid is not flowing through (not shown). For instance, they may be positioned on surfaces of the chamber lacking ports or other components through which liquid can flow in and out of the chamber and/or may be positioned such that liquid flows across (e.g., instead of through) them.
  • a membrane is capable of performing and/or configured to perform both cross-filtration and through-filtration (e.g., liquid may flow both through the membrane and across the membrane, and both types of flow may provide some filtration effect).
  • cross-filtration and through-filtration e.g., liquid may flow both through the membrane and across the membrane, and both types of flow may provide some filtration effect.
  • FIG. 11 depicts another example of a chamber in which oscillating flow is capable of being performed and/or is configured to be performed.
  • FIG. 11 further shows a system in which such a chamber may be positioned.
  • a system may comprise air chambers that may be filled to apply pressure to a liquid present in the system and emptied to relieve such pressure. Filling and emptying such air chambers may cause oscillating flow.
  • the chamber through which oscillating flow occurs may comprise a channel (e.g., a channel having a microfluidic volume). Without wishing to be bound by any particular theory, it is believed that flow through such channels may occur at relatively high flow rates, which may reduce fouling of cell filters present in such systems.
  • the mixing element takes the form of two diaphragms that form two opposing walls of a chamber (e.g., a mixing chamber, a cell-culture chamber).
  • the diaphragms may be actuated (e.g., back and forth) in order to mechanically mix liquid present in the chamber.
  • a diaphragm may be positioned externally to a wall of a chamber, such as externally to a wall that can be deformed by the diaphragm, and/or for a diaphragm to be positioned inside a wall of a chamber. As shown in FIG.
  • mixing elements that take the form of diaphragms are compatible with the flow of liquid (e.g., perfusion) through the chamber and/or with the use of cell filters (e.g., filters and/or membranes).
  • cell filters e.g., filters and/or membranes.
  • FIGs. 13-15 A slight variation on the mixing element shown in FIG. 12 is depicted in FIGs. 13-15.
  • two diaphragms may be positioned on a common side of a chamber (e.g., an upper side). Such diaphragms may be laterally separated.
  • one diaphragm may be positioned above one part of a chamber (e.g., an upstream part) and one diaphragm may be positioned above a different part of the chamber (e.g., a downstream part).
  • such diaphragms may form parts of a common wall of a chamber (e.g., they may form two parts of a common upper wall thereof).
  • a diaphragm that forms part of a mixing element may comprise a side having a triangular shape (e.g., a side that forms a part of a wall of a chamber). It is also possible for a diaphragm to comprise a side having a shape that is substantially triangular (e.g., that is close to a triangle, but for which one or more vertices have been replaced with short sides, such as sides having a length that is at most 10% of the length of one or more other sides present in the shape). Without wishing to be bound by any particular theory, it is believed that diaphragms that are triangular and/or substantially triangular may desirably cause improved mixing and/or reduced circulation time compared to diaphragms that have other shapes.
  • a chamber e.g., a mixing chamber, a cell-culture chamber
  • an active mixing element positioned internal to the chamber.
  • suitable active mixing elements include mechanical mixers, such as stir bars (e.g., magnetically driven stir bars), impellers (e.g., mechanically driven impellers, micro-impellers), and the like.
  • the chamber shown in FIG. 16 can also allow liquid flow (e.g., perfusion) therethrough and/or be employed in conjunction with a cell filter (e.g., a filter and/or membrane).
  • a cell filter e.g., a filter and/or membrane
  • FIG. 17 shows one embodiment of an exemplary chamber (e.g., a mixing chamber, a cell-culture chamber) comprising an active mixing element and a portion of a system in which it is positioned.
  • the chamber comprises an active mixing element that takes the form of a magnetically driven stir bar that can move within the chamber to mix liquid therein.
  • the chamber shown in FIG. 17 further comprises two ports on two opposing sides of the chamber, each in fluidic communication with a diaphragm pump.
  • the chamber (e.g., a mixing chamber, a cell-culture chamber) shown in FIG. 17 also comprises two membranes, one positioned on top of the chamber and one positioned adjacent one of the ports.
  • the membrane positioned on top of the chamber is an elastic gas filtration membrane that is capable of expanding and contracting to accommodate volume loss (and configured to perform the same).
  • the membrane positioned adjacent to one of the ports is capable of retaining and configured to retain suspended cells.
  • This membrane is also capable of performing and configured to perform cross-filtration (e.g., an active mixing element, such as a magnetically driven stir bar, can create flow parallel to the membrane, causing cross -filtration; in some embodiments, this cross-filtration occurs simultaneously with through-filtration).
  • Each diaphragm pump shown in FIG. 17 comprises a plurality of pump-valves that are capable of being employed and/or configured to be employed to be actuated in a peristaltic fashion to pump fluid. Additionally, each such diaphragm pump is further in fluidic communication with a valve, and each such valve is in fluidic communication with a variety of ports.
  • FIG. 18 shows one non-limiting example of a method of operating a system comprising the chamber shown in FIG. 16 to remove waste from the chamber, deliver reagents thereto, and mix the reagents in the chamber.
  • the top panel of FIG. 18 shows the use of an active mixing element (a magnetically driven stir bar) to mix liquid in the chamber.
  • an active mixing element a magnetically driven stir bar
  • waste may be pumped out of the chamber by actuating the right diaphragm pump, closing the pump-valve in the left diaphragm port closest to the chamber, and employing the valve downstream from the right diaphragm port to place the right diaphragm port and a waste chamber in fluidic communication.
  • liquid in the chamber may flow through the membrane positioned proximate to the end of the chamber closest to the right diaphragm pump, retaining suspended cells in the chamber and allowing liquid comprising waste to flow out of the chamber. The liquid can then flow through the right diaphragm pump, the valve, and into the waste chamber.
  • the bottom panel of FIG. 18 shows the introduction of a reagent into the chamber. This is accomplished by closing the pump-valve in the right diaphragm port closest to the chamber, and employing the valve upstream from the left diaphragm pump to place the left diaphragm pump in fluidic communication with a source of a reagent. When this performed, a liquid comprising the reagent can flow through the valve, the right diaphragm pump, and into the chamber.
  • the valves shown in FIG. 18 are also in fluidic communication with further ports whose operation is not shown in FIG. 18, but which may be present in some embodiments and/or operated in some embodiments.
  • valves may be in fluidic communication with more than one port in fluidic communication with a source of a reagent (e.g., like the valve in fluidic communication with the left diaphragm pump shown in FIG. 18), a port in fluidic communication with a port through which cell seeding and/or cell recovery may be performed (e.g., like the valve in fluidic communication with the left diaphragm pump shown in FIG. 18), a port in fluidic communication with a component (e.g., a sensor) that can determine the amount of IgG in a liquid (e.g., like the valve in fluidic communication with the right diaphragm pump shown in FIG.
  • a component e.g., a sensor
  • a port in fluidic communication with a source of a sample e.g., like the valve in fluidic communication with the right diaphragm pump shown in FIG. 18.
  • the locations of these ports may be as shown in FIG. 18 or may differ therefrom (e.g., they all may be in direct fluidic communication with a single valve and/or may be apportioned between the valves differently than shown in FIG. 18).
  • a system comprises a mixing component that is a flow path configured to promote mixing.
  • the flow path may comprise one or more components that agitate the liquid, thereby causing it to mix, such as one or more pumps and/or one or more mixers (e.g., one or more active mixing element, one or more static mixers).
  • a flow path configured to promote mixing comprises and/or passes through a cell-culture chamber and/or a fluidic channel in fluidic communication therewith (e.g., that is capable of supplying a liquid, such as a liquid flowing through a flow path, to a cell-culture chamber and/or that is configured to do so).
  • the flow path may comprise a component capable of retaining and/or configured to retain cells within the flow path while allowing liquid to enter and/or exit the flow path, such as a cell filter. It is also possible for a single flow path to both be configured to promote mixing and take the form of a cell filter. Further details regarding exemplary flow paths configured to promote mixing are provided below.
  • FIG. 19 depicts one exemplary flow path that is configured to promote mixing.
  • the flow path shown in FIG. 19 comprises a peristaltic pump, and a rotary valve. It should be understood that analogous flow paths are contemplated that comprise different types of pumps and/or valves. Additionally, some embodiments may comprise a flow path like that shown in FIG. 19 but further comprise a cell-culture chamber and/or may be in fluidic communication with a cell-culture chamber. As shown in FIG. 19, a flow path may pass through the pump and the valve to form a loop through which liquid can flow. The liquid may undergo mixing and/or may become suspended when doing so. As shown in FIG. 19, the flow path may comprise a first portion that passes through the valve. This first portion may place upstream and downstream portions thereto in fluidic communication and may lack a membrane or other type of cell filter.
  • FIG. 20 shows one manner in which some liquid may be removed from the flow path shown in FIG. 19 and replaced with a different liquid. More specifically, FIG. 20 shows a manner in which liquid comprising waste may be removed from the flow path and fresh medium may be introduced thereinto. These two actions may be accomplished while retaining suspended cells in the flow path. In some embodiments, the process shown in FIG. 19 may be employed to perform perfusion.
  • the process shown therein may be initiated by selecting a second position for the valve that modifies the flow path such that it no longer comprises the initial portion that passes through the valve (i.e., the first portion) and instead comprises a new, different portion (i.e., a second portion) that passes through the valve (leftmost panel).
  • This second position for the valve may result in a membrane (and/or filter) positioned on the valve being placed upstream of the second portion of the flow path that flows through the valve.
  • liquid being pumped by the pump will encounter the membrane prior to flowing through the valve, and so liquid will flow through the valve while cells suspended therein will be prevented from doing so by the membrane.
  • the valve may be positioned in a third position (middle panel).
  • the third position may place the second portion of the flow path in fluidic communication with a source of a liquid (e.g., a source of a liquid comprising a reagent, a source of liquid cell-culture media) and a waste chamber. Additionally, it may position the second portion of the flow path such that the membrane is the downstream-most portion thereof (or such that substantially all of the second portion of the flow path is upstream from the membrane).
  • the orientation of the second portion with respect to fluid flow may be opposite from its orientation with respect to fluid flow when the valve is in the second position (i.e., the upstream and downstream directions may be opposite from when the valve is in the second position).
  • Flow through the second portion of the flow path may thus cause liquid present therein to flow through the membrane and into a waste chamber.
  • suspended cells present in this liquid may be retained by the membrane.
  • the liquid flowing to the waste chamber may be replaced by liquid originating from the source of the liquid. Such flow may be effectuated by a further pump (not shown).
  • the flow path shown in FIG. 19 may no longer comprise any portion that passes through the valve. In other words, it may not form a flow loop during when the valve is in the third position.
  • action of the pump may be halted while the valve is in the third position and flow may cease (e.g., until the valve is positioned in a different position, such as the first position or the second position).
  • the first and second flow portions of the flow path may take the form of “dead space” (also referred to herein as “dead volume” and “dead zones”).
  • This dead space may have a known volume, which may facilitate the removal of a known volume of liquid from the flow loop (e.g., a known volume of liquid comprising waste) and/or the introduction of a known volume of liquid to the flow loop (e.g., a known volume of fresh cell-culture media).
  • the process shown in FIG. 20 may then comprise positioning the valve back in the second position (rightmost panel).
  • the liquid present in the second portion of the flow path may be flowed into the flow loop (e.g., under the operation of the pump). Liquid may continue to flow until the liquid present in the second portion of the flow path is delivered to the flow loop and/or the second portion of the flow path is filled with liquid comprising waste.
  • the process shown in FIG. 20 may be repeated a number of times.
  • the valve may be once again positioned in the first position.
  • FIG. 21 shows one manner in which cell seeding and cell recovery may be performed for the flow path shown in FIG. 19.
  • cell seeding and cell recovery may be performed by selecting a position for the valve (e.g., a fourth position) that transforms the flow path so that it is no longer a flow loop.
  • the flow path may be in fluidic communication with a portion of the system (e.g., a port) through which cell seeding and/or recovery may be performed.
  • Cell seeding may be performed by actuating the pump such that a liquid comprising cells is flowed from this portion of the system, through the valve (e.g., through the first portion of the flow path), and into the flow path. Liquid initially present in the flow path at this point in time may flow out of the flow path through a different flow path present in the valve (e.g., through the second portion of the flow path).
  • a cell filter such as a membrane, may be present to prevent cells from flowing out of the flow path with the liquid.
  • Cell recovery may be performed by actuating the pump so that the fluid flows in the opposing direction (i.e., out of the flow path and to the portion of the system through which cells may be recovered). Liquid removed from the flow path by this process may be replaced by liquid flowing into the flow path (e.g., through the second portion of the flow path).
  • the liquid flowing into the flow pay may comprise one or more reagents and/or fresh cell-culture media.
  • FIG. 22 shows another example of a flow path that is configured to promote mixing.
  • the flow path shown in FIG. 22 passes through a valve, two pressurizable membranes, and two pinch valves.
  • the two pressurizable membranes may be pressurized to cause flow through the flow path.
  • the pinch valves may allow for cell seeding and/or cell collection. Liquid flowing through the path shown in FIG. 22 may undergo mixing as it flows.
  • the flow path shown in FIG. 22 may comprise a cell-culture chamber and/or be in fluidic communication with a cell-culture chamber.
  • the valve when liquid is flowing through the flow path, the valve may be in a first position in which a first portion of the flow path passing through the valve is positioned such that liquid passes therethrough. This first portion may place upstream and downstream portions thereto in fluidic communication and may lack a membrane or other type of cell filter.
  • FIG. 23 shows one non-limiting example of a manner in which some liquid may be removed from the flow path shown in FIG. 23 and replaced with a different liquid (e.g., by which perfusion may be performed).
  • the manner in which this is performed may comprise selecting a set and ordering of positions for the valve that mirrors that shown in FIG. 20.
  • the valve may be positioned in the second position and the second portion of the flow path may be filled with liquid originating from the flow path but from which suspended cells have been removed by a membrane (leftmost panel).
  • the valve may be positioned in a third position in which the second portion of the flow path is arranged such that the membrane is the downstream-most portion thereof (or such that substantially all of the second portion of the flow path is upstream from the membrane).
  • the second portion When in this position, the second portion may be supplied with liquid originating from a source (and, possibly comprising a reagent and/or fresh cell-culture media) and/or liquid initially present in the second portion may be flowed into a waste chamber.
  • the valve may be again positioned in the second position, in which the liquid introduced into the second portion may be supplied to the flow path and cells present in the flow path may be prevented from flowing into the second portion by the membrane. As in FIG. 20, these steps may be repeated multiple times and/or the valve may be again positioned in the first position at the conclusion of this process.
  • FIG. 24 shows a third example of a flow path that is configured to promote mixing.
  • the flow path that is shown in FIG. 24 takes the form of a loop that passes through a cellculture chamber, a pump, and a hollow-fiber membrane. Liquid flowing through this loop may be mixed during the flow.
  • the flow may be caused by one or more pumps.
  • a pump in fluidic communication with the cell-culture chamber but not part of the flow path may cause liquid to flow into the cell-culture chamber (e.g., liquid comprising a reagent, liquid comprising fresh cell-culture media).
  • liquid comprising a reagent e.g., liquid comprising a reagent, liquid comprising fresh cell-culture media.
  • the pump through which the flow path passes e.g., the middle pump in FIG.
  • a pump downstream from the hollow-fiber membrane may cause liquid to flow through the hollow-fiber membrane and out of the flow loop.
  • the hollowfiber membrane may allow liquid to flow therethrough out two different outlets but only allow cells suspended in the liquid to flow out of one such outlet.
  • the outlet through which suspended cells cannot flow may allow liquid flowing therethrough to exit the flow loop and/or the outlet through which the suspended cells can flow may supply the flow loop.
  • the liquid flowing through the hollow-fiber membrane and out of the flow loop may comprise waste and/or other components that it would be desirable to remove from the flow loop.
  • the suspended cells may continue to flow through the flow loop.
  • FIG. 24 depicts a hollow-fiber membrane
  • other types of cell filters e.g., membranes and filters
  • flow paths having the design shown in FIG. 24 instead of and/or in addition to a hollow-fiber membrane.
  • FIGs. 25-27 show further examples of flow paths similar configured to promote mixing similar to that shown in FIG. 24.
  • FIG. 25 shows that the hollow-fiber membrane may pass through and/or be present in a perfusion chamber.
  • FIG. 25 also shows that the cellculture chamber depicted therein (labeled as a “growth chamber”) may further comprise one or more sensors.
  • FIG. 26 presents a three-dimensional schematic view of a flow path similar to the flow paths shown in FIGs. 24 and 25.
  • FIG. 26 also shows that liquid may flow into a perfusion chamber that liquid is flowing out of (e.g., a perfusion chamber that a hollow-fiber membrane passes through).
  • such liquid may comprise liquid cellculture media (e.g., fresh liquid cell-culture media) and/or one or more reagents (e.g., one or more reagents present in a liquid and/or originating from a source).
  • a cell-culture chamber (labeled as a “growth chamber”) may further comprise one or more components that allow for and/or facilitate gas exchange.
  • FIG. 27 presents a three-dimensional schematic view of a flow path similar to the flow paths shown in FIGs. 24-26.
  • the perfusion chamber has a cylindrical design instead of the cuboid design shown in FIG. 26.
  • cylindrical perfusion chambers may be capable of providing similar functionality to cuboid perfusion chambers having larger volumes.
  • FIG. 28 shows a fourth example of a flow path that is configured to promote mixing.
  • the flow path shown in FIG. 28 is similar to the flow paths shown in FIGs. 24-27 but includes a spiral inertial separator instead of a hollow-fiber membrane. Accordingly, fluid may flow through the flow path shown in FIG. 28 in a manner similar to that described in the preceding paragraph with respect to the flow path depicted in FIGs. 24-27.
  • the spiral inertial separator may comprise two outlets: one that it directs suspended cells to flow through and that is a portion of the flow loop shown in FIG. 28 and one that it directs suspended cells away from and that flows out of the flow path shown in FIG. 28.
  • Liquid flowing through the flow path shown in FIG. 28 may thus be divided into two portions: one that continues to flow through the flow path (and, possibly, comprises suspended cells) and one that exits the flow path after flowing through the spiral inertial separator.
  • FIG. 29 shows a fifth example of a flow path that is configured to promote mixing.
  • the flow path shown in FIG. 29 comprises both a spiral inertial separator and a hollow-fiber membrane.
  • the same symbols shown in FIGs. 24-28 to depict pumps, hollow-fiber membranes, spiral inertial separators, and cell-culture chambers are employed in FIG. 28 to depict these same features.
  • the flow path shown in FIG. 29 is similar to that shown in FIG. 28, but includes a further flow loop downstream from the spiral inertial separator. As shown in FIG. 29, this flow loop comprises a further pump and a hollow-fiber membrane. Liquid flowing out of the spiral inertial separator through the outlet for the liquid that lacks suspended cells is flowed into this flow loop. Upon entering the second flow loop of FIG.
  • the hollow-fiber membrane shown in FIG. 29, may comprise two outlets: one through which liquid lacking suspended cells may flow and the other through which liquid either comprising or lacking suspended cells may flow.
  • the liquid flowing through the flow path depicted in FIG. 29 may be subjected to a further filtering step in comparison to liquid flowing through the flow path depicted in FIG. 28. Any suspended cells not removed by the spiral inertial separator may exit the hollow-fiber membrane and be recirculated back to the cell-culture chamber via the second flow loop while liquid may exit the second flow loop via the hollow-fiber membrane.
  • FIG. 29 depicts a hollow-fiber membrane
  • other types of cell filters e.g., membranes and/or filters
  • flow paths having the design shown in FIG. 29 instead of and/or in addition to a hollow-fiber membrane.
  • FIG. 30 shows a sixth example of a flow path that is configured to promote mixing.
  • the flow path shown in FIG. 30 passes through a cell-culture chamber (labeled in FIG. 30 as a “growth chamber”), a spiral inertial separator (shown in the top left portion of FIG. 30 as a spiral), either a hollow-fiber membrane or a channel parallel thereto, a pump, and a chamber comprising a static mixing element. Liquid flowing through this flow path may be mixed during the flow and/or by the static mixing element.
  • liquid exiting the spiral inertial separator may be divided into liquid that is relatively rich in suspended cells and liquid that is relatively poor in suspended cells. The former liquid may flow directly into a pump that causes fluid to flow along the flow path.
  • the latter liquid may pass through a hollow-fiber membrane, in which it is further divided into liquid that is even poorer in suspended cells and liquid that contains some or all of the suspended cells in the latter liquid.
  • the liquid that is even poorer in suspended cells may flow out of the flow path and to a waste chamber.
  • the other liquid may flow into the pump.
  • one or more branches into which a flow path divides comprises a component that directs a higher volume of fluid to flow along that branch and/or comprises a component that directs a higher volume of fluid to flow along the other branch.
  • FIG. 30 depicts a flow restrictor (represented by a narrowing of the channel) downstream from the hollow-fiber membrane. The flow restrictor increases the resistance along that branch of the flow path, thereby directing a higher percentage of the liquid to flow through the branch parallel thereto (i.e., the branch accessed by the outlet of the spiral inertial separator that outputs a liquid that is fairly rich in suspended cells).
  • liquid may flow into the flow path depicted therein, such as via a port and/or a junction positioned between the pump and the chamber comprising the static mixing element.
  • a flow path passes through a pump.
  • the flow path shown in FIG. 30 passes through a peristaltic pump.
  • one or more portions of the flow path e.g., the portion of the flow path passing through a pump
  • a material e.g., tubing
  • the flow path may be enclosed by a gas-permeable membrane (e.g., semipermeable membrane that allows one or more gases to cross but does not allow liquid to cross).
  • a material that allows gas exchange and/or is configured to promote gas exchange and encloses a portion of a flow path is further enclosed by a chamber that controls gas exchange, such as a sealed pressure chamber.
  • FIG. 30 depicts a hollow-fiber membrane
  • other types of cell filters e.g., membranes and/or filters
  • FIG. 30 depicts a static mixing element
  • a flow path it is also possible for a flow path to have a design similar to that shown in FIG. 30 but to, additionally or alternatively, comprise an active mixing element.
  • cell filters other than a spiral inertial separator could be employed in a flow path having some aspects of the design shown in FIG. 30.
  • FIGs. 31 and 32 depict further possible aspects of the flow path shown in FIG. 30.
  • FIG. 31 further shows that an IgG line can be present (e.g., to recover a liquid for analysis of its IgG content, such as via an IgG sensor).
  • FIG. 32 also depicts other components of this flow path with more particularity. It should be understood that some flow paths may have one or more features that are similar to those shown in FIG. 31 but that differ from FIG. 32 in one or more ways.
  • FIG. 33 shows a seventh example of a flow path that is configured to promote mixing.
  • the flow path shown in FIG. 33 passes through a cell-culture chamber (labeled in FIG. 33 as a “growth chamber”) that comprises cell filters (e.g., membrane filters, such as microfiltration membranes) proximate to its two points of fluidic communication with the flow path.
  • cell filters e.g., membrane filters, such as microfiltration membranes
  • liquid can be flowed through this flow path in two directions (e.g., through the cell-culture chamber from left-to-right and through the cell-culture chamber from right- to-left).
  • the direction of flow can be selected by selecting positions of valves in the flow path.
  • liquid can enter the flow path and be directed by a valve to either flow on either a left or a right conduit emanating from a branching point upstream of the cellculture chamber.
  • Liquid can then flow into the cell-culture chamber through the cell filter fed by the relevant conduit, through the cell-culture chamber, and out the other membrane.
  • Liquid can then continue to flow towards a valve that directs the liquid to continue to circulate in the flow path (e.g., back to the first cell filter) or out of the flow path (e.g., to waste, for IgG analysis).
  • this may allow for liquid present in and/or flowing through the cell-culture chamber (e.g., liquid cell-culture media) to be backflushed through each cell filter, which may assist with reducing the fouling of these filters.
  • flow through a flow path like that shown in FIG. 33 may be driven by a pump, such as the piston pump shown in FIG. 33.
  • FIG. 34 shows one non-limiting example of a suitable staging well.
  • the staging well may comprise an outlet that supplies a flow path.
  • the staging well may also comprise one or more inlets.
  • the inlet(s) may comprise one or more inlet(s) suitable for receiving liquid(s) (e.g., liquids comprising reagents, liquid cell-culture media) and/or one or more inlet(s) suitable for receiving gas(es) (e.g., a gas to be dissolved in a liquid and/or undergo exchange with a liquid).
  • liquid(s) e.g., liquids comprising reagents, liquid cell-culture media
  • gas(es) e.g., a gas to be dissolved in a liquid and/or undergo exchange with a liquid.
  • one or more gases and one or more liquids may undergo exchange in the staging well, which may allow for the introduction of liquid into the flow path that comprises a desirable amount of gas. This exchange may occur across a gas-liquid interface (e.g., the gas inlet(s) may be positioned above the liquid inlet(s), which may result in the formation of a gas-liquid interface in which the gas is above the liquid).
  • a gas-liquid interface e.g., the gas inlet(s) may be positioned above the liquid inlet(s), which may result in the formation of a gas-liquid interface in which the gas is above the liquid).
  • a staging well further comprises a mixing element.
  • the staging well depicted therein comprises a magnetically driven stir bar.
  • FIG. 35 shows an eighth example of a flow path that is configured to promote mixing. Like the seventh flow path shown in FIG. 34, this flow path can allow for liquid to flow therethrough in opposing directions.
  • the left-hand side of FIG. 35 shows liquid flow through this flow path in a first direction.
  • liquid may enter the flow path on the right-hand side, flow into a cell-culture chamber, through a cell filter positioned therein (e.g., a membrane filter, as shown in FIG. 35), into a pump, and then out of the flow path. It is also possible for liquid to enter the flow path on the left-hand side, flow through the pump, through the cell filter, and out of the flow path. This direction of flow is shown on the righthand side of FIG. 35.
  • the direction of flow may be determined by actuating the pump.
  • the pump may be actuated to cause the liquid present in the flow path to flow back and forth (e.g., through the cell filter), which may promote mixing of the liquid (and, possibly, associated mixing of any components therein, such as reagents, and/or suspension of cells therein).
  • Such flow may also cause backflushing of the cell filter, which may assist with preventing and/or reducing fouling thereof.
  • a flow path having such a design may comprise a pump that is a piston pump and/or may comprise a pump comprising an air pocket.
  • the presence of an air pocket between one or more portions of the pump (e.g., a piston in a piston pump) and liquid flowing in the flow path may desirably reduce and/or prevent contamination of such portion(s) of the pump.
  • a piston pump may cause liquid to flow by actuating the piston up and down to affect the pressure in the flow path.
  • the flow path depicted therein may comprise pinch valves (e.g., to the left of the pump and the right of the cell-culture chamber).
  • pinch valve(s) may be capable of opening and closing and/or configured to be opened and closed. Open valves may permit liquid to flow therethrough (e.g., into or out of the flow path) and closed valves may prevent liquid flow therethrough (e.g., when it is desirable to retain the liquid present in the flow path therein).
  • FIG. 35 also shows that the cell-culture depicted therein may be covered by a gasexchange membrane (e.g., a semipermeable membrane) and/or may comprise a cell filter that retains cells in the cell-culture chamber.
  • the former may promote and/or allow for gas exchange.
  • Liquid may be supplied to the flow path shown in FIG. 35 from a source (e.g., a source of liquid cell-culture media, a source of a reagent).
  • a source e.g., a source of liquid cell-culture media, a source of a reagent.
  • FIG. 36 shows a further example of flow through the flow path shown in FIG. 35 in which liquid is flowed from the right to the left side and liquid is introduced into the flow path.
  • liquid may be introduced into the flow path via a dispensing well proximate the right side of the flow path.
  • the right pinch valve may be opened to allow for flow from the dispensing well into the cell-culture chamber.
  • the pump may be employed to reduce the pressure in the flow path (e.g., by raising the piston for a piston pump).
  • Liquid may be introduced into the dispensing well via a reagent-dispensing head or in any other suitable manner.
  • the dispensing well may have a design such that, during and/or subsequent to introducing liquid thereinto, the liquid does not fill the entirety of the dispensing well. This may result in an air gap between the reagent-dispensing head and the liquid in the dispensing well during the liquid introduction process.
  • the liquid may comprise reagents, cell-culture media, and/or cells.
  • FIG. 37 shows a further example of flow through the flow path shown in FIG. 35 in which liquid is flowed from the right to the left side and liquid is removed from the flow path.
  • liquid may be removed from the flow pay by flowing it past the left pinch valve (which may be opened during this process).
  • Such liquid may then be flowed to a waste chamber and/or to a further system component for analysis of IgG content (e.g., with the use of an IgG sensor).
  • the right pinch valve may be closed and the pressure in the flow path may be increased (e.g., by raising the piston of a piston pump). This may apply positive pressure to the liquid, causing it to flow out of the flow path as shown in FIG. 37.
  • FIG. 38 shows a ninth example of a flow path that is configured to promote mixing.
  • the flow path shown in FIG. 38 passes beneath a pathway along which a roller can roll at two locations (e.g., the flow path may have a U-shape for which opposing ends of the U are positioned beneath such a pathway).
  • the roller may be rolled across one of these locations, compressing a conduit enclosing the flow path and thereby applying a pressure to the liquid present in the flow path. This pressure may cause liquid in the flow path to flow through the flow path away from the roller (e.g., towards the other location passing beneath the pathway along which the roller can roll).
  • flow through the flow path can be initiated by rolling the roller and the direction of flow can be selected by the location over which the roller is rolled.
  • Flow through the flow path caused in this manner may cause the flowing liquid to mix and/or for cells to be suspended in the flowing liquid.
  • liquid mixed in this manner may take the form of a slug that occupies less than the full spatial extent of the flow path.
  • the flow path shown in FIG. 38 may be enclosed by a conduit that can be deformed upon the application of pressure thereto by a roller. As shown in FIG. 38, this may take the form of an elastic material, such as a silicone membrane. In some such embodiments, the elastic material (and/or another material) may allow for gas exchange as described elsewhere herein. As also shown in FIG. 38, the flow path may pass through a growth chamber.
  • the flow path may also comprise one or more ports through which one or more liquids may be delivered (e.g., a liquid comprising a reagent, liquid cell-culture media), one or more ports through which gas (e.g., air) may be removed from the flow path, and/or one or more ports through which waste may be removed (e.g., one or more ports in fluidic communication with a conduit in fluidic communication with a waste chamber).
  • one or more liquids e.g., a liquid comprising a reagent, liquid cell-culture media
  • gas e.g., air
  • Some flow paths configured to promote mixing have a fixed volume.
  • liquid being removed therefrom e.g., to a waste chamber
  • another fluid e.g., another liquid
  • such liquid is replaced by liquid cell-culture media (e.g., fresh liquid cellculture media).
  • liquid cell-culture media e.g., fresh liquid cellculture media
  • the amount of liquid entering and exiting a flow path makes up less than 10%, less than 7.5%, less than 5%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, or less than 0.2% of the total amount of liquid in the flow path (e.g., and greater 0.1%, greater than 0.2%, greater than 0.5%, greater than 0.75%, greater than 1%, greater than 2%, greater than 5%, or greater than 7.5% of the total amount of liquid in the flow path).
  • Some flow paths configured to promote mixing may undergo one or more processes prior to the introduction of cells thereinto.
  • a flow path may be cleaned, primed, and/or degassed before the introduction of cells thereinto.
  • a system comprises two or more flow paths that are configured to promote mixing.
  • the two or more flow paths may be in fluidic communication with one or more common system components.
  • a system may comprise two or more flow paths that are in fluidic communication with a common component that assists with cell retention and/or a common component through which one or more liquids and/or reagents (e.g., fresh cell-culture media) may be delivered to the flow paths.
  • one or more of the flow paths may lack such components.
  • designs comprising two or more flow paths that are configured to promote mixing may facilitate multiplexing and/or reduce the need for multiple, duplicative components in multiplexed designs.
  • each flow path comprises a cell-culture chamber (labeled as a “Growth chamber”) and a pump (e.g., a peristaltic pump). Operation of the pumps causes liquid present in the flow paths to flow around the flow paths through the cell-culture chambers and the pump.
  • a cell-culture chamber labeled as a “Growth chamber”
  • a pump e.g., a peristaltic pump
  • each flow path depicted therein is in fluidic communication with two ports, one of which places the relevant flow path in fluidic communication with a hollow-fiber membrane upstream thereof and one of which places the relevant flow path in fluidic communication with the hollow-fiber membrane downstream therefrom.
  • liquid flowing through both of the flow paths depicted therein can thus be removed from the relevant flow path through the first port, passed through the hollow-fiber membrane, and then resupplied to the relevant flow path (or to the other flow path).
  • a variety of suitable ports may be employed, one non-limiting example of which is a rotary shear valve.
  • cells that are suspended therein may be retained in the liquid that is resupplied to one of the two flow paths while a portion of the liquid in which they are suspended is removed through an alternate portion of the hollow-fiber membrane. This separation may allow for the removal of liquid waste, as this liquid may be directed to a waste chamber.
  • the ports depicted therein that place the flow paths in fluidic communication with liquid flowing out of the hollow-fiber membrane are also in fluidic communication other flow paths present in the system.
  • Such flow paths may be in fluidic communication with sources of liquids, such as sources of reagents and/or cell-culture media. Accordingly, these ports may also be employed to supply the flow paths depicted in FIG. 39 with such liquids.
  • FIG. 39 shows an exemplary piston pump that may be employed for this purpose. It is also possible for other types of pumps, such as other pumps described herein, to be present instead of and/or in addition to a piston pump. Similarly, although a hollow-fiber membrane is depicted in FIG. 39, it is also possible for other types of cell filters (e.g., membranes and/or filters) to be employed instead of or in addition to hollowfiber membranes. As shown in FIG. 40, systems having the design shown in FIG. 39 (and/or a design like that shown in FIG. 39) may be divided into two or more modules. With reference to FIG.
  • the system depicted therein comprises a filtration module comprising the hollowfiber membrane and the piston pump and two modules that each comprise a cell-culture chamber (labeled therein as “Growth chambers”).
  • a filtration module comprising the hollowfiber membrane and the piston pump and two modules that each comprise a cell-culture chamber (labeled therein as “Growth chambers”).
  • systems comprising multiple modules may be capable of flowing liquid between and/or through different modules therein and/or configured to perform either or both such functions.
  • the system may further comprise one or more components that reduce cross-contamination between different liquids flowing primarily in different flow paths that also at least partially flow through a common module and/or system component.
  • common modules and/or system components (and/or the system as a whole) may be capable of being sterilized and/or configured to be sterilized. The sterilization may be performed subsequent to the flow of a liquid from one flow path through the relevant module and/or system component and prior to the flow a liquid from a different flow path through that module and/or system component.
  • FIG. 41 depicts one non-limiting example of a module present in the systems shown in FIGs. 39 and 40.
  • the module may comprise one or more ports through which liquid can be introduced into the module.
  • a module may comprise a seeding port (e.g., a well and/or a reservoir) through which cells can be seeded and/or one or more ports (e.g., as depicted in FIG. 41, two ports) in fluidic communication with another module (e.g., a filtration module).
  • the port(s) in fluidic communication with other module(s) may allow for liquid to be transferred between modules.
  • FIG. 41 also depicts a pump that can be employed to cause flow through the module. As noted in FIG.
  • FIG. 41 shows a gas-permeable membrane (referred to therein as a semipermeable membrane).
  • the gas-permeable membrane may allow for any gases dissolved in liquid present in the module to be removed therefrom and/or for gas present in an environment external to the module to diffuse through the gas-permeable membrane and into the liquid.
  • FIGs. 42-43 depict one non-limiting example of a manner in which liquid may be flowed through the cell-culture chamber while retaining cells therein.
  • old liquid cell-culture media is flowed out of the cell-culture chamber and replaced with fresh liquid cell-culture media while retaining the cells therein.
  • the old liquid cell-culture media may be flowed out of a growth chamber, out of the module therein via a port, and into the hollow fiber membrane (labeled therein as a “cross flow filter” and “filter”).
  • the cells in the old liquid cell-culture media may be retained by the hollow fiber membrane and subsequently returned to the module from which they originated (and the growth chamber therein).
  • the old liquid cell-culture media may exit the hollow fiber membrane via a different path and be pumped out of the system and/or to a waste chamber by a pump (e.g., the piston pump depicted in FIG. 42).
  • a pump e.g., the piston pump depicted in FIG. 42.
  • fresh liquid cell-culture media may be added to the module by flowing it from a source thereof and into the module (e.g., in some embodiments, like the embodiment shown in FIG. 42, through a port and/or through a valve placing the module in fluidic communication with the source of the liquid cell-culture media).
  • the fresh liquid cell-culture media may comprise one or more reagents.
  • a system may have one or more features that facilitates the above-described liquid cell-culture media exchange.
  • a system may comprise one or more components that are capable of accommodating different volumes of liquid and/or are capable of undergoing a volume change in response to a change in the volume of liquid therein.
  • the cell-culture chamber has this property.
  • a system may comprise one or more components that can achieve this functionality because they are formed from a material that is flexible (and/or comprise one or more portions that are formed from such a material).
  • some cells may initially be retained outside of a cell-culture chamber (labeled in FIG. 43 as a “growth chamber”) but still in a flow path comprising the cell-culture chamber. Such cells may be reintroduced into the cell-culture chamber by flowing a liquid through the hollow-fiber membrane and into the cell-culture chamber (e.g., via a port).
  • the liquid employed for this purpose is old liquid cell-culture media. For instance, some old liquid cell-culture media may be pumped back by the pump it was initially pumped towards, through the hollow-fiber membrane, and into the cell-culture chamber.
  • sterilization may be performed by flowing one or more fluids (e.g., one or more cleaning fluids, such as one or more cleaning solvents) through one or more components of the system that can be subject to flow originating in and/or terminating at more than one module.
  • sterilization may be performed by flowing one or more fluids through a hollow-fiber membrane and/or one or more conduits and/or valves. The fluid may sterilize and/or assist with sterilizing the component(s) through which it flows.
  • fluids e.g., one or more cleaning fluids, such as one or more cleaning solvents
  • liquid growth media e.g., fresh liquid growth media
  • cell-culture media may be flowed through some or all such components.
  • FIGs. 45 and 46 depict further examples of a system comprises multiple flow paths that are configured to promote mixing. More specifically, FIG. 45 depicts an example of a system that comprises multiple flow paths, each having the design shown in FIG. 41. As can be seen from FIG. 45 and FIG. 41, each such flow path may have two locations at which liquid can be supplied to the flow path and/or removed from the flow path: upstream and downstream from the pinch valves. FIG. 45 shows that, if such flow paths are arranged appropriately, they can be positioned so that these locations are aligned for each flow path (i.e., that, for each flow path, the pinch valves proximate the pump are aligned and the pinch valves proximate the cell-culture chamber are aligned).
  • sources of liquid e.g., sources of liquid cell-culture media, sources of reagents
  • conduits in fluidic communication with a waste chamber.
  • sources of liquid e.g., sources of liquid cell-culture media, sources of reagents
  • Such access may be provided as desired when it is desired to supply a particular liquid to a particular flow path or remove waste therefrom.
  • Such access may be further facilitated by one or more components that allow for facile movement of sources of liquids and conduits in fluidic communication with a waste chamber, such as motorized components.
  • liquid may be dispensed into a flow path via a liquiddispensing head, such as a reagent-dispensing head.
  • a liquiddispensing head such as a reagent-dispensing head.
  • an air gap between a reagent-dispensing head and any liquid in a dispensing well may facilitate the dispensing of reagents among multiple flow paths from a single reagent-dispensing head without appreciable contamination thereof.
  • FIG. 46 depicts an example of a system related to that shown in FIG. 45. It shows that, in some embodiments, a bracket may be employed to hold and/or seal one or more system components (e.g., pumps in a plurality of flow paths).
  • a system may comprise a conduit in fluidic communication with a system component that is capable of determining an IgG content of a liquid recovered from a flow path and/or configured to determine such an IgG content (e.g., an IgG sensor).
  • a system component that is capable of determining an IgG content of a liquid recovered from a flow path and/or configured to determine such an IgG content (e.g., an IgG sensor).
  • a system comprises a mixing component that is a component capable of being rotated and/or configured to be rotated (in other words, a rotatable component).
  • the rotation may be performed by another component of the system (e.g., a component capable of rotating the rotatable component and/or configured to rotate the rotatable component).
  • the rotation may cause liquid present in the rotatable component to be mixed.
  • a rotatable component comprises one or more other system components described elsewhere herein (e.g., a cell-culture chamber, a cell filter, a flow path).
  • a cell-culture chamber is rotatable, which may render it both a mixing component (i.e., a rotatable component) and a cell-culture chamber. It is also possible for the system, as a whole, to be rotatable (i.e., for the system to consist of a rotatable component) and/or to consist essentially of a rotatable component.
  • a rotatable component may comprise two or more orientations that are mechanically stable.
  • the rotatable component may comprise two or more orientations that the rotatable component may stably rest in indefinitely unless an external force is applied (e.g., a rotational force).
  • the mechanical stability may be due to the distribution of weight and/or the shape of the rotatable component (e.g., alone) and/or may be facilitated by the presence of one or more system components also present in the system (e.g., one or more grooves, tabs, and/or other features that enhance the mechanical stability of the rotatable component when positioned appropriately).
  • Rotating a rotatable component may comprise rotating the rotatable component between mechanically stable orientations.
  • the rotatable component may be positioned in the first mechanically stable orientation during cell culture and positioned in the second mechanically stable orientation during the performance of optical measurements.
  • the rotatable component may be rotated therebetween during a transition from cell culture to optical measurement and vice versa.
  • this may facilitate performing cell culture while the rotatable component is in a mechanically stable orientation that facilitates doing so (e.g., in which it is relatively facile to flow liquid through the cell-culture chamber) while also allowing for the cell-culture chamber therein to be readily subject to optical measurements that might require and/or be facilitated by a different geometry (e.g., a geometry that is more compatible with an optical detector but less suitable for cell-culture).
  • FIGs. 47 and 48 show one non-limiting example of a rotatable component comprising two mechanically stable orientations.
  • the rotatable component may comprise one orientation that is suitable for flow therethrough and/or perfusion (e.g., the orientation shown in FIG. 47) and one other orientation (e.g., the orientation shown in FIG. 48).
  • the other orientation may solely be suitable for mixing liquid present in the rotatable component or may be suitable for one or more further purposes.
  • the other orientation may be suitable for imaging liquid present in the rotatable component.
  • FIG. 47 shows one example of the flow of liquid through an exemplary rotatable component.
  • liquid may be introduced into the rotatable component via one or more ports and may be flowed therethrough upon the application of pressure (e.g., supplied by another port, such as a port supplying a pressurized gas or liquid). The flow rate may be adjusted by adjusting the amount of pressure applied.
  • the flow of liquid through the rotatable component may comprise flow in multiple directions, including upwards, downwards, and/or laterally.
  • the liquid Upon flowing out of the rotatable component, the liquid may be directed to a waste chamber (as shown in FIG. 47) and/or to a component for determining an IgG content of the liquid (e.g., an IgG sensor).
  • FIG. 48 shows one example of a rotatable component through which liquid is not flowing. In some instances, such as during imaging of a chamber present in a rotatable component, it may be desirable for liquid not to be flowing therethrough.
  • rotatable components may further comprise additional mixing components.
  • a rotatable component may further comprise a mixing element, such as a magnetically driven stir bar.
  • a rotatable component may also have one or more structural features that promote mixing.
  • the rotatable component comprises a partition that divides liquid flowing thereinto. When liquid flows thereinto and/or the rotatable component is rotated, liquid may flow up through the central conduit defined by the partition and/or down through one or both side conduits defined by the partition. This may promote convection and/or rotating flow in the rotatable component, which may promote mixing.
  • FIG. 50 shows a second example of a rotatable component. It should be noted that this component is also depicted in FIG. 38 and is also a flow path as described with respect to FIG. 38.
  • a rotatable component is capable of being rotated and/or configured to be rotated upon application of a centrifugal force. Application of such a force may cause liquid therein to migrate away from the axis around which the rotatable component is being rotated. When application of such a force is combined with the opening of a port through which liquid may be removed from the rotatable component, liquid present in the rotatable component may be so removed. This is shown schematically in the right-hand side of FIG.
  • the application of such force may be employed to remove old liquid cell-culture media from the rotatable component. It is also possible for cells to remain in the rotatable component during such removal. This may be facilitated by a cell filter that retains cells in the rotatable component and/or by the formation of a cell pellet that is too large to be removed from the rotatable component. Such a cell pellet may be formed upon the application of the centrifugal force if it is sufficiently high. Alternatively, centrifugal force may be applied to create a cell pellet without the associated removal of liquid from a rotatable component.
  • Such centrifugal force may be applied while all ports in the rotatable component are closed and/or to push liquid away from a port through which liquid may be removed from the rotatable component, creating a cell pellet away from such a port. After the application of centrifugal force, some or all of the liquid not present in the cell pellet may then be removed via such a port (e.g., via aspiration). The cells present in the cell pellet may be retained in the rotatable component during such removal.
  • a second port may also be opened during liquid removal (e.g., a port that allows the venting of air). In such embodiments, air may flow into the rotatable component through this second port while the liquid is being removed therefrom.
  • two or more rotatable components may be positioned such that they can be rotated upon the application of a single force (e.g., a single centrifugal force).
  • a single force e.g., a single centrifugal force
  • FIG. 51 a system comprising such rotatable components is shown schematically in FIG. 51.
  • a plurality of rotatable components is disposed on a single substrate that, as a whole, can be rotated. This may desirably allow for multiplexing, the facile delivery of liquids (e.g., liquids comprising one or more reagents, fresh liquid cellculture media) to the rotatable components and/or the facile removal of liquid from the rotatable components simultaneously.
  • the rotation may be effectuated by a motor, such as a spindle motor.
  • the substrate may take the form of a CD.
  • systems having the above-described design may further comprise one or more components that allow for the liquid present in the rotatable component to be monitored, such as one or more optical fibers and/or optical sensors (not shown).
  • a single component employed in a system having the design shown in FIG. 51 may be capable of performing more than one function and/or configured to do so.
  • a system may comprise a single motor that can cause the substrate to rotate, cause one or more rollers to be translated (thereby causing mixing of liquid in one or more of the flow paths), cause one or more reagent-dispensing heads to be translated and/or dispense a liquid, and/or cause one or more optical fibers to be translated.
  • a rotatable component in which a rotatable component is configured to undergo rotation under a centrifugal force having a high magnitude, it may be desirable to seal the rotatable component so that liquid is not removed therefrom through any leaks therein.
  • a rotatable component may be fabricated in a sealed chip. When the rotatable component is sealed, it may be desirable to break the seal in order to supply liquid thereto and/or remove liquid therefrom. This may be accomplished by the use of a seal that can be opened and then resealed to allow liquid introduction and/or removal followed by subsequent sealing.
  • FIG. 52 One example of a design for a seal that can be opened and then resealed is shown in FIG. 52.
  • a flow path as shown in FIG. 52 may comprise a port sealed in this manner, such as the “Growth chamber access port” labeled therein.
  • a port is sealed by two components: an upper component (labeled therein as the “Dispense needle seal”) and a lower component that is disposed on a spring.
  • the upper component may take the form of a resilient material that can be pierced by a needle and/or pipette but reseal (e.g., a rubber septum).
  • Application of force to the lower component e.g., by a needle and/or pipette containing a fluid to be introduced into the rotatable component
  • the spring may recover and cause the component to reseal the port.
  • liquid may be dispensed through a seal having a design like that shown in FIG. 52 by performing the following series of steps.
  • the upper component may be pierced by a needle and/or pipette.
  • a small volume of fluid may be dispensed through the needle and/or pipette and into the space between the upper and lower components (e.g., a volume close to the volume sufficient to fill the space between these components).
  • the lower component may be depressed, allowing for the dispensed liquid to be imbibed into the rotatable component.
  • Introducing liquid into a rotatable component in this manner may advantageously allow for its introduction in a manner that results in low dead volume and/or without a displacement interface.
  • FIG. 53 shows one non-limiting example of the filling of a sealed component having a seal as shown in FIG. 52.
  • the sealed component may initially start in a state that is sealed and free from liquid. As liquid is introduced thereinto through the opened seal, it fills the component. During this process, other ports (e.g., all other ports) may be sealed. In some such embodiments, the extra volume introduced into the sealed component is accommodated by the expansion of a membrane (e.g., the membrane shown at the bottom of the sealed component). It is also possible for liquid to be introduced into a sealed component that already comprises a liquid (e.g., liquid comprising one or more reagents and/or fresh liquid cell-culture media may be introduced into a sealed component that already comprises a liquid).
  • liquid e.g., liquid comprising one or more reagents and/or fresh liquid cell-culture media may be introduced into a sealed component that already comprises a liquid).
  • FIG. 54 depicts one non-limiting embodiment of such a method. It should be noted that the method shown in FIG. 54, and methods shown in other FIGs. herein, depict one possible order for performing the steps shown therein. However, it should also be understood that the steps may be performed in other orders and/or that two or more steps may be performed during periods of time that partially or fully overlap.
  • the method shown in FIG. 54 includes step 5424 of flowing a liquid (e.g., cell-culture media) through a cell-culture chamber. It further comprises step 5426, which comprises retaining cells in the cell-culture chamber.
  • the cellculture chamber may contain cells (e.g., while the step 5424 is performed).
  • Steps 5424 and 5426 may be performed simultaneously (e.g., liquid may be flowed through a cell-culture chamber while cells are retained therein).
  • a method comprises flowing a liquid through a cell-culture chamber in a manner such that, after the flow has ceased, at least some cells are retained in the cell-culture chamber (possibly after being removed from the cell-culture chamber and returned thereto during the liquid flow).
  • the method shown in FIG. 54 may further include one or more optional steps, some of which are shown in FIG. 54.
  • a method like that shown in FIG. 54 may comprise the optional step of employing a pump to cause the liquid to flow through the cell-culture chamber. This step is depicted schematically in FIG. 54 as step 5428. It also possible, additionally or alternatively, for another system component to be employed to cause the liquid to flow and/or for a force to be applied by a component other than a pump to cause the liquid to flow (e.g., a system component and/or a force described elsewhere herein).
  • a component e.g., a system component and/or a force described elsewhere herein.
  • Flow through a cell-culture chamber as described herein may comprise flow to the cell-culture chamber from a variety of system components (e.g., one or more system components described elsewhere herein.
  • a method may comprise flowing a liquid into a cell-culture chamber.
  • liquid is flowed into a cell-culture chamber is flowed thereinto from a port.
  • liquid may flow through a port and into a cell-culture chamber.
  • the liquid may originate from a source thereof (e.g., a source of a reagent, a source of cell-culture media).
  • liquid is flowed out of a cell-culture chamber and into (e.g., through) a port. It is also possible for the liquid to flow through a valve (e.g., a valve positioned upstream from the port, a valve positioned fluidically between the port and the cell-culture chamber).
  • a valve e.g., a valve positioned upstream from the port, a valve positioned fluidically between the port and the cell-culture chamber.
  • Flow through a cell-culture chamber as described herein may comprise flow from the cell-culture chamber to a variety of system components (e.g., one or more system components described elsewhere herein.
  • a method may comprise flowing a liquid out of a cell-culture chamber.
  • the liquid may flow into a waste chamber and/or into a component comprising a sensor for a biological product (e.g., a component that can determine the amount of IgG in a liquid, such as an IgG sensor).
  • the liquid flowing out of the cell-culture chamber may flow through a port and/or a valve (e.g., a valve positioned between the cell-culture chamber and the port, a valve positioned downstream from the port).
  • a single valve may be employed to direct the liquid through a first port into the cell-culture chamber and through a second port (e.g., a port different from the first port) out of the cell-culture chamber.
  • the valve may simultaneously place a source of a reagent in fluidic communication with the cell-culture chamber via the first valve and a waste chamber and/or a component that can determine the amount of IgG in a liquid in fluidic communication with a second port.
  • the optional step 5434 comprises mixing a liquid. This mixing may be performed prior to flowing the liquid into the cell-culture chamber (e.g., it may be performed upstream from the cell-culture chamber). This mixing may be performed as described elsewhere herein. For instance, it may be performed in a mixing chamber and/or in a flow path configured to promote mixing. Similarly, it may be performed by a mixing element described herein (e.g., an active mixing element, a static mixer) and/or may comprise mechanical mixing via one or more such mixing elements.
  • a mixing element described herein e.g., an active mixing element, a static mixer
  • the optional step 5434 or an analogous step thereto, to be performed in the cell-culture chamber (e.g., subsequent to flowing the liquid into the cell-culture chamber and/or prior to flowing the liquid out of the cell-culture chamber).
  • the optional step 5434 may be performed during periods of time during which one or more other steps shown in FIG. 54 are not performed (e.g., during which liquid is not pumped into, out of, and/or through a cell-culture chamber). Without wishing to be bound by any particular theory, it is believed that performing such steps separately may improve the accuracy of the other steps.
  • the optional step 5436 comprises flowing a gas through a gas-permeable membrane.
  • the gas-permeable membrane may be positioned in the cell-culture chamber (e.g., it may form a wall thereof).
  • flowing a gas through a gas-permeable membrane may allow for gas exchange between liquid present in the cell-culture chamber and an environment external thereto.
  • the gas may flow into the cell-culture chamber (e.g., from such an environment) and/or out of the cell-culture chamber (e.g., to such an environment).
  • the gas may comprise a reagent (e.g., a gaseous reagent) and/or oxygen.
  • the optional step 5438 comprises performing one or more measurements on the liquid. Such measurements may be performed while the liquid is present in the cell-culture chamber, before the liquid has entered the cell-culture chamber, and/or after the liquid has left the cell-culture chamber. Such measurements may be performed with one or more sensors and/or optical detectors.
  • the optional step 5440 comprises introducing cells into the cell-culture chamber and the optional step 5442 comprises removing cells from the cell-culture chamber. Cells may be introduced into the cell-culture chamber to be cultured and/or removed therefrom at the conclusion of culturing and/or to assess one or more properties thereof.
  • FIG. 55 shows one nonlimiting example of such a method.
  • the method shown in FIG. 55 may be performed as part of the method shown in FIG. 54 (e.g., some or all of the steps shown in FIG. 55 may be performed as part of a method comprising some or all of the steps shown in FIG. 54 and/or the step 5434 in FIG. 54 may comprise some or all of the steps shown in FIG. 55) or may be performed separately (e.g., some or all of the steps shown in FIG. 55 may be performed without performing any of the steps shown in FIG. 54).
  • the method shown in FIG. 55 comprises the optional step 5544, in which a fluid is pumped into a mixing chamber.
  • the fluid may be pumped into a mixing chamber through a port and/or a valve (and/or from a cell-culture chamber and/or a source of reagents) as described elsewhere herein.
  • the method shown in FIG. 55 further comprises the optional step 5546.
  • the optional step 5546 comprises pumping a second fluid into a mixing chamber.
  • both optional steps 5544 and 5546 they may be performed simultaneously and/or sequentially.
  • two or more fluids may be pumped into a single mixing chamber simultaneously and/or sequentially.
  • the fluids may be pumped into the mixing chamber via a single port (e.g., the fluids may be sequentially pumped through the port or may be mixed upstream from the port and then pumped into the mixing chamber) and/or via two different ports (e.g., the first fluid may be pumped into the mixing chamber through a first port and the second fluid may be pumped into the mixing chamber through a second port).
  • the first and second fluids may both flow through a common valve (e.g., taking different pathways therethrough, originating from different locations prior to entering the valve and/or exiting the valve to flow into different ports).
  • the method shown in FIG. 55 further comprises the optional step 5548.
  • the optional step 5548 comprises pumping a third fluid into a second mixing chamber.
  • This fluid may flow through a valve (e.g., a valve through which the first and/or second fluids flow into the first mixing chamber) and/or through a port.
  • the third fluid is a mixture of the first and second fluids (e.g., after being mixed in the first mixing chamber).
  • the third fluid may be a different fluid (e.g., a fluid flowing from a source of a reagent).
  • two mixing chambers may be employed simultaneously (and, possibly, with the use of a common valve) to mix different fluids.
  • FIG. 56 shows one non-limiting example of such a method.
  • the method shown in FIG. 56 may be performed as part of the method shown in FIG. 54 (e.g., some or all of the steps shown in FIG. 56 may be performed as part of a method comprising some or all of the steps shown in FIG. 54 and/or the step 5438 in FIG. 54 may comprise some or all of the steps shown in FIG. 56) or may be performed separately (e.g., some or all of the steps shown in FIG. 56 may be performed without performing any of the steps shown in FIG. 54).
  • the method shown in FIG. 56 comprises the optional step 5650.
  • the optional step 5650 comprises performing an optical measurement.
  • the optical measurement may be performed with the use of an optical detector (possibly comprising an optical sensor) and/or a light source as described elsewhere herein.
  • the optical measurement is performed on the cell-culture chamber and/or a liquid present therein.
  • an optical measurement may be performed on a system component other than the cellculture chamber (e.g., on a component configured to determine the amount of IgG in a liquid flowing out of the cell-culture chamber) and/or on a liquid in another such system component.
  • a variety of suitable optical measurements may be performed, one non-limiting example of which is a measurement of the optical density of a liquid (e.g., a liquid present in the cell-culture chamber, a liquid flowing out of the cell-culture chamber).
  • the optional step 5652 comprises determining an amount of cells present in the liquid based on the optical measurement. For instance, if the optical measurement is a measurement of the optical density of the liquid, the amount of cells present in the liquid may be determined from the optical density.
  • FIG. 57 shows another non-limiting example of a method in which one or more measurements are performed on a liquid.
  • the method shown in FIG. 57 may be performed as part of the method shown in FIG. 54 (e.g., some or all of the steps shown in FIG. 57 may be performed as part of a method comprising some or all of the steps shown in FIG. 54 and/or the step 5438 in FIG. 54 may comprise some or all of the steps shown in FIG. 57) or may be performed separately (e.g., some or all of the steps shown in FIG. 57 may be performed without performing any of the steps shown in FIG. 54).
  • the method shown in FIG. 57 comprises the optional step 5754.
  • the optional step 5754 comprises measuring a property of a liquid.
  • the measurement is performed on a liquid present in a cell-culture chamber. It is also possible for a measurement to be performed on a liquid present in a system component other than the cell-culture chamber (e.g., on a liquid present in a component configured to determine the amount of IgG in a liquid flowing out of the cell-culture chamber, a liquid obtained from the cell-culture chamber).
  • liquid properties include dissolved-oxygen content, pH, an amount of a biological product (e.g., IgG) and/or other species (e.g., carbon dioxide, nutrients, metabolites such as lactate and/or glucose) present in the liquid, cell concentration, cell viability, temperature, pressure, and/or viscosity. Liquid properties may be measured with sensors described elsewhere herein.
  • the method shown in FIG. 57 further comprises the optional step 5756.
  • the optional step 5756 comprises delivering a reagent to the liquid based on the measurement of the property of the liquid.
  • reagents include oxygen, acids (e.g., hydrochloric acid), bases (e.g., sodium bicarbonate), and cell-culture media.
  • the reagent may be delivered to the liquid at the location at which the measurement was performed (e.g., a cell-culture chamber) and/or to a location upstream or downstream therefrom (e.g., to liquid flowing out of a cell-culture chamber, to a cell-culture chamber based on a measurement performed on liquid flowing out of a cell-culture chamber).
  • the optional steps 5754 and 5756 may be performed together in order to adjust one or more properties of a liquid and/or to keep one or more properties of a liquid in a desired range.
  • a method may comprise delivering a cell-culture medium having a dissolved-oxygen content within a range (e.g., pre-conditioned cell-culture media) determined based on the measurement of the dissolved-oxygen content.
  • a method comprises measuring the pH of a liquid and delivering an amount of an acid and/or a base determined based on the measurement of the pH (e.g., an amount sufficient to cause the liquid to have a pH in a desirable range). It is possible for the acid and/or base to be in a liquid that may also have one or more properties that are desirable for inclusion in a cell-culture chamber.
  • a method may comprise delivering a cell-culture medium having a pH content within a range determined based on the measurement of the pH (e.g., comprising a sufficient amount of acid and/or base to have such a pH).
  • FIG. 58 shows a non-limiting example of a method in which cells may be introduced to and/or recovered from a cell-culture chamber.
  • the method shown in FIG. 58 may be performed as part of the method shown in FIG. 58 (e.g., some or all of the steps shown in FIG. 58 may be performed as part of a method comprising some or all of the steps shown in FIG. 54 and/or the steps 5440 and/or 5442 in FIG. 54 may comprise some or all of the steps shown in FIG. 58) or may be performed separately (e.g., some or all of the steps shown in FIG. 58 may be performed without performing any of the steps shown in FIG. 54).
  • some or all of the method shown in FIG. 58 may be performed as the first step in the method shown in FIG. 54 or prior to the performance of the method shown in FIG. 54. For instance, cells may be introduced into a cell-culture chamber at the beginning of liquid flow therethrough and/or prior to liquid flow therethrough.
  • the method shown in FIG. 58 comprises the optional step 5858.
  • the optional step 5858 comprises delivering cells into a port and/or a reservoir.
  • the cells are delivered in the presence of a liquid (e.g., they may be suspended in a liquid that is delivered into the port and/or reservoir).
  • the port and/or reservoir may be a port and/or reservoir through which cell seeding may be performed.
  • the port and/or reservoir is sealed (e.g., by a septum, such as a rubber septum and/or a silicone septum).
  • delivering the cells may comprise piercing the septum (e.g., with a needle and/or pipette) and delivering the cells through the piercing (e.g., through the needle and/or pipette). It is also possible for the port and/or reservoir to be covered (and sealed or unsealed) by a lid. In such embodiments, delivering the cells may comprise removing the lid. In some embodiments, cells may be delivered in an automated manner (e.g., that does not require piercing of a septum or removal of a lid). Such automated delivery may comprise the use of one or more pumps.
  • the method shown in FIG. 58 further comprises the optional step 5860.
  • the optional step 5860 comprises flowing the cells from the port and/or reservoir into the cell-culture chamber. This may be accomplished by pumping the cells (and/or a liquid in which they are present) along a fluidic pathway connecting the port and/or reservoir and the cell-culture chamber.
  • the method shown in FIG. 58 further comprises the optional step 5862.
  • the optional step 5862 comprises flowing the cells from the cell-culture chamber to a second port. This step, too, may be accomplished by pumping the cells (and/or a liquid in which they are present) along a fluidic pathway connecting the second port and/or reservoir and the cellculture chamber.
  • the second port and/or reservoir may be a different port and/or reservoir from a port and/or reservoir from which the cells are flowed into the cell-culture chamber. For instance, it may be a port and/or reservoir through which cell recovery may be performed.
  • the method shown in FIG. 58 further comprises the optional step 5864.
  • the optional step 5864 comprises removing the cells from the second port and/or reservoir.
  • the cells are recovered in the presence of a liquid (e.g., they may be suspended in a liquid that is recovered from the port and/or reservoir).
  • the port and/or reservoir may be a port and/or reservoir through which cell recovery may be performed.
  • the port and/or reservoir is sealed (e.g., by a septum).
  • recovering the cells may comprise piercing the septum (e.g., with a needle and/or pipette) and recovering the cells through the piercing (e.g., by withdrawing the cells through the needle and/or pipette). It is also possible for the port and/or reservoir to be covered (and sealed or unsealed) by a lid. In such embodiments, recovering the cells may comprise removing the lid. In some embodiments, cells may be recovered in an automated manner (e.g., that does not require piercing of a septum or removal of a lid). Such automated delivery may comprise the use of one or more pumps.
  • FIG. 59 schematically depicts a method by which cells may be introduced into a cellculture chamber (e.g., a method of cell seeding).
  • the introduction of cells may be performed manually and/or in an automated manner. In some embodiments, such introduction may result in a relatively uniform distribution of cells in the cell-culture chamber.
  • cells may be introduced into a cell-culture chamber from a seeding port.
  • a pump may be employed to cause liquid to flow as desired.
  • the cell-culture chamber and/or other system components may be primed by flowing liquid therethrough (e.g., liquid supplied by a pump and/or from a source of a reagent, such as cell-culture media).
  • a valve e.g., the seeding port valve shown in FIG. 59, a turning valve
  • one or more valves e.g., including the seeding port valve shown in FIG. 59
  • This may seal the cell-culture chamber and/or system, which may be beneficial during incubation.
  • cells may be removed from a cell-culture chamber by recovering cells from the seeding port shown in FIG. 59 and/or by recovering cells from a different seeding port (e.g., a seeding port positioned on an opposite side of a different seeding port valve).
  • Cells may be removed from a cell-culture chamber for further analysis, the performance of one or more assays thereon, and/or further cell-culture (e.g., in a larger chamber and/or vessel).
  • Cell-culture chambers may have a variety of suitable designs.
  • a cell-culture chamber comprises a channel, consists essentially of a channel, and/or consists of a channel.
  • such a channel may be a microfluidic channel.
  • a cell-culture chamber has a microfluidic volume.
  • a cell-culture chamber may have a volume of less than or equal to 500 microliters, less than or equal to 400 microliters, less than or equal to 300 microliters, less than or equal to 200 microliters, less than or equal to 100 microliters, less than or equal to 75 microliters, or less than or equal to 50 micro liters.
  • Cells present in a cell-culture chamber may be retained with relatively high efficiencies.
  • cells are retained in a cell-culture chamber (e.g., during fluid flow therethrough) with an efficiency of greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 99%.
  • cells are retained in a cell-culture chamber (e.g., during fluid flow therethrough) with an efficiency of less than or equal to 100%, less than or equal to 99%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, or less than or equal to 75%.
  • a cell-culture chamber is sterilizable (e.g., via an autoclave, exposure to gamma radiation, exposure to x-ray radiation, exposure to ethylene oxide, dry heat sterilization, and/or chemical sterilization, such as via exposure to hydrogen peroxide vapor, bleach, and/or alcohol).
  • Such cell-culture chambers may change color and/or deform (or not change color and/or not deform) during sterilization.
  • a gas-permeable membrane proximate to a cell-culture chamber facilitates desirable gas exchange therein because it reduces the distance from the cell-culture chamber to and from which gas must diffuse to be exchanged.
  • a system for performing cell-culture comprises one or more sensors (e.g., two or more sensors). Such sensors may be positioned in one or more locations in which it may be desirable to sense a property of the system and/or a property of a liquid present in the system. For instance, sensors may be present in cell-culture chambers, mixing chambers, and/or flow paths configured to promote mixing. It is also possible for a sensor to be positioned at a location in fluidic communication with one or more such locations. In such embodiments, liquid may be removed from the relevant portion of the system and supplied to the location comprising the sensor.
  • sensors e.g., two or more sensors.
  • sensors may be positioned in one or more locations in which it may be desirable to sense a property of the system and/or a property of a liquid present in the system.
  • sensors may be present in cell-culture chambers, mixing chambers, and/or flow paths configured to promote mixing. It is also possible for a sensor to be positioned at a location in fluidic communication with one or more such locations.
  • a system may comprise an optical sensor (e.g., an optical detector and/or a portion of an optical detector), an electrochemical sensor, an electrical sensor, and/or an acoustic sensor.
  • an optical sensor e.g., an optical detector and/or a portion of an optical detector
  • an electrochemical sensor e.g., an electrochemical sensor
  • an electrical sensor e.g., an acoustic sensor
  • one or more of these types of sensors may be employed to sense one or more of the species described in the preceding paragraph.
  • an optical sensor may be employed to perform an optical measurement from which the amount of a biological product is determined. It is also possible for the species described in the preceding paragraph to be sensed directly (e.g., by sensors that detect binding between the relevant species and a sensor component). Further examples of types of sensors that may be employed to sense different properties are described below.
  • pH sensors may be optical sensors and/or electrochemical sensors (e.g., ion-sensitive field-effect transistors, solid-state polymer membranes, capacitive sensors, sensors comprising carbon nanotubes, and/or enzymatic sensors).
  • Optical sensors may measure the pH by measuring the optical properties of a pH- sensitive indicator dye and calculating the pH therefrom.
  • Electrochemical sensors may measure the pH by measuring the potential, current, and/or capacitance of a liquid and calculating the pH therefrom.
  • a pH sensor is capable of and/or configured to measure a pH value in a particular range (e.g., between 5.5 and 8.5).
  • Dissolved-oxygen sensors may comprise optical sensors (e.g. fluorescence sensors, colorimetric sensors, and/or surface plasmon resonance sensors) and/or electrochemical sensors (e.g., microfabricated Clark-type electrodes, thin-film platinum electrodes, screen- printed electrodes, and/or polymer electrolyte -based electrodes).
  • Optical sensors may measure the dissolved-oxygen content by measuring the absorption, fluorescence, and/or refractive index of a liquid and calculating the dissolved oxygen content therefrom.
  • Electrochemical sensors may measure the dissolved oxygen content by measuring the potential, current, and/or capacitance of a liquid and calculating the dissolved oxygen content therefrom.
  • a dissolved-oxygen sensor is capable of and/or configured to measure a dissolved-oxygen value in a particular range (e.g., between 0% and 100%).
  • Sensors for carbon dioxide may comprise optical and/or electrochemical sensors (e.g., potentiometric and/or amphoteric sensors, ion-sensitive field-effect transistors, solid-state electrolyte sensors, and/or enzymatic sensors).
  • Optical sensors may measure the carbon dioxide content by measuring the absorption, fluorescence, and/or refractive index of a liquid and calculating the carbon dioxide content therefrom.
  • Electrochemical sensors may measure the carbon dioxide content by measuring the potential, current, and/or capacitance of a liquid and calculating the carbon dioxide content therefrom.
  • Sensors for cell concentration may comprise optical (e.g., optical density sensors, bioreflectance sensors, fluorescence sensors, and/or near-infrared spectroscopy sensors), electrical (e.g., electrical impedance sensors, dielectric spectroscopy sensors, RF impedance sensors, and/or capacitance-based sensors), and/or acoustic sensors (e.g., surface acoustic wave sensors, bulk acoustic wave sensors). Such sensors may measure the optical, electrical, and/or acoustic properties of the liquid and calculate the cell concentration therefrom.
  • optical e.g., optical density sensors, bioreflectance sensors, fluorescence sensors, and/or near-infrared spectroscopy sensors
  • electrical e.g., electrical impedance sensors, dielectric spectroscopy sensors, RF impedance sensors, and/or capacitance-based sensors
  • acoustic sensors e.g., surface acoustic wave sensors, bulk acoustic wave sensors.
  • Sensors for cell viability may comprise optical (e.g., fluorescence, reflectance) and/or electrical (e.g., electrical impedance sensors, capacitance-based sensors, potentiometric sensors, and/or conductivity sensors). Such sensors may measure the optical and/or electrical properties of the liquid and calculate the cell concentration therefrom.
  • Sensors for nutrients and metabolites may comprise optical (e.g., fluorescent NADH sensors and/or fluorescent NADPH sensors) and/or electrochemical (e.g., ion-selective electrodes, conductivity sensors, amperometric glucose sensors, and/or amperometric and/or potentiometric lactate sensors). Such sensors may measure the optical and/or electrochemical properties of the liquid and calculate the amount of a nutrient and/or metabolite therein therefrom.
  • Sensors for biological products may comprise optical (e.g., surface plasmon resonance sensors, fluorescence sensors, surface-enhanced Raman scattering sensors, interferometric sensors, colorimetric sensors, and/or luminescence sensors) and/or electrical (e.g., field-effect transistors, electrochemical impedance spectroscopy sensors, dielectric spectroscopy sensors, capacitive sensors, electrical impedance sensors, and/or sensors comprising carbon nanotubes) sensors.
  • optical e.g., surface plasmon resonance sensors, fluorescence sensors, surface-enhanced Raman scattering sensors, interferometric sensors, colorimetric sensors, and/or luminescence sensors
  • electrical e.g., field-effect transistors, electrochemical impedance spectroscopy sensors, dielectric spectroscopy sensors, capacitive sensors, electrical impedance sensors, and/or sensors comprising carbon nanotubes
  • Temperature sensors may comprise optical (e.g., fluorescence sensors and/or luminescence sensors) and/or electrical (e.g., resistance temperature detectors) sensors. Such sensors may measure the optical and/or electrical properties of the liquid and calculate the temperature therefrom.
  • optical sensors e.g., fluorescence sensors and/or luminescence sensors
  • electrical sensors e.g., resistance temperature detectors
  • Pressure sensors may comprise optical (e.g., optical pressure sensors) and/or electrical (e.g., capacitive pressure sensors) sensors.
  • Humidity sensors may comprise optical (e.g., absorbance sensors and/or reflectionbased sensors) and/or electrical (e.g., capacitive sensors, resistance sensors, conductivity sensors, and/or dielectric sensors).
  • optical sensors e.g., absorbance sensors and/or reflectionbased sensors
  • electrical e.g., capacitive sensors, resistance sensors, conductivity sensors, and/or dielectric sensors.
  • Viscosity sensors may comprise electrical sensors (e.g., capacitance-based sensors and/or electrical impedance sensors).
  • optical sensors e.g., transmittance sensors and/or reflectance sensors
  • electrical sensors e.g., conductivity sensors and/or capacitance-based sensors
  • Optical detectors may comprise a variety of components that facilitate performing optical detection.
  • Non-limiting examples of such components include optical sensors, light sources, and components that facilitate imaging (e.g., microscopes).
  • Light sources present in optical detectors may supply light in a variety of suitable manners, one non-limiting example of which is fluorescence (e.g., a light source may be a source of fluorescent light).
  • an optical detector takes the form of and/or comprises a plate reader.
  • the sensors described herein may have a variety of suitable designs, two non-limiting examples of which include sensor plugs and sensor patches.
  • a sensor comprises a sensor plug that can receive an optical fiber (e.g., a polymer optical fiber, an optical fiber having a 1 mm diameter).
  • the optical fiber may be capable of and/or configured to transmit an optical signal to a location where it can be detected.
  • valves may comprise valves.
  • suitable valves include pneumatic valves, turning valves, rotary valves (e.g., rotary membrane valves), pinch valves, solenoid valves, shape-memory alloy valves, and membrane valves.
  • a valve employed in a system described herein has a design such that it places one port in fluidic communication with and upstream from an inlet to a pump and another port in fluidic communication with and downstream from an outlet of the pump.
  • these ports can be selected arbitrarily (i.e., any port with which the valve is in fluidic communication may be placed in fluidic communication with and upstream from an inlet to a pump and any port with which the valve is in fluidic communication may be placed in fluidic communication with and downstream from an outlet of the pump, and/or any pairs of ports may be selected for these two positions).
  • this may facilitate fluid flow through the system between a variety of components positioned therein that is driven by a pump that is only capable of pumping in a single direction.
  • the direction of pumping of fluid through the system may be capable of being selected by the valve (e.g., even if the pump is only capable of pumping in a single direction).
  • FIGs. 60-66 show one non-limiting example of a valve having the above-described design and an associated system in which the valve is positioned.
  • FIG. 60 depicts the valve pusher. From FIG. 60, it can be seen that the valve pusher has two open positions, which may allow for the selection of ports that are in fluidic communication with a pump (e.g., the fluidic communication may occur via the open positions).
  • FIG. 61 shows one non-limiting embodiment of a portion of a system comprising such a valve and further comprising a pump. As can be seen from FIG. 61, the pusher may be capable of being rotated such that a variety of different ports are in fluidic communication with and upstream or downstream from the pump.
  • FIG. 62 shows one example of a position of the above-described valve that places a port (that is in fluidic communication with a source of reagents) in fluidic communication with and upstream from an inlet to the pump and places a port (that is upstream from and in fluidic communication with a mixing chamber) in fluidic communication with and downstream from an outlet of the pump.
  • the arrow in FIG. 62 shows the flow of fluid from the port in fluidic communication with the source of reagents, through the valve, through the pump, through the valve again, and then to a chamber (e.g., a cell-culture chamber).
  • FIGs. 63-65 show further examples of manners in which fluid may flow through the valve depicted therein.
  • the direction of flow through the valve and/or pump may be adjusted to cause flow in a number of different directions, to a number of different locations, and/or from a number of different locations.
  • FIG. 66 depicts the base of the system in which the above-described valve is positioned.
  • various components of the system may have static positions (e.g., they may not move and/or they may be mechanically coupled to a substrate present in the system) even though flow thereto, therethrough, and/or therefrom may be selected as desired.
  • a valve in fluidic communication with ports that are in fluidic communication with a source of a fluid other than a reagent (e.g., a source of a sample) and/or a system component other than a source (e.g., with a mixing chamber, with a pump, with a waste chamber) and/or that are in fluidic communication with a system component other than a port (e.g., with a mixing chamber, with a pump, with a waste chamber).
  • a source of a fluid other than a reagent e.g., a source of a sample
  • a system component other than a source e.g., with a mixing chamber, with a pump, with a waste chamber
  • a system component other than a port e.g., with a mixing chamber, with a pump, with a waste chamber
  • a system comprises a reservoir through which cell seeding and/or cell recovery may be performed.
  • reservoirs when present, may be sealed by septa and/or may comprise lids that may be pierced and/or removed, respectively, to allow for cell seeding and/or recovery.
  • FIG. 67 depicts one non-limiting example of a system having this design.
  • the system shown in FIG. 67 comprises a first valve (a tuning valve) in fluidic communication with a port through which cell seeding may be performed and/or cell recovery may be performed.
  • This system also comprises a second valve (a rotary valve) in fluidic communication with a plurality of ports comprising a port in fluidic communication with a source of reagents and a port in fluidic communication with a waste chamber.
  • a first valve a tuning valve
  • a second valve a rotary valve
  • 67 further comprises a cell-culture chamber for which a gas-permeable membrane forms one wall (labeled therein as a “Growth chamber” and a “Gas channel,” respectively), an oxygen sensor, a filter, and a rotary pump.
  • the pumps described herein may have a volume per pump cycle of greater than or equal to 100 nL, greater than or equal to 200 nL, greater than or equal to 500 nL, greater than or equal to 750 nL, greater than or equal to 1 microliter, greater than or equal to 2 microliters, or greater than or equal to 5 microliters.
  • the pumps described herein may have a volume per pump cycle of less than or equal to 10 microliters, less than or equal to 5 microliters, less than or equal to 2 microliters, less than or equal to 1 microliter, less than or equal to 750 nL, less than or equal to 500 nL, or less than or equal to 200 nL.
  • Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 100 nL and less than or equal to 10 microliters, greater than or equal to 100 nL and less than or equal to 1 microliter, greater than or equal to 5 microliters and less than or equal to 10 microliters). Other ranges are also possible.
  • each pump may independently have a volume per pump cycle in one or more of the above-referenced ranges.
  • the pumps described herein may have a maximum flow rate of greater than or equal to 10 nL/s, greater than or equal to 20 nL/s, greater than or equal to 50 nL/s, greater than or equal to 75 nL/s, greater than or equal to 100 nL/s, greater than or equal to 150 nL/s, greater than or equal to 200 nL/s, greater than or equal to 300 nL/s, or greater than or equal to 400 nL/s.
  • the pumps described herein may have a maximum flow rate of less than or equal to 500 nL/s, less than or equal to 400 nL/s, less than or equal to 300 nL/s, less than or equal to 200 nL/s, less than or equal to 150 nL/s, less than or equal to 100 nL/s, less than or equal to 75 nL/s, less than or equal to 50 nL/s, or less than or equal to 20 nL/s. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 10 nL/s and less than or equal to 500 nL/s). other ranges are also possible.
  • each pump may independently have maximum flow rate in one or more of the above-referenced ranges.
  • Some pumps described herein may be capable of withstanding, configured to withstand, and/or withstand high negative pressure. Similarly, some such pumps may be capable of delivering, may be configured to deliver, and/or may deliver predictable volumes of fluid under high pressure (e.g., high negative pressure). Without wishing to be bound by any particular theory, it is believed that pumps may be subjected to relatively high negative pressure when employed to pump fluid through a gas-permeable membrane (e.g., from a cellculture chamber though a gas-permeable membrane that forms a portion or all of a wall thereof).
  • a gas-permeable membrane e.g., from a cellculture chamber though a gas-permeable membrane that forms a portion or all of a wall thereof.
  • a pump comprises one or more valves. Fluid flow through such pumps may comprise flow through one or more valves.
  • FIG. 68 shows an example of a pump that may be suitable for use in the systems described herein and associated valves.
  • a liquid being pumped may flow into a pump through an input valve and out through an output valve. In between, the liquid may flow through a diaphragm valve.
  • a pump may further comprise one or more air channels associated with its valves. Such air channels may facilitate pumping action.
  • a pump may comprise more than one inlet valve and/or may comprise one or more routing valves. Routing valves may be suitable for routing a liquid to a diaphragm valve internal to the pump and/or to a different location in the system.
  • the presence of more than one routing valve and/or more than one inlet valve may allow for a single pump to supply liquid originating from multiple sources and/or locations in the system (e.g., one or more sources of reagents, one or more mixing chambers, one or more cell-culture chambers) to a single outlet valve (e.g., that supplies a mixing chamber, a cell-culture chamber, and/or a waste chamber).
  • a single pump to supply liquid originating from multiple sources and/or locations in the system (e.g., one or more sources of reagents, one or more mixing chambers, one or more cell-culture chambers) to a single outlet valve (e.g., that supplies a mixing chamber, a cell-culture chamber, and/or a waste chamber).
  • more than one pump may be associated with a common air channel.
  • the diaphragm valve and the outlet valve share air channels with other pumps.
  • a common air pressure clock may be employed to drive actuation of all pumps associated with the common air pressure channel.
  • routing valves may be closed on selected pumps to prevent pumping by such pumps (and associated addition of the reagent to the relevant cell-culture chambers).
  • routing valves may be capable of being independently controlled.
  • liquid may flow through a pump in multiple directions (e.g., in opposite directions).
  • flow through a pump in one direction may result in the flow of liquid to a cell-culture chamber and flow through a pump in another direction may result in the flow of liquid to a waste chamber.
  • a system comprises more than one pump.
  • FIG. 69 shows one non-limiting example of such a system.
  • the system shown in FIG. 69 also includes other exemplary components discussed elsewhere herein (e.g., two pluralities of ports, a waste chamber, and a cell-culture chamber).
  • FIGs. 70 and 71 show another non-limiting example of such a system.
  • the system shown in FIGs. 70 and 71 include two pumps, each driven by a manifold that may also be employed to drive pumps associated with other systems (e.g., other systems positioned on a common substrate, as described in further detail below).
  • a system described herein is positioned on a substrate. It is also possible for some, but not all, components of a system described herein to be positioned on a substrate.
  • a system may comprise a cell-culture chamber, one or more mixing components, and/or a valve that is positioned on a substrate and further comprise a pump that is positioned off the substrate.
  • a system may comprise one or more cell-culture chambers and/or mixing components that are positioned on a substrate and further comprise a valve and a pump that are positioned off the substrate.
  • Other system components described herein, such as sensors (e.g., optical detectors) may be positioned on and/or off such substrates.
  • suitable such substrates include chips, such as microfluidic chips.
  • one or more system components comprise volumes that have been removed (e.g., etched) from a substrate. Some or all such system components may be positioned in a single substrate and/or a system may comprise a single substrate from which one or more portions have been removed to form system component(s).
  • a system comprises a substrate from which various volumes have been removed and on which two components are disposed.
  • various channels and ports take the form of volumes that have been removed from the substrate.
  • a valve pin and a pump pin are disposed on the substrate, and/or a membrane may be positioned between the substrate and these components. It is also possible for several volumes to be removed from the membrane. These volumes may take the form of through- holes disposed beneath the valve pin that form a membrane valve and a channel disposed beneath the pump pin that forms a membrane pump.
  • the valve pin may rotate to place various ports on the substrate in fluidic communication with the channel taking the form of a volume removed from the substrate.
  • the pump pin may rotate to cause the pump to deliver fluids to this channel and/or to remove fluids therefrom.
  • Some systems may be thermally bonded together. Such bonding may advantageously be performed in the absence of solvents and/or laser welding.
  • Substrates may have volumes removed through which one or more sensors may be inserted.
  • Substrates may be assembled in a variety of suitable manners.
  • one or more materials may be machined to form a substrate and/or one or more layers thereof.
  • one or more materials may be laser cut to form a substrate and/or one or more layers thereof.
  • a substrate e.g., the chip, the microfluidic chip
  • a substrate may comprise a surface that is transparent to at least one wavelength of light. Such a surface may extend across an entirety of the substrate or may only extend across a portion of the substrate.
  • the substrate may comprise one or more portions that, as a whole, are transparent to at least one wavelength of light. It is also possible for the entirety of the substrate to be transparent to at least one wavelength of light.
  • the wavelengths of light to which the substrate may be transparent and the levels of transparency may be in one or more of the ranges provided above with respect to the transparency of a channel to wavelengths of light.
  • a substrate on which a system is positioned has one or more features that facilitate performing cell culture therein.
  • a substrate may be biocompatible and/or sterilizable (e.g., as described elsewhere herein with respect to cell-culture chambers). It is also possible for a substrate to be disposable. In such instances, some system components (e.g., those positioned off the substrate and/or those capable of being removed from the substrate, such as valves and/or pumps) may be reusable.
  • a substrate is formed from and/or comprises a plastic, such as polystyrene, PMMA, a cyclic olefin copolymer, polycarbonate, polysulfone, polyetherimide (e.g., Ultem), polyurethane, and/or a 3D-printed resin.
  • a substrate is formed from and/or comprises PDMS.
  • a substrate is formed from and/or comprises glass.
  • a system comprises a substrate and further comprises one or more additional components. Such components may be positioned above or below the substrate.
  • a system may further comprise a bottom cap disposed beneath a substrate and/or a cover layer disposed on a substrate.
  • an adhesive may be employed to adhere the substrate to such components.
  • FIG. 72 shows one non-limiting embodiment of a system having this design.
  • a cover layer disposed on the substrate comprises a membrane valve and pump and/or a silicone.
  • a system may comprise one or more components that fit into a volume that has been removed from a substrate (e.g., a pump that fits into such a volume, a sensor that fits into such a volume, an optical cable that fits into such a volume, an optical transmitter that fits into such a volume, and/or a valve that fits into such a volume).
  • a pump that fits into such a volume
  • a sensor that fits into such a volume
  • an optical cable that fits into such a volume
  • an optical transmitter that fits into such a volume
  • a valve that fits into such a volume
  • a system may comprise one or more additional components that are not placed on a substrate and/or below a substrate.
  • a system may comprise one or more motors (e.g., one or more motors for one or more pumps, one or more sensors, one or more optical cables, one or more optical transmitters, and/or one or more valves) that are placed in such locations.
  • FIGs. 73 and 74 show examples of a system that comprises some components that are placed on a substrate and some components that are not placed on that substrate (e.g., they may be a part of a manifold that can integrate with the first substrate). In FIGs. 73 and 74, these components are placed on a manifold (such as a manifold placed on a different, second substrate). The system shown in FIGs.
  • 73 and 74 comprises a substrate A66 on which a cellculture chamber, a dissolved-oxygen sensor, a pH sensor, and a cell filter are placed and a manifold A68 on which a plurality of valves, pumps, sources of reagents (taking the form of reservoirs that are capped or uncapped), and associated conduits are disposed.
  • the substrate A66 shown in FIGs. 73 and 74 further comprises a seeding port.
  • ports 73 and 74 further comprises ports that are configured to connect and/or are capable of connecting to an external air supply, a port that is configured to connect and/or is capable of connecting to a waste chamber and/or a conduit in fluidic communication therewith, and a port that is configured to connect and/or is capable of connecting with a source of a liquid that may be employed to clean the cell-culture chamber and/or a sample therein.
  • Ports present on a substrate and/or a manifold may comprise fittings (e.g., luer-barb connectors, threaded connectors).
  • FIG. 73 shows a configuration in which the substrate is separated from the manifold and FIG. 74 shows a configuration in which the substrate is nested in the manifold.
  • systems having a design in which there are two substrates may comprise one or more features that facilitate such nesting.
  • the first and manifold may comprise one or more components (e.g., one or more tabs, fasteners, a gasket, etc.) that mechanically couple such components when the substrate is nested in the manifold.
  • FIG. 75 shows one example of another view of the system shown in FIGs. 73 and 74 arranged so that the substrate is nested in the manifold.
  • the components positioned on the manifold may be capable of interfacing and/or configured to interface with the components on the substrate via a plurality of ports disposed on the manifold and/or a plurality of ports disposed on the substrate.
  • Such ports may be supplied with liquid (e.g., liquid comprising a reagent, media) and/or pressure (e.g., positive pressure, negative pressure). Pressure supplied to a port may cause liquid to flow into, out of, and/or through the port and any system components in fluidic communication therewith (e.g., a cell-culture chamber disposed on the substrate).
  • the substrate may be in electrical communication with the manifold (e.g., the sensors placed on the substrate may be controllable and/or may supply data to one or more components placed on the substrate via electrical communication).
  • a system comprising components disposed on two substrates may comprise one substrate that is reusable and one substrate that is disposable.
  • the substrate on which the cell-culture chamber is placed may be disposable and the other substrate may be reusable. This may allow for cell culture to be performed in a disposable, replaceable system while not requiring the disposal of other, more expensive and/or difficult to replace, system components (e.g., pumps, valves, and/or sources of reagents).
  • the appropriate substrate may be disposed of and then the same components on the manifold may be employed to perform cell culture in a new cell-culture chamber placed on a new substrate.
  • FIG. 76 shows one example of a disposable substrate (e.g., that may be employed in combination with a second, non-disposable substrate).
  • the disposable substrate may comprise a cell-culture chamber in direct fluidic communication with two conduits and in which a dissolved-oxygen sensor and a pH sensor are disposed. It is also possible for the cell-culture chamber to be in direct fluidic communication with further conduits that are not shown.
  • the disposable substrate may further comprise a plurality of ports that are capable of being placed in fluidic communication with and/or are configured to be placed in fluidic communication with a second, non-disposable substrate (e.g., via a plurality of ports on the second, non-disposable substrate). As also shown in FIG.
  • the disposable substrate may comprise a gas-permeable membrane (labeled therein as a “Silicone Membrane”), a seeding port (labeled therein as a “Sample Well”), and an air chamber that may be employed to cause mixing within the cell-culture chamber (described in further detail elsewhere herein).
  • the disposable substrate shown in FIG. 76 also comprises a conduit that places the seeding port in fluidic communication with a port in the plurality of ports, through which it may be supplied to a conduit in direct fluidic communication with the cell-culture chamber (and, possibly, to the cell-culture chamber).
  • the flow path of liquid from the seeding port to the cell-culture chamber may pass through the manifold (e.g., from one port in the plurality of ports on the disposable substrate, through a portion of the manifold, and to a second port in the plurality of ports on the disposable substrate).
  • a system like that shown in FIGs. 73-75 possibly comprising a disposable substrate like that shown in FIG. 76
  • Use of the system may commence by performing a priming operation (e.g., controlled by a controller, such as a controller to which the system is plugged in). Then, cell-culture media and/or cells may be flowed into the cell-culture chamber (e.g., via a seeding process). Subsequently, the system may be sealed and/or placed in an instrument (as described in further detail below). Sealing the system may allow for it to be transported to an instrument without undue spillage and/or flow through different system components. After introduction of the system into the instrument, the seeding port may be cleaned (e.g., manually) and/or cells may be recovered through the seeding port and/or a different port. Recovered cells may be further analyzed (e.g., by one or more components on the manifold, by the instrument).
  • a priming operation e.g., controlled by a controller, such as a controller to which the system is plugged in.
  • cell-culture media and/or cells may be flowed into the cell
  • FIG. 77 shows one example of liquid flow through the system shown in FIGs. 73-75 during priming.
  • Priming may comprise flowing a liquid through some or all of the system components (e.g., through one or more conduits therein). Priming may prepare the subsequent flow of further liquid therethrough.
  • liquid flowing through the manifold depicted therein may flow into, out of, and/or through one or more reservoirs (e.g., one or more reservoirs serving as sources of reagents) and/or through one or more valves.
  • FIG. 78 shows one example of liquid flow through the system shown in FIGs. 73-75 subsequent to priming but prior to the introduction of cells into a cell-culture chamber.
  • This may comprise an initial filling of a cell-culture chamber with a liquid prior to introducing cells thereinto.
  • liquid may flow from a source thereof (e.g., positioned on a manifold) and into the cell-culture chamber (e.g., positioned on a substrate).
  • the liquid may flow therebetween via one or more ports and/or one or more conduits.
  • the liquid flowing through the cell-culture chamber may comprise cellculture media.
  • a port through which cleaning liquids may be supplied (shown in FIG. 78 as a “Sample Cleaning Port”) is not in fluidic communication with the rest of the system (e.g., it is isolated therefrom via a valve).
  • FIG. 79 shows one example of liquid flow through the system shown in FIGs. 73-75 during the introduction of cells thereinto (e.g., during cell seeding).
  • a liquid comprising cells may be introduced from a seeding port, flow through a port in the plurality of ports on the substrate into the manifold, flow through the manifold and back into the substrate via another port in the plurality of ports on the substrate, and flow into the cell-culture chamber.
  • the liquid may then flow out of the cell-culture chamber and into the manifold (and, possibly, out of the manifold and into a waste chamber and/or receptacle).
  • a port through which cleaning liquids may be supplied (shown in FIG. 79 as a “Sample Cleaning Port”) is not in fluidic communication with the rest of the system (e.g., it is isolated therefrom via a valve).
  • a cell filter positioned in the cell-culture chamber allows for liquid to flow out of the cellculture chamber while retaining cells therein.
  • FIG. 80 shows one example of liquid flow through the system shown in FIGs. 73-75 during perfusion.
  • fresh liquid media may flow into the cellculture chamber and old liquid media may flow out of the cell-culture chamber while cells are retained therein (e.g., by a cell filter).
  • FIG. 81 shows one example of liquid flow through the system shown in FIGs. 73-75 during cleaning of the conduits that fluidically connect a seeding port disposed on the manifold and the cell-culture chamber.
  • liquid may flow into an open (e.g., unplugged) port through which cleaning liquids may be supplied and through these conduits.
  • the liquid flowing during this process may comprise, consist of, and/or consist essentially of a wash liquid and/or a liquid comprising one or more cleaning agents.
  • FIG. 82 shows one example of liquid flow through the system shown in FIGs. 73-75 during the recovery of cells therefrom.
  • cells may be recovered from the seeding port.
  • liquid may flow into the cell-culture chamber (e.g., from a source of liquid media disposed on a manifold) and through the cell-culture chamber.
  • the liquid may flow out of the cell-culture chamber through a conduit that is in fluidic communication (e.g., via ports and/or conduits disposed on the substrate and/or the manifold) with the seeding port.
  • the cell-culture chamber may comprise a cell filter that is positioned to allow cells to flow through this pathway (e.g., it may be positioned proximate and/or across a conduit that places the cell-culture chamber in fluidic communication with a different port on the substrate, such as a port through which perfusion may occur).
  • the cells may accordingly flow with the liquid to the seeding port, from which they may be recovered.
  • the port through which cleaning liquids may be supplied may not be in fluidic communication with the rest of the system (e.g., it is isolated therefrom via a valve).
  • a system is positioned on a substrate (e.g., a chip) that further comprises one or more additional systems.
  • the additional systems may comprise some or all of the same components present in the system (e.g., a substrate may comprise a plurality of systems, each having at least one component that is the same). It is also possible for the additional systems to differ from the system (e.g., a substrate may comprise a plurality of systems, and some or all of the systems may differ from each other in one or more ways).
  • a substrate comprises two or more modules.
  • Each such module may be a system as described herein and/or two or more modules together may be part of a common system.
  • each module may comprise an identical set of components.
  • Such identical components may comprise a pump, a valve, a cell-culture chamber, and/or a sensor.
  • two or more modules present in a substrate may differ in one or more ways and/or comprise differing components.
  • two or more modules on a substrate share one or more components (e.g., they may share a pump, a valve, a mixing chamber, and/or a sensor).
  • liquid may flow from a module, through some or all of the common components, and/or back to the module as desired.
  • a substrate comprising two or more systems and/or two or more modules has a size that is similar to that of a standard laboratory consumable, such as a 96-well plate. Without wishing to be bound by any particular theory, it is believed that this may facilitate handling of the substrate with existing laboratory instrumentation.
  • FIGs. 83 and 84 show two non-limiting examples of substrates comprising pluralities of modules.
  • the raised components may comprise motors (e.g., that may be capable of driving and/or configured to drive valves and/or pumps).
  • FIGs. 85-91 show further views of the substrate shown in FIG. 83.
  • such modules may be in fluidic communication with a common chip manifold.
  • a plurality of systems and/or modules are arranged horizontally on a substrate (e.g., they pass through and/or be disposed on a single substrate). Without wishing to be bound by any particular theory, it is believed that this arrangement may facilitate high throughput.
  • a single second substrate may be supplied with a plurality of substrates (e.g., a plurality of substrates, each taking the form of a module).
  • FIG. 94 shows one non-limiting embodiment of a system having this design.
  • the system may comprise a plurality of similar and/or identical components placed on the second substrate (e.g., the second substrate may comprise a plurality of modules, each configured to interface and/or capable of interfacing with a substrate).
  • the second substrate may comprise one, common, set of components that are configured to interface and/or are capable of interfacing with a plurality of modules (e.g., comprising a single, common pump that can supply pressure to two or more modules; a single, common source of a reagent that can supply the reagent to two or more modules, etc.).
  • modules e.g., comprising a single, common pump that can supply pressure to two or more modules; a single, common source of a reagent that can supply the reagent to two or more modules, etc.
  • some embodiments relate to instruments that comprise a system described herein and/or are otherwise associated with an instrument described herein.
  • the system may fit into a component present in the instrument (e.g., a chip holder and/or tray).
  • a component present in the instrument e.g., a chip holder and/or tray.
  • an incubator e.g., a carbon dioxide incubator.
  • some or all of a system described herein is positioned inside an instrument that is an incubator.
  • a cell-culture chamber is positioned inside an incubator.
  • a system is positioned inside an incubator that is itself positioned inside an optical detector (e.g., an optical microscope).
  • a system can interface mechanically and/or electronically with an instrument.
  • a system is capable of being “plugged into” an instrument electronically and/or mechanically.
  • the instrument may be capable of providing and/or configured to supply pressure to one or more components of the system (e.g., positive and/or negative), which cause liquid to flow into, out of, and/or through the system.
  • pressure is supplied by flowing air (e.g., compressed air) into a component to pressurize it.
  • the instrument may comprise one or more air lines that can be mechanically coupled to the system.
  • an instrument serves as and/or comprise a source of one or more liquids (e.g., a liquid comprising a reagent, liquid media) and/or a receptacle to which one or more liquids can be transported (e.g., as waste, as liquid to be analyzed by the instrument).
  • a source of one or more liquids e.g., a liquid comprising a reagent, liquid media
  • a receptacle e.g., as waste, as liquid to be analyzed by the instrument.
  • an instrument comprises a controller that provides and/or receives signals (e.g., electrical signals) to and/or from the system.
  • a system comprises one or more conduits that take the form of tubing, such as PEEK tubing.
  • a cell-culture chamber contains cells.
  • Such cells may comprise adherent cells and/or suspended cells.
  • the suspended cells may be suspended in a liquid also contained in the cell-culture chamber.
  • suitable adherent cell types include HeLa cells, HEK293 cells, iPSC cells, and hMSC cells (human mesenchymal stem cells).
  • suitable suspended cell types include CHO cells, iPSC aggregates, T cells, HEK293S cells, and Jurkat cells.
  • liquids may be contained in the cell-culture chambers described herein and/or flowed therethrough (and/or contained in and/or flowed through other system components described herein).
  • some such liquids may comprise cells (e.g., suspended cells) and/or reagents.
  • Such liquids may be aqueous.
  • Liquid flow through cell-culture chambers and other system components may be intermittent and/or continuous. It may be flowed as part of a batch process, a fed-batch process, and/or a perfusion process (e.g., employing continuous and/or bolus addition, and/or allowing for the delivery of reagents in between additions).
  • a perfusion process e.g., employing continuous and/or bolus addition, and/or allowing for the delivery of reagents in between additions).
  • a method comprises flowing liquid through one or more system components to clean the component(s). For instance, in some embodiments, 10% bleach and/or 70% alcohol may be flowed through a system component (e.g., sequentially) to clean it. After cleaning, the system component may be prepared for cell-culture by flushing with a wash liquid (e.g., a PBS wash).
  • a wash liquid e.g., a PBS wash
  • one or more method steps described herein may be automated. For instance, flow through a cell-culture chamber, the introduction of reagents and/or cells into a cell-culture chamber, the removal of waste from a cell-culture chamber, and/or the removal of samples and/or cells from a cell-culture chamber may be automated. Automation may comprise performing the relevant method step at particular time points and/or in response to particular sensed conditions (e.g., pH, oxygen content).
  • particular sensed conditions e.g., pH, oxygen content
  • FIG. 95 shows a further exemplary system.
  • the system shown in FIG. 95 comprises a cell-culture chamber (labeled as a “growth chamber”) comprising a cell filter, a port through which a sample can be introduced and/or removed (e.g., via cell seeding and/or recovery), two sources of air (labeled “L air” and “R air”), a waste chamber (labeled “Waste”), three sources of reagents (labeled “L 5mL,” “R 5mL,” and “ImL”), a plurality of pumps, a plurality of valves, and a plurality of ports.
  • a cell-culture chamber labeled as a “growth chamber”
  • L air two sources of air
  • R air waste chamber
  • three sources of reagents labeleled “L 5mL,” “R 5mL,” and “ImL”
  • the ports in the plurality of ports serve to allow for fluidic communication of the cell-culture chamber with the sources of reagents upstream therefrom and the filter with the waste chamber downstream therefrom.
  • a pump and a valve positioned fluidically between each port and each reagent source or the waste chamber.
  • These pumps can serve to pump fluid to and/or from the ports with which they are in fluidic communication.
  • These valves can allow for such pumping to occur when open and can prevent such pumping from occurring when closed.
  • the port through which a sample can be introduced and/or removed is in fluidic communication with the cell-culture chamber.
  • FIG. 96 shows another exemplary system.
  • the system shown in FIG. 97 is similar to that shown in FIG. 95, except that the plurality of ports includes only a single port that allows for fluidic communication between the cell-culture chamber and two of the sources of reagents (those labeled “L 5mL” and R mL”) and that it includes further fluidic components associated with the source of reagents labeled “ImL.”
  • the sources of reagents are separated by separate valves from a single pump that is in fluidic communication with a single port. This pump, when pumping, can pump reagents from the sources of reagents with which it is in fluidic communication via an open valve.
  • a first valve (labeled “Valve 5”) is positioned between a port and a branching flow path.
  • the branching flow path includes a first branch that is in fluidic communication with a pump and the source of reagents labeled “ImL” and a second branch is in fluidic communication with the waste chamber via a second valve (labeled “Valve 4”).
  • This branching flow path may allow for reagents to be pumped from the source of reagents labeled “ImL” to the cell-culture chamber (if Valve 5 is opened and Valve 4 is closed).
  • FIG. 97 shows yet another exemplary system.
  • the system shown in FIG. 97 includes two modules, each comprising a cell-culture chamber (labeled as a “growth chamber”) comprising a cell filter, a port through which a sample can be introduced and/or removed (labeled “S”; e.g., via cell seeding and/or recovery), and a plurality of ports.
  • the system further comprises two different types of sources of reagents: those that are not temperature- controlled (labeled “#1 5mL” and “#2 5mL”) and those that are cooled (those labeled “#3” and “#4”).
  • a waste chamber (labeled “Waste”), a plurality of pumps, and a plurality of valves.
  • a pump is positioned between each source of a reagent and a junction, and can pump reagents from these sources of reagents to the junction.
  • On the other side of the junction are two valves (labeled “Valve 1” and “Valve 3”), each in fluidic communication with a port on a module.
  • a module’s valve When a module’s valve is open, reagents pumped from one or more sources of reagents into the junction can further flow into the cell-culture chamber through the port. Waste can flow out of each module through the filter, through a port, and to the waste chamber.
  • there are two further pumps (labeled “Pump 1” and “Pump 6”) that can pump waste accordingly.
  • There is also a further valve (labeled “Valve 2”) that is positioned between the junction and the waste chamber.
  • the non-temperature-controlled sources of reagents shown in FIG. 97 may be present in a manifold.
  • the temperature-controlled reagents shown in FIG. 97 may be stored separately (e.g., in a cold reservoir).
  • the sources of reagents may be purgeable.
  • FIG. 98 shows yet another exemplary system.
  • the system shown in FIG. 98 is similar to that shown in FIG. 97 except that it includes a different number of reagent tanks, includes two waste chambers (each labeled “Waste Well”), and includes flow cells positioned between the pumps that can pump waste and the waste chambers.
  • a system like that shown in FIG. 98 but including a valve instead of Pump 5 is also contemplated.
  • the system shown in FIG. 98 could be employed to perform perfusion by pumping Pump 5 at a speed twice that of the speeds of Pumps 1 and 4. Perfusion may be paused to supply one or more reagents from one or more of the sources of reagents to the cell-culture chambers.
  • FIG. 99 shows yet another exemplary system.
  • the system shown in FIG. 99 shows a system similar to that shown in FIG. 98, but in which many of the components are disposed on a substrate suitable for inclusion in an incubator (labeled “Incucyte Tray”). The waste chambers are not disposed on this substrate.
  • FIG. 100 shows yet another exemplary system.
  • various components are disposed on a substrate, including three manifolds (one for fluid pumps, one for fluid valves, one for air mixers), various ports, two consumables that each comprise a cell-culture chamber (labeled “Growth Chamber Consumable”), and one consumable comprising sources of reagents (labeled “Reagent Tank Consumable”).
  • the manifolds may be integrally connected to the substrate, the consumables comprising the cell-culture chambers may be removable, and/or the consumable comprising sources of reagents may be removable.
  • Systems described herein may include temperature sensors at a variety of suitable locations.
  • suitable locations include on and/or in manifolds, on and/or in ports in fluidic communication with sources of reagents (which may be particularly suitable for measuring the temperatures of reagents being supplied through such ports), and on and/or in sources of reagents.
  • sources of reagents which may be particularly suitable for measuring the temperatures of reagents being supplied through such ports
  • a temperature sensor may monitor the temperature a location that is being temperature-controlled (e.g., a refrigerator, a warming reservoir in fluidic communication with a refrigerator).
  • a system described herein may comprise one or more sensors (e.g., temperature sensors, other sensors, such as pH and/or dissolved oxygen sensors) that are translatable (e.g., with respect to one or more other components of the system, such as a cell-culture chamber therein).
  • sensors may advantageously be capable of measuring, be configured to measure, and/or measure a property at one or more locations in the system (e.g., one or more locations in a cell-culture chamber, two or more different cell-culture chambers).
  • the translation may be effectuated by use of a motor.
  • the motor may be controlled automatically (e.g., via software and/or electrical signals) and/or manually (e.g., by an operator).
  • the sensors have a design such that they can measure a feature of a system without contact (e.g., they may be optical sensors). Sensors that are translatable may be particularly suitable for performing multiplexing.
  • a system comprises a component comprising two sensors that are translatable together (e.g., that are mechanically coupled, that are integrally connected, that cannot be separated without the use of specialized tools, that are mounted on the same component and/or holder).
  • FIG. 101 depicts one non-limiting example of a block diagram for an exemplary translatable component comprising two sensors.
  • the two sensors shown in FIG. 101 are a pH patch sensor and a dissolved oxygen patch sensor (labeled “DO patch sensor”) that are both optical sensors comprising optical fibers. These sensors are mounted on a single holder and are controlled by separate electro-optical OEM module boards that are themselves in electrical communication with a computer.
  • FIG. 102 is a photograph showing the location of the component shown in FIG. 101 within an exemplary system. In this photograph, it is labeled as “PreSens pH patch sensor” and is positioned to make a measurement of a cell-culture chamber.
  • FIG. 103 depicts a second block diagram of the component shown in FIGs. 101 and 102.
  • the pH and dissolved oxygen sensors are in electrical communication with an optical transmitter, which itself is in electrical communication with a board that can supply power and can communicate with the sensors.
  • the optical transmitter is also in electrical communication with a PC, which may be employed to trigger cell imaging and/or analysis, provide information, and/or detect and/or control location and/or movement of the component (e.g., position, whether the component is moving, whether the component is idle).
  • the PC is in electrical communication with a computer controlling an incubator in which the component may be positioned (labeled “Incucyte controller computer”).
  • FIG. 104 shows a cross-sectional view of a portion of an exemplary system.
  • the system includes a silicone gasket (identified by the arrow). This silicone gasket seals the components that it is positioned between (which, in FIG. 104, are formed from polycarbonate).
  • the system shown in FIG. 104 further depicts screws, but systems otherwise similar to those shown in FIG. 104 but lacking screws are also contemplated (e.g., that are heat-staked together).
  • Some systems like those shown in FIG. 104 may be facilely injection molded and/or may be sealed such that leaks, contamination, and/or bubbles are limited or avoided. Some such systems may desirably exhibit relatively low dead volumes.
  • a system described herein comprises a component that is capable of delivering a gas (e.g., air, oxygen, nitrogen), configured to deliver a gas, and/or delivers a gas to a cell-culture chamber.
  • a gas e.g., air, oxygen, nitrogen
  • Such a component has been referred to elsewhere herein as an “Air Mixer Manifold” and “air mixer manifold.”
  • FIG. 105 One exemplary air mixer manifold is shown in FIG. 105.
  • FIG. 106 depicts a system diagram for an exemplary air mixer manifold.
  • Air mixer manifolds may be fluidically connected to a cell-culture chamber described herein via one or more ports. It is also possible for such a component to be a mixing element as described elsewhere herein. In such instances, an air mixer manifold may be capable of delivering a gas, configured to deliver a gas, and/or deliver a gas to a cell-culture chamber in a manner that causes the contents of the cell-culture chamber to mix. It is also possible for an air mixer manifold to supply a fresh gas to the cell-culture chamber while causing minimal or no such mixing. The gas supplied from the air mixer manifold may diffuse through a gas- permeable membrane that forms a portion (or all) of a wall of a cell-culture chamber.
  • the amount of dissolved oxygen in a cell-culture chamber supplied with gas by an air mixer manifold may be capable of being controlled, may be configured to be controlled, and/or may be controlled independently from the amount of mixing effectuated by the air mixer manifold. This may be achieved by selecting the gas composition to deliver a desired amount of oxygen and an amount of gas (e.g., at a particular pressure and/or flow rate) to deliver a desired amount of mixing and/or applying a positive pressure at two or more ports (e.g., at both a port supplied by an air mixer manifold and a vent port).
  • an air mixer manifold may be present in a system comprising a cell-culture chamber containing adherent cells.
  • an air mixer manifold comprises a pump, such as a piezo pump.
  • the pump may draw fresh air in from the atmosphere and/or may vent waste air back to the atmosphere (e.g., through the gas-permeable membrane described above).
  • the pump maintains a high pressure reservoir.
  • placing the air mixer manifold in fluidic communication with a cell-culture chamber e.g., by opening a valve positioned therebetween
  • a single air mixer manifold may be capable of causing mixing in and/or supplying air to two or more cell-culture chambers, may be so configured, and/or may cause mixing and/or supply air to two or more cell-culture chambers. Fluidic communication between the cell-culture chamber(s) and the air mixer manifold may be started by opening a valve positioned therebetween and stopped by closing such a valve.
  • Air mixer manifolds described herein may be operated via automation (e.g., via software and/or electrical signals) and/or manually (e.g., via an operator).
  • Air mixer manifolds described herein may have a size and/or shape such that they can be positioned in a system described herein and/or in an incubator described herein.
  • a system described herein has a design that facilitates cell sampling.
  • FIGs. 107 and 108 illustrate one portion of such a system.
  • a system may include a bracket that, in a depressed state, seals a membrane to the other system components (FIG. 107), but that, in a raised state, allows the membrane to deform upwards (FIG. 108).
  • FIG. 108 When the membrane is deformed upwards, fluid can flow through the position where the membrane was formerly located and to a location at which it can be removed from the system (e.g., from a cell-culture chamber).
  • This location may be a port through which cell recovery may be performed and/or may allow for a capillary tube, pipette, and/or collection vessel into which fluid can enter via capillary action and/or aspiration to be inserted thereinto.
  • the bracket can serve as a valve that is typically locked in the closed position (i.e., the depressed state), but that can be opened (i.e., allowed to assume the raised state). The opening can be effectuated by pressing on a snap feature.
  • the system shown in FIGs. 107 and 108 may prevent direct fluidic communication between a cell-culture chamber and an environment external to the system, which may reduce contamination of the cell-culture chamber. Such a system may also, additionally or alternatively, exhibit reduced dead volume.
  • FIGs. 109 and 110 depict another example of a design that facilitates cell sampling.
  • FIG. 109 is a top view and FIG. 110 is a side view.
  • the design shown in FIGs. 109 and 110 includes two chambers positioned in series between a port through which a sample can be introduced and/or removed (e.g., via cell seeding and/or recovery) and a cell-culture chamber: a first chamber that acts as a membrane valve (e.g., that exhibits zero dead volume), and a second chamber that acts as a pump that is capable of pumping, configured to pump, and/or pumps a known sample volume therethrough (e.g., from the port to the cell-culture chamber, from the cell-culture chamber to the port).
  • a first chamber that acts as a membrane valve (e.g., that exhibits zero dead volume)
  • a second chamber that acts as a pump that is capable of pumping, configured to pump, and/or pumps a known sample volume therethrough (e.g., from the port to
  • a membrane e.g., a silicone membrane passes through these chambers. This membrane may be raised and lowered to affect the pressure in these chambers and their fluidic communication with each other, the cell-culture chamber, and the port.
  • the system shown in FIGs. 109 and 110 further comprises a channel passing through the second chamber and into the cell-culture chamber.
  • Membrane valves such as those shown in FIGs. 109 and 110 may benefit from the application of a positive pressure to maintain desired seals.
  • FIG. I l l depicts a method that may be performed in the system shown in FIGs. 109 and 110 by which a sample may be transferred from the port to the cell-culture chamber.
  • the portions of the membrane spanning the two chambers may be raised and lowered to cause fluid to flow out of the port, through the two chambers, into the channel, and into the cell-culture chamber.
  • the channel may further contain a buffer with which the sample may mix prior to flowing into the cell-culture chamber.
  • the port may be flushed with liquid that is directed to a waste chamber and/or cleaned.
  • FIG. 112 depicts another example of a design that facilitates cell sampling.
  • the system shown in FIG. 112 includes a cell-culture chamber (labeled “Growth Chamber”) comprising a filter, a port through which a sample can be introduced and/or removed (labeled “Seed”), a waste chamber (labeled “Waste”), various sources of reagents (labeled “L 5mL,” “R 5mL,” and “ImL”), and various pumps and valves.
  • the valves may, when open, allow for fluidic communication therethrough, and, when closed, prevent fluidic communication therethrough.
  • the pumps may be capable of pumping, configured to pump, and/or pump fluid through the system in the directions of the arrows shown in FIG. 112.
  • the waste chamber may receive waste from the port (through Valve 1, Valve 8, and Pump 1), and/or from the cell-culture chamber (through Filter, Valve 2, and Pump 1).
  • a port through which a sample can be introduced and/or removed may be capable of serving, configured to serve, and/or serve as a vent port (e.g., after priming and/or by remaining open).
  • a valve positioned between the cell-culture chamber and this port may subsequently be closed and this port and any associated flow paths may be cleaned via a sterilization cycle.
  • FIGs. 113-114 depict yet another example of a design that facilitates cell sampling.
  • FIG. 113 depicts a larger portion of a system having such a design and FIG. 114 depicts a capillary and the portions of this system associated with the capillary.
  • FIG. 114 depicts a capillary and the portions of this system associated with the capillary.
  • These Figures depict spring-loaded mechanical components that together are configured to actuate, capable of actuating, and actuates the filling and removal of a capillary tube.
  • the top panel in each Figure depicts the presence of a plug on the end of a capillary. As shown by the middle panels, this plug may be removed (e.g., via a first plunger), which causes the capillary to be filled to a specified volume by fluid originating from the system component to the left thereof (e.g., a cell-culture chamber).
  • a second plunger is actuated, which causes the capillary to be moved so that it is no longer positioned to receive fluid from the location from which it previously receiving fluid. This may be accomplished by, for instance, removing the capillary from that location, such as by pulling it out of a septum.
  • a system having a design like that shown in FIGs. 113-114 may be capable of storing multiple capillaries, configured to store multiple capillaries, and/or store multiple capillaries.
  • FIG. 115 shows a cell-culture chamber (labeled “Growth Chamber”) that stores multiple capillaries (shown as lines extending downwards therefrom).
  • a system like that shown in FIG. 115 may be preloaded with a plurality of capillary tubes that can be employed to perform cell sampling during cell culture as desired. Preloading all of the capillaries that are anticipated to be needed during a particular cell culture process may desirably allow for the entire system to be sterilized together.
  • FIG. 115 further depicts a source of a cleaning fluid (labeled “Clean”) that may be supplied to a port through which a sample can be introduced and/or removed (labeled “Seed”) and/or the cell-culture chamber.
  • a source of a cleaning fluid labeled “Clean”
  • Seed a sample can be introduced and/or removed
  • a system described herein has a design that facilitates performing cell counting.
  • FIG. 116 depicts one non-limiting example of such as system.
  • This system includes a flow cell having a dimension that is sufficiently small such that cells cannot flow therethrough in a manner where they overlap along that dimension (e.g., such that the cells form a monolayer, such that, if the dimension is a height, cells cannot flow above or below each other, such that a projection of the flow cell in the plane perpendicular to the direction does not include any cells that overlap spatially).
  • This may desirably allow for facile cell counting, which may be employed to determine cell density.
  • FIG. 116 also depicts a process by which a sample may be retrieved from a cellculture chamber (labeled “Growth Chamber”).
  • Pump 1 may be employed to pull a sample out of the cell-culture chamber through Valve 3.
  • Pump 2 may be employed to pump fresh cell-culture media into the cell-culture chamber through Valve 3, thereby flushing any cells positioned between Pump 2 and Valve 3 (e.g., those upstream from the junction between Valve 3 and Valve 4 and those on the Valve 3 branch of that junction) into the cell-culture chamber. Any remaining cells present between Pump 2 and Valve 4 (e.g., those on the Valve 4 branch of that junction) may be pumped through the flow cell by Pump 1 and/or Pump 2.
  • Samples may be introduced into the cell-culture chamber shown in FIG. 116 from a port through which a sample can be introduced and/or removed (labeled “Seed”) by opening Valves 2 and 3 and employing Pump 1 to cause fluid to flow from this port, through Valve 1, through Valve 3, through the cell-culture chamber, through the filter, through Valve 2, through the flow cell, and through Pump 1 to the waste chamber (labeled “Waste”).
  • a port through which a sample can be introduced and/or removed labeled “Seed”
  • Pump 1 to cause fluid to flow from this port, through Valve 1, through Valve 3, through the cell-culture chamber, through the filter, through Valve 2, through the flow cell, and through Pump 1 to the waste chamber (labeled “Waste”).
  • FIGs. 117 and 118 depict two different designs for manners in which valves may be sealed. Both designs include a pusher (which may be plastic) that can be pushed downwards to apply force to a component of the valve to cause a seal to form.
  • FIG. 117 depicts a valve including a sealing rib and a diagonal channel disposed in a hard plastic.
  • FIG. 118 depicts a valve including two silicone components between which a membrane is disposed.
  • a valve comprises a spring-loaded pusher such that the pusher is closed unless force is applied to compress the spring.
  • the valves shown in FIGs. 117 and 118 may be opened and/or closed manually (e.g., by an operator) and/or in an automated manner (e.g., in response to electrical signals sent by software and/or an electrical controller).
  • Rotating valves positioned between a cell-culture chamber and a port through which a sample can be introduced and/or removed may allow for samples to be dosed in precise volumes and exhibit simple designs that could be operated by solenoids.
  • Membrane valves (such as those shown in FIGs. 109-111) may exhibit reduced dead volume, linear fluidics that reduce risk of contamination, a compact structure, and allowing for facile sample recovery from ports.
  • Components configured to actuate, capable of actuating, and/or that actuate capillary tubes (such as those shown in FIGs. 113 and 114) may be mechanically straightforward and may be capable of being used with existing fluidics without modifying such fluidics.
  • Flow cells like that shown in FIG. 116, may allow for quick and/or accurate cell counting and may have a very low likelihood of introducing contamination into the system.
  • FIG. 119 is a photograph of an exemplary cross-slit valve through which a capillary can be inserted.
  • FIG. 120 includes mechanical drawings showing the valve with an inserted capillary and without. Capillaries inserted through a cross-slit valve may form a radial seal with a silicone gasket present in a system described herein.
  • Some systems described herein comprise a chip having a substrate formed from relatively few components.
  • some systems comprise a chip having a substrate that is formed a silicone layer (e.g., a cast silicone layer) positioned between two plastic pieces.
  • a silicone layer e.g., a cast silicone layer
  • FIG. 121 A top view of one chip having such a design is shown in FIG. 121 and a side view of this chip is shown in FIG. 122.
  • Such chips may be sealed by a compressive face seal along the edges of any open portions therein (e.g., any chambers and/or channels).
  • one or more such open portions e.g., one or more chambers and/or channels
  • either or both of the plastic pieces comprises one or more positioning ribs that assist with forming a seal.
  • the silicone layer may also comprise one or more relatively thin portions that form one or more membranes described elsewhere herein.
  • FIG. 123 depicts such a portion with an arrow.
  • FIGs. 124 and 125 depict a further example of a chip having a substrate that is formed a silicone layer positioned between two plastic pieces.
  • the chip shown in FIGs. 124 and 125 comprises a cell-culture chamber comprising top and bottom hexagonal walls.
  • One such hexagonal wall comprises a portion that is formed from cast silicone and comprises a plurality of protrusions that extend towards the opposing hexagonal wall.
  • Such protrusions may form a seal with this opposing hexagonal wall and/or apply a compressive force thereto.
  • the spaces between such protrusions may take the form of channels through which fluid may flow.
  • Such flow may comprise flow over a filter positioned in the cell-culture chamber and/or flow over one or more sensors (e.g., over a pH sensor and/or a dissolved oxygen sensor).
  • a system described herein may be suitable for performing RNA transcription and/or a method may comprise performing RNA transcription in a system described herein. In some embodiments, RNA transcription may be performed in a cellculture chamber described herein.
  • RNA transcription is a process by which a DNA sequence is copied into a strand of messenger RNA.
  • the process comprises three steps: initiation, elongation, and termination.
  • Initiation comprises binding an RNA polymerase to a DNA promoter sequence on double-stranded DNA and then opening the DNA.
  • Elongation comprises growing an RNA strand with the assistance of the RNA polymerase.
  • one strand of the opened, double- stranded DNA serves as a template for the growing RNA strand.
  • nucleotides may be added to the RNA strand complementary to the template DNA strand.
  • Termination occurs at the end of RNA transcription. Termination may be triggered by the formation of terminator sequences in the growing RNA strand. This causes the release of the RNA from the RNA polymerase.
  • RNA transcription process may require the presence of one or more reagents.
  • reagents may be introduced into a system and/or instrument described herein (and/or into one or more components thereof) from sources thereof. It is also possible for a system and/or instrument described herein to comprise a source of a reagent.
  • Non-limiting examples of such reagents include DNA templates (i.e., double- stranded DNA that serves as a template for growing RNA), an RNA polymerase, nucleoside triphosphates (also referred to as "NTPs"), cofactors, Mg 2+ , capping reagents, acids, and bases, RNase inhibitors, and pyrophosphatase.
  • NTPs may serve as the precursors undergo a reaction during which they are incorporated into the growing RNA strand
  • cofactors are proteins that may bind to the DNA being transcribed and control the rate of RNA transcription
  • Mg 2+ (which may be provided in the form of a salt, such as MgCh) may assist with stabilizing the structure of the RNA polymerase during transcription
  • capping reagents may assist with forming a cap on the growing RNA
  • acids and bases may be employed to perform pH control
  • RNase inhibitors may reduce RNA degradation by inhibiting RNAse
  • pyrophosphatase may remove pyrophosphates formed during RNA synthesis by catalyzing their hydrolysis to orthophosphates.
  • one or more reagents are provided in a buffer and/or are added to a fluid comprising a buffer (e.g., a transcription buffer).
  • RNA polymerase One non-limiting example of an RNA polymerase is T7.
  • Non-limiting examples of suitable NTPs include adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).
  • suitable cofactors include general transcription factors (GTFs), mediator complexes, chromatin-modifying enzymes, RNA-binding proteins (RBPs), transcriptional activators, transcriptional repressors, and transcription elongation factors.
  • Non-limiting examples of suitable capping reagents include reagents suitable for enzymatic capping and/or post-transcriptional capping (e.g., vaccinia capping enzyme (VCE), faustovirus capping enzyme (FCE)), and co-transcriptional capping enzymes (e.g., cap analogs).
  • VCE vaccinia capping enzyme
  • FCE faustovirus capping enzyme
  • co-transcriptional capping enzymes e.g., cap analogs
  • Non-limiting examples of suitable acids include acetic acid and citric acid.
  • Non-limiting examples of suitable bases include sodium hydroxide (NaOH), potassium hydroxide (KOH), and sodium bicarbonate (NaHC03).
  • a method of performing RNA transcription comprises performing one or more steps that involve allowing RNA to be synthesized from a DNA template, monitoring the progress of RNA synthesis, and/or collecting RNA synthesized during an RNA transcription process.
  • Such methods may comprise measuring the amount of RNA in a fluid (e.g., present in a cell-culture chamber described herein) and/or adjusting the fluid composition based on such a measurement.
  • the fluid composition may be maintained if the measured amount of RNA indicates that RNA transcription is proceeding in an appropriate and/or expected manner.
  • the fluid composition may be adjusted if the measured amount of RNA indicates that RNA transcription is occurring at a rate that is too fast or too slow.
  • a method may comprise, in addition to or instead of measuring the amount of RNA in the fluid and adjusting the fluid composition based on the measured amount of RNA, measuring the amount of a reagent present in the fluid and/or adjusting the fluid composition based on the measured amount of the reagent.
  • a method may comprise measuring an amount of NTP, Mg 2+ , and/or a capping reagent and maintaining or adjusting the fluid composition based on the measurement.
  • the fluid composition may be adjusted if the measurement indicates that the fluid includes too little or too much of such reagents for RNA synthesis to proceed at a desired rate.
  • adjusting the composition of a fluid may comprise adding a reagent to the fluid, such as from a source.
  • the adjustment may comprise adding NTP, Mg2+, and/or a capping reagent to the fluid.
  • an adjustment of the composition of a fluid may comprise adding a buffer (e.g., a transcription buffer) lacking any reagents to the fluid in order to dilute the concentration of reagents in the fluid.
  • This Example depicts an exemplary system and its use in an incubator.
  • FIG. 126 shows various photographs of an exemplary system described herein and its placement in an incubator.
  • This Example describes the performance of HeLa cell culture in a system described herein.
  • FIGs. 127-129 show micrographs obtained at various time points during the culturing of HeLa cells in a cell-culture chamber in an exemplary system disposed in a humidified chamber. As can be seen from these micrographs, it is possible to proliferate cells in such cell-culture chambers.
  • FIGs. 130-133 show micrographs obtained prior to perfusion on the second day of cell-culture (FIG. 130), immediately subsequent to perfusion (FIG. 131), and several days later (FIGs. 132 and 133).
  • the cell culture was performed substantially as described above.
  • Perfusion comprised removing the cell-culture chamber from the humidified chamber and then removing waste to a waste chamber, adding fresh liquid media, and mixing. As can be seen from these Figures, it was possible to perform perfusion and still continue to proliferate cells in the cell-culture chamber.
  • FIGs. 134 and 135 shows micrographs obtained on the eighth day of cell culture after performing perfusion on the second and seventh days and a micrograph obtained on the eighth day of cell culture performed without perfusion.
  • the cell culture and perfusion were performed substantially as described in above. As can be seen from these Figures, performing perfusion resulted in a higher degree of cell proliferation.
  • This Example describes the performance of iPSC cell culture in a system described herein.
  • FIG. 136 shows micrographs obtained at various time points. As can be seen from these micrographs, iPSC cells were capable of being cultured under such conditions.
  • This Example describes the performance of CHO cell culture in a system described herein.
  • Example 2 Cell culturing was performed substantially as described in Example 2. Perfusion was also performed substantially as described in Example 2. Perfusion comprised removing waste for 20 seconds and then adding fresh liquid media for 20 seconds. Perfusion was performed every eight hours for the first 6.5 days of cell culture, and was performed every four hours thereafter. Additionally, during perfusion, the pump was operated at 22 rpm for the first two days of cell culture, and was operated at 11 rpm thereafter.
  • FIG. 137 shows confluency as a function of elapsed time (in hours)
  • FIG. 138 shows a micrograph obtained on the first day of cell culturing
  • FIG. 139 shows a micrograph obtained on the ninth day of cell culturing.
  • CHO cells were capable of being cultured under the above conditions.
  • This Example describes the performance of HeEa cell culture in a system described herein.
  • a sample comprising HeEa cells were introduced into a system comprising a cellculture chamber described herein from a port by pumping the sample from the port to the cell-culture chamber. Air-containing syringes were then employed to dispense air into the system on opposing sides of the cell-culture chamber, causing flow within the cell-culture chamber and mixing of the sample. Then, cell culture was performed. At certain time points during cell culture, the cell-culture media was removed from the cell-culture chamber and replaced with fresh cell-culture media.
  • FIG. 143 shows confluency as a function of time and FIG. 141 shows the dissolved oxygen as a function of time.
  • the arrow in FIG. 140 indicates a time point at which cells were accidentally removed from the cell-culture chamber.
  • the arrows in FIG. 141 indicate time points at which fresh media was added.
  • confluency increased as a function of time and dissolved oxygen content decreased as a function of time, indicating cell proliferation. Additionally, the introduction of fresh media caused the dissolved oxygen to increase.
  • optical micrographs were also taken of various positions in the cellculture chamber as a function of time during cell culture. These optical micrographs indicate that the cells were well-mixed and were dispersed relatively evenly across the cell-culture chamber.
  • This Example describes the performance of HeLa cell culture after transfection in a system described herein.
  • a sample comprising HeLa cells was introduced into a system comprising a cellculture chamber and cultured for 24 hours as described in Example 5. Then, the HeLa cells were transfected by GFP mRNA and jetRNA+ mix. After transfection, cell culture continued and fluorescent images of the cell-culture chamber were obtained at various time points.
  • FIG. 142 Two sets of these images are shown in FIG. 142. As can be seen from FIG. 142, transfection was successful.
  • the live cell counts and the average intensity of green fluorescence present in the fluorescent images were determined and compared to live cell counts and average intensity of green fluorescence present in HeLa cells transfected in the same manner but cultured instead in wells in a microwell plate.
  • the live cell counts and the average intensity of green fluorescence were comparable for the fluorescent images obtained from the cell-culture chamber and those obtained from the microwell plates, further indicating the suitability of the system for culturing HeLa cells.
  • This Example describes the performance of T cell culture in a system described herein.
  • PBMC cells were thawed, concentrated, and then introduced to the cell-culture chamber of a system described herein. After being introduced into the cell-culture chamber, these cells were mixed and cultured as described in Example 5. Additionally, PBMC cells were prepared in the same manner but, instead of being introduced into a cell-culture chamber, were cultured in wells in a microwell plate. Optical micrographs of the cell-culture chamber and microwell plate were obtained at various time points and samples of the cells were removed from the cell-culture chamber and microwell plate at various time points.
  • the optical micrographs indicated that the confluency of the PBMC cells cultured in the cell-culture chamber was comparable to the confluency of the PBMC cells cultured in the wells of the microwell plate. This is shown in FIG. 146, where the arrow indicates the introduction of a bubble that artificially decreased further confluency measurements. Additionally, the visually observed color of the cell-culture chamber was comparable to that of the wells in the microwell plate, indicating comparable T cell expansion.
  • the viability of cells in the samples of cells removed from the cell-culture chamber was comparable to the viability of the cells removed from the wells of the microwell plate (80- 85%).
  • This Example describes the performance of iPSC cell culture in a system described herein.
  • Samples comprising iPSC cells were introduced into humidified cell-culture chambers in a system as described in Example 5. Subsequent to such introduction, the samples were mixed and then cultured as described in Example 5. Optical micrographs were obtained during cell culture at various time points, from which live cell counts were obtained. These live cell counts are shown in FIG. 144, which shows that cell culture was performed successfully.
  • FIG. 145 shows phase object counts per image of cell-culture chambers supplied with cell-culture media in this manner. As can be seen from FIG. 145, the use of a manifold in this manner did not result in appreciable cytotoxicity.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

L'invention concerne de manière générale des systèmes pour effectuer une culture cellulaire et des procédés de réalisation d'une culture cellulaire. Certains de ces systèmes comprennent une ou plusieurs caractéristiques qui facilitent la performance de culture cellulaire d'une manière qui est relativement avantageuse. Par exemple, de tels systèmes peuvent comprendre une ou plusieurs caractéristiques qui facilitent la réalisation d'une culture cellulaire conjointement avec un composant fourni en tant que partie du système ou externe à celui-ci, tel qu'un incubateur, un système d'imagerie et/ou des sources externes de réactifs. À trois autres titres exemples, de tels systèmes peuvent comprendre une ou plusieurs caractéristiques qui facilitent l'utilisation du système d'une manière traversante, facilitent la détection en temps réel d'une ou de plusieurs propriétés du liquide dans lequel les cellules sont cultivées, et/ou permettent au système (et/ou à un ou plusieurs composants de celui-ci) d'avoir un volume relativement petit. Dans certains modes de réalisation, un système a une combinaison d'une partie ou de la totalité des avantages décrits ci-dessus qui facilite la réalisation d'expériences multiplex pour évaluer l'impact de différentes conditions de culture cellulaire sur la croissance cellulaire. Certaines de ces expériences peuvent être réalisées de manière souhaitable sans nécessiter de réactifs et/ou de cellules appréciables, et/ou peuvent être avantageusement réalisées dans un incubateur unique. Certains procédés présentement décrits peuvent concerner la réalisation d'une culture cellulaire dans un ou plusieurs systèmes présentement décrits.
PCT/US2025/024411 2024-04-12 2025-04-11 Systèmes et procédés de culture cellulaire Pending WO2025217607A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20020025547A1 (en) * 2000-08-14 2002-02-28 Govind Rao Bioreactor and bioprocessing technique
US20060199260A1 (en) * 2002-05-01 2006-09-07 Zhiyu Zhang Microbioreactor for continuous cell culture
US20140099717A1 (en) * 2006-07-07 2014-04-10 University Of Miami Enhanced oxygen cell culture platforms
US20180135006A1 (en) * 2015-05-07 2018-05-17 Bayer Aktiengesellschaft Modular system and process for the continuous, microbe-reduced production and/or processing of a product
US20180149593A1 (en) * 2016-11-30 2018-05-31 Scientific Industries, Inc. Apparatus for Detecting pH and Dissolved Oxygen

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025547A1 (en) * 2000-08-14 2002-02-28 Govind Rao Bioreactor and bioprocessing technique
US20060199260A1 (en) * 2002-05-01 2006-09-07 Zhiyu Zhang Microbioreactor for continuous cell culture
US20140099717A1 (en) * 2006-07-07 2014-04-10 University Of Miami Enhanced oxygen cell culture platforms
US20180135006A1 (en) * 2015-05-07 2018-05-17 Bayer Aktiengesellschaft Modular system and process for the continuous, microbe-reduced production and/or processing of a product
US20180149593A1 (en) * 2016-11-30 2018-05-31 Scientific Industries, Inc. Apparatus for Detecting pH and Dissolved Oxygen

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