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WO2025166414A1 - Dispositif microfluidique à faible débit - Google Patents

Dispositif microfluidique à faible débit

Info

Publication number
WO2025166414A1
WO2025166414A1 PCT/AU2025/050086 AU2025050086W WO2025166414A1 WO 2025166414 A1 WO2025166414 A1 WO 2025166414A1 AU 2025050086 W AU2025050086 W AU 2025050086W WO 2025166414 A1 WO2025166414 A1 WO 2025166414A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluid
flow
microfluidic
cell
microfluidic device
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/AU2025/050086
Other languages
English (en)
Inventor
Jeremy Gilbert Elliot Thompson
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.)
Cell Tech Holding Pty Ltd
Original Assignee
Cell Tech Holding Pty Ltd
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
Priority claimed from AU2024900270A external-priority patent/AU2024900270A0/en
Application filed by Cell Tech Holding Pty Ltd filed Critical Cell Tech Holding Pty Ltd
Publication of WO2025166414A1 publication Critical patent/WO2025166414A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • B01L3/50273—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 the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • 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
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    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M29/10—Perfusion
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    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/14—Pressurized fluid
    • C—CHEMISTRY; METALLURGY
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1894—Cooling means; Cryo cooling
    • 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
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0638—Valves, specific forms thereof with moving parts membrane valves, flap 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
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/28—Constructional details, e.g. recesses, hinges disposable or single use
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/30—Constructional details, e.g. recesses, hinges biodegradable
    • 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
    • C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas

Definitions

  • the technical field generally relates to low flow microfluidic devices, low flow microfluidic systems and methods adapted for use in a variety of cell or tissue culture environments.
  • Microdevices enable the manipulation of cells and their culture environments, giving rise to new therapies, products, and processes, many of which are still in their infancy.
  • New microdevices and improvements to existing microdevices such as microfluidic devices, 'lab on a chip' and 'organ on a chip' technologies, microscaffolds and micromanipulation devices have given rise to new approaches to 3D tissue engineering, stem cell differentiation, and assisted reproductive technologies as well as advances in the success rates of these techniques.
  • organ culture systems for various embryonic tissues now enable the cultivation of embryonic brain, retina, cardiac and vascular tissue, limb bud, lung, kidney, salivary gland, hair follicle, and tooth cell lines.
  • organ culture systems for various embryonic tissues now enable the cultivation of embryonic brain, retina, cardiac and vascular tissue, limb bud, lung, kidney, salivary gland, hair follicle, and tooth cell lines.
  • the application of new tissue engineering techniques, and the microdevices that enable these techniques promote new and improved approaches to a number of different therapies.
  • the present disclosure enables the user to provide multiple media treatments.
  • the only way to achieve a certain number of different media treatments would involve the same number of different dishes, or different wells in a well plate.
  • the disclosed cradle level control permits multiple media treatments within a single dish, loading only one set of reagents.
  • microfluidic system is designed so that a specific operation of valves and onboard pumps can deliver microfluidic flow and fluids to an individual, or to multiple cell cradles. Resulting in each cradle being able to receive its own unique flow and media profile.
  • cryoprotectants are more viscous than water-based media and require greater pressure or larger channels for equivalent flow rates.
  • Microfluidic channels for delivery of cryoprotectants are optimised for the delivery of higher viscosity fluids to the culture chambers.
  • Microfluidic supports described herein may comprise an upper microfluidic support surface and a lower microfluidic support surface and each of the one or more valves comprises a pump.
  • a preferred pump may be a pneumatic pump.
  • Valves according to the present disclosure modulate the flow of fluid through the microfluidic device, preferably, upon the upper microfluidic support surface.
  • the microfluidic device is adapted for culturing one or more cells thereon.
  • the flow of fluid within the microfluidic device to cell cradles comprising one or more cells therein may be stopped at predetermined positions to allow for static (no flow in either direction) conditions as required.
  • microfluidic devices may comprise mixing channels or chambers to ensure that solution equilibrium is achieved. Thereafter, exposure to biological elements may commence.
  • Microfluidic devices and systems disclosed herein may comprise a network of tubing and 2PP printed channels with thin, mechanically flexible membranes spanning the channels.
  • microfluidic devices and systems disclosed herein may comprise a pneumatic supply and control system.
  • the present disclosure relates to methods for use of microfluidic devices and systems.
  • the timing of the repeated programmed sequences of such methods can be modified to achieve higher or lower flow rates.
  • the one or more valves of the microfluidic devices, systems and methods disclosed herein may actuate the flow of fluid through the microfluidic device by a singular pneumatic channel (i.e. a channel containing fluids driven by pressurised gas), allowing for multiple embedded pumps to run simultaneously on a singular, shared programmed sequence, or the cessation of fluidic flow to all single cell holding devices.
  • a singular pneumatic channel i.e. a channel containing fluids driven by pressurised gas
  • Incubators used for cell culture are typically supplied with a premix of reduced air comprising various concentrations of oxygen, carbon dioxide, nitrogen or air gas (either separately or mixed in various proportions) for the purpose of modulating the level of oxygen dissolved in the media and the regulation of the pH of the media.
  • Gases are typically provided under pressure where they can be harnessed to drive a dedicated pump. Further, pressurised ambient gases such as incubator gases may be used to drive additional pumps and/or to actuate the valves described here. This is termed 'incubator supply'.
  • Incubator supply pressure can be used to drive the pumps and/or valves described herein or they can be driven by an additional source of pressurised fluid.
  • the pressure that drives the pumps and actuates valves is the 'pneumatic supply' whether it is sourced from the 'incubator supply' or some independent source of gas pressure.
  • Incubator supply pressure is one source of 'pneumatic supply' which may be utilised to drive the pumps and/or actuate the valves described herein.
  • pneumatic supply comprises incubator supply, preventing changes in gas concentration from impacting the osmolarity or pH of the fluid or media in the event of valve pneumatic leakage (gas from pneumatic channels impacting on the fluidic channels).
  • the pneumatic supply of preferred methods is preferably the same as the incubator supply, preventing changes in gas concentration from impacting the osmolarity or pH of the fluid or media in the event of valve pneumatic leakage (and gas from pneumatic channels impacting on the fluidic channels).
  • Valves may be either closed, or open in the fluidic design to allow for fail safe or fail secure functionality.
  • the valves described herein are open in their inactive state and can be actuated to close, however a valve that is closed in its inactive state is envisaged and is preferred.
  • the valves described herein may be actuated by one of several means, for example, they may be actuated by solenoids.
  • the system may be primed by the operation of the on-board diaphragm pump(s) and squeeze valve(s), or optionally by use of manually applied driving pressure from an external system.
  • the design of the one or more fluid conduits and/or a network of fluid conduits of the microfluidic devices of the systems described herein comprise a large reservoir within which components of the device or system are maintained.
  • the components within the large reservoir comprise one or more cells which may, for example, be maintained in a cell cradle or similar.
  • the large reservoir acts as a passive sink through which media can be changed, media formulations can be altered, or samples can be taken for further testing.
  • the size and shape of devices and systems may be scaled to achieve a flow rate that mimics the environment of the cell or tissue type.
  • Devices and systems may aim to replicate somatic vessels, for example blood vessels, lymphatic vessels, ventricular vessels, urinary vessels and the like, or reproductive vessels for example the fallopian tubes.
  • the flow rate of devices and systems configured to provide an interstitial flow rate deliver fluid at approximately 0.07 - 3.2 pL/sec.
  • the flow rate of devices configured to provide a capillary flow rate deliver fluid at approximately 25 - 75 pL/sec.
  • the flow rate of devices configured to provide a venule or arteriole flow rate deliver fluid at a velocity of at approximately 0.12 - 2.55 microL/sec.
  • the flow rate of devices and systems may be configured to provide fluids having similar physical properties to blood at an interstitial flow rate whereby fluid is delivered at a velocity of 0.0001 - 0.004 mm/sec.
  • fluid is delivered at a velocity of 0.5 - 1.5 mm/sec.
  • venule or arteriole flow rate fluid is delivered at a velocity of 0.5 - 10 mm/sec.
  • the flow of fluids within devices and systems described herein emulate conditions within the fallopian tube. Fluid flow rates are very similar to interstitial flow rates.
  • the preferred flow rate of devices and systems for oocyte or embryo culture occur at a velocity of 0.0001 - 0.004 mm/sec.
  • systems and devices deliver fluids in a pulsatile rhythm to mimic flow with any one of a number of somatic or reproductive conduits.
  • Pumps and valves such as evaporative, diaphragm or peristaltic pumps, may be selected which achieve pulsatile flow.
  • the very small scale of the devices and systems described herein have a lower impact on the environment that the alternatives available.
  • the very small scale of devices means that they may be manufactured and sanitised as single use devices without the need to reuse and sanitise prior to each use.
  • biocompatible materials from which systems and devices are manufactured are readily degraded and have minimal environmental impact upon disposal.
  • the present disclosure provides a single-cell housing which allows for the separation, identification and tracking of single cells throughout culture processes.
  • Single cell 'cradles' consist of 2PP printed structures that may be connected to a microfluidic system.
  • the structures will have geometries that support a range of single cell systems from approximately 10 pm to 500 pm. Structures may be modular, removable and are optionally compatible with multiple systems.
  • any surface of the devices and systems described herein may be surface treated either chemically or physically.
  • embodiments described herein are plasma treated to modify surface wettability to emulate the natural surface of a somatic or reproductive channel. It is envisaged that surface treatment to alter surface hydrophobicity or surface tension will be necessary for certain uses. Typically, the surface modification required will be determined according to the cell type and the conditions present in the natural environment in which it occurs.
  • Any surface of the microfluidic device may be surface treated or modified, in particular, the one or more conduits, reservoirs or cradle surfaces. Surfaces may undergo chemical or physical surface treatment or functionalisation to improve biocompatibility, material stability, cell loading and cell retrieval, as well as to facilitate connection and disconnection from a larger microfluidic system.
  • the one or more reservoirs described herein may comprise at least one single cell housing.
  • the at least one single cell housing may have different geometries for connection and disconnection from a larger microfluidic system, or different geometries to help achieve desired fluid concentrations and gradients.
  • Cradle arrays may also be stacked vertically to create arbitrarily large two- dimensional or three-dimensional arrays of single cell housing devices for creating large micro factories wherein each cradle contains one cell in contact with fluid flow.
  • network of tubing and printed channels feeds into one, or many distinct, large two-dimensional arrays of single cell holding devices or multi cell holding devices.
  • the loading of a large array of single cell holding devices is achieved by loading the array separately and docking it with a larger fluidic system and / or by delivering cells down fluidic channels that bind to specialised sites in the individual compartments of the single cell holding devices.
  • Embodiments may comprise a host system that allows for the live interchange of a large two-dimensional cradle array without interruption to other cradle arrays.
  • Embodiments may comprise 2PP printed channels in communication with a single cell housing comprising either an embedded pump; that can optionally be addressed independently without impacting other 2PP printed channels. Further embodiments may comprise or one or more embedded valves to isolate the channel from changes in the system, or a combination thereof wherein a single embedded pump can feed multiple channels, that are independently isolated with embedded valves. Such embodiments may be formed with or without individual monitoring.
  • the present disclosure describes a feature of the loading step of use in a specific single cell housing case that allows for an oocyte with a cumulus layer to be stripped away, as the aperture at the bottom of a conical funnel shape, is the nominal diameter of the oocytes.
  • the fluid that exits the single cell housing device may be extracted via an automated pumping mechanism or a syringe mechanism to generate a fluid sample.
  • the fluid sample may be delivered to a device that will perform analytical tests on the media, including, but not limited to UV-Vis spectroscopy, FTIR spectroscopy, High Pressure Liquid Chromatography - Mass Spectroscopy (HPLC-MS), Gas Chromatography - Mass Spectroscopy (GC-MS), Non Invasive Preimplantation Genetic Testing (NIPGT).
  • the results of the analysis of the fluid sample may then be used to modify the programmed sequence that delivers the dynamic flow.
  • the flow and flow rate of cell culture fluids may be modulated, mixed or adjusted upon operator request or based upon results of any morphological assessment, media sampling, or any sensors attached to the device.
  • the direction and speed of the flow may be controlled to divert fluid to a premixing chamber to achieve a desired concentration gradient for delivery to one or many single cell holding devices.
  • the tubing or channels may also undergo chemical or physical surface modification to improve flow control.
  • embedded pumps and valves may be actuated to achieve controlled levels of fluid flow to precise locations, either to one channel in isolation or to multiple channels in parallel.
  • 2PP printed structures that are actuated using pneumatic pressure, using diaphragms and squeeze valves to open and close fluidic channels.
  • An embedded pump is a series of valves operating in a repeated programmed sequence to drive fluid flow in a direction using a peristaltic motion.
  • Certain forms comprise a cradle with a bi-funnel shape, allowing for the cell complex to be held at the neck, and microfizidica lly accessed from the dynamic flow system, such that delivery of active biological components may be accomplished.
  • a mechanism for extracting a sub-microlitre volume fluid sample from a location near the single cell housing such as a syringe or thin tube connected to a pump to generate vacuum
  • a mechanism for delivering that fluid sample to an analytical device such as pumping source, or robotics to move the fluid sample inside of a capsule or other vessel
  • a mechanism for analysing the sample and delivering the results to an operator or as the input of an evaluation algorithm.
  • aspects described herein may comprise one or more high pressure microfluidic channels capable of flowing liquified gases, and/or modified microfluidic channels capable of flowing viscous cryoprotectants.
  • cradles to hold oocytes and embryos that are separated from liquified gases via membranes but that are connected to the cryoprotectant microfluidic channels may also be comprised in the low flow microfluidic devices described herein.
  • Cryoprotective fluids are more viscous than water-based media and require greater pressure or larger channels for equivalent flow rates.
  • Cryo-microfluidic (cryoprotectant carrying) channels may be optimised for the delivery of higher viscosity fluids to the culture chambers by increasing their diameter thereby reducing their resistance.
  • Methods for modulating the flow of fluids in the culture of a single cell or small cell mass comprising the steps of; obtaining the microfluidic device of the present disclosure, adding a cell to be cultured to a cell culture chamber, adding a culture medium to a fluid reservoir, actuating the one or more pumps, modulating the flow of fluid through the one or more fluid conduits by opening and closing the one or more valves.
  • the step of modulating the flow of fluid through the one or more fluid conduits by opening and closing the one or more valves in the present disclosure may comprise the modulation of the flow of fluid through the one or more conduits to a flow rate of between approximately 0.0001 and 0.004 mm/sec, between approximately 0.5 and 1.5 mm/sec, or between approximately 0.5 and 10 mm/sec.
  • the step of actuating the one or more pumps according to the methods of the present disclosure may be undertaken in pulses to affect the pulsatile flow of fluid through the one or more fluid conduits.
  • Cryoprotective fluids may require an external pumping source, as well as a dedicated cryoprotectant channel. Segmentation from the main microfluidic system would be achieved using embedded valves in a closed state, near to the single cell housing location.
  • cryoprotective fluids would be pumped in a gradient, rather than a sequential series of baths of decreasing hydration.
  • Figure 15 provides side perspective view of a housing structure of a low flow microfluidic device according to an embodiment of the present disclosure.
  • Figure 16 provides a side perspective sectional view of a housing structure through section H-H of Figure 11 of a low flow microfluidic device according to an embodiment of the present disclosure.
  • Microfluidic device 100 generally comprises a microfluidic support 107 having four fluid reservoirs 104 formed thereon.
  • each medium comprises stage of development-specific formulations, for example, for the maturation of oocytes, for the fertilisation of mature oocytes, for supporting pre compaction growth and for supporting post compaction development. Greater or fewer fluid reservoirs may be provided depending on the needs of the cell culture environment.
  • Microfluidic device 100 further comprises one or more of fluid conduits which provide for the communication of fluids to and from fluid reservoirs 107, to and from cell culture chamber 103, and they provide for the communication of fluids between any one of fluid reservoirs 107 and cell culture chamber 103.
  • the various fluid conduits of microfluidic device 100 comprise a combination of one or more tubes and one or more channels either formed within microfluidic support 107 or on the surface of microfluidic support 107.
  • Cell culture media 101 is, in turn, transferred to cell culture chamber 103 via fluid channel 106.
  • the transfer of cell culture media 101 is directed from reservoir 104 to cell culture chamber 103 by one or more valves directing and/or modulating the flow of fluid along fluid channel 106.
  • the coordination and control of valves and pumps 102 and further independently actuated valves 401 placed along various fluid conduits, including fluid channel 106, supply cell culture media 101 to a cradle array 108 containing the desired cell type for culturing.
  • Individual cradles 604 (shown in Figure 6) within the cradle array 108 are fluidically connected to cell culture chamber 103 via a connector such as a barb connector (not shown).
  • International Patent Application Number PCT/AU2020/051318 describes the form of cradles 604 and cradle arrays 108 in detail. While the low flow microfluidic device 100 may be suitable for culturing any cell type, the low flow microfluidic device described herein and depicted at 100 is adapted for culturing single cells and small cell clusters, for example embryos.
  • FIG. 6 provides a side sectional view of the microfluidic device 100 through section D-D of microfluidic support 107 shown in Figure 1.
  • Bifurcated supply line 503 provides two channels through which to direct the flow of fluids; one comprising waste valve 601 to direct fluid to waste reservoir (not shown) and the other comprising supply valve 502 to direct and/or modulate the flow of cell culture media 101 to cradle 604.
  • Waste media emanating from cradle 604 is transferred from cradle 604 through waste line 501 to waste reservoir (not shown) and fresh supply media (cell culture media 101) is directed to cradle 604 for further culture of cells therein.
  • Figure 7 shows independently actuated valve 401 comprising inlet port 701 provided to enable the transfer of incoming gases which pressurise valve chamber contained therein, thereby restricting the flow of fluid through neighbouring outlet channel 702.
  • Figures 8, 9 and 10 provide side sectional views of microfluidic device 100 across sections E-E, F-F and G-G of independently actuated valve 401 shown in Figure 7.
  • Figures 8- 10 show embedded channel 801 which is pressurised with gas and thereby deforms the surrounded channel 802 to restrict the flow of fluid passing therethrough.
  • FIG 11 shows the general form of squeeze valve 1101, produced and actuated with a pneumatic pump (not shown) formed from 2PP printed UpFlow; an acrylate-based resin available commercial from UpNano, Vienna, Austria.
  • Squeeze valve 1101 consists of a thinned, rectangular central fluid chamber 1102 flanked by two rectangular pressure chambers 1103, providing two thin membranes on either side of fluid chamber 1102. The membranes provide fluid deflection when pressure is applied.
  • Figure 12 shows the location of each pressure chamber 1103 on microfluidic support 107 which is serviced by air line inlet 1201 and air channel 1202 for flushing, connected to the pressure chambers 1103 at the top and bottom of the pressure chambers 1103 respectively.
  • Attachment nozzle 1203 at one end of air line inlet 1201 allows for the connection of tygon tubing (not shown) to permit the flushing the air channel 1202 and fluid channel 1207, and connection of air channel 1202 to pneumatic pump (not shown).
  • Air outlet 1204 may be released to enable the flushing of air channel 1202 and fluid channel 1207 but must be remain closed to pressurize air channel 1202.
  • a connector on fluid outlet 1205 allows tubing to be held in place for flushing, and for liquid to be added into media reservoir 1206.
  • a second tubing connection 1301 may optionally be fitted to permit a manual valve 1302 to be added.
  • Manual valve 1302 may be released and closed to pressurize air channel 1202 and/or fluid channel 1207.
  • Figure 14 provides a side sectional view of diaphragm valve 1301 showing its general form.
  • Diaphragm valve 1301 provides an alternative to squeeze valve 1101 to provide a larger diameter membrane for culture conditions that may require a lower flow rate.
  • Diaphragm valve 1301 is implemented by replacement of squeeze valve 1101 in the microfluidic device described in Figures 11-13, with diaphragm valve 1301. Membrane 1302 is maintained in positioned between control chamber 1303 and fluid chamber 1304 as illustrated in Figure 14a.
  • the construction of diaphragm valve 1301 is based on the work of prior authors; namely, Gong et al. Lab Chip, 2016, 16, 2450-2458.
  • tygon tubing (not shown) is connected to the air lines inlet 1201. Tygon tubing is connected at one end to a mitos pump and at the other end it is closed with a manual diaphragm valve for modulation of flow rate. Upon actuation of diaphragm valve 1301, air flow and fluid flow are driven in parallel as shown in Figure 14b.
  • Such embodiments may administer an ultra-low flow rate perfusion, which enables the automation of media delivery and achieves dynamic culturing without excessive disturbance to the embryo environment.
  • Culture conditions may be adapted or modified by persons skilled in the art to optimise fluid flow rate according to the nutritional needs of the embryo.
  • Optimal media flow rates for the culture of embryos, or other cell types will typically mimic the flow rates of the environment in which these cells naturally occur. Therefore, depending on the cell type under culture, fluid flow rates may be modified to mimic interstitial flow rates, capillary flow rates, venule flow rates and arteriole flow rates.
  • the methods described herein for use of the present embodiments may be adapted for housing oocytes, and then subsequently embryos, by utilising low flow microfluidic devices to mimic the fluidic conditions in vivo.
  • the methods described herein allow for the dynamic culturing of these cells without disturbance to the local environment, while meeting their metabolic needs throughout the duration of cell culture.
  • low flow microfluidic devices may be adapted to dynamically exchange media and cryoprotectants gradually; in a controlled manner.
  • Integrated valves and pumps 102 may be modified to facilitate flow, in instances wherein a housing structure 1501 is being utilised to separate and identify embryos.
  • Methods involving cryo-microfluidics may be adopted to allow for the low flow of cryopreservation fluids, optionally, together with use of single cell cradle arrays that ease the recovery of cells cultured therein.
  • Additional devices may be adopted that remove the dependency on operator skill for the handling of liquid nitrogen, by automating the selection of media concentrations and/or the flow rate of cryoprotectants and, thereafter, the application of liquid nitrogen. Methods that utilise such devices together with low flow microfluidic devices may result in the minimisation of oocyte and embryo handling and consequently a reduction of stress on the cultured cells.
  • Methods may comprise additional devices that assist in reducing manual handling during the procedures described above and automating the process of extracting homologous (mother's) ovarian-stem cell sourced mitochondria.
  • the process of injecting mitochondria from a homologous source (preferably ovarian) in conjunction with or without other "rejuvenating compounds” may additionally be automated using the devices described above together with mitochondrial "sieves".
  • Low flow microfluidic devices may be utilised in conjunction with the methods described herein to pass COC cells through a sorting system for visualisation, collection, handling with cell cradles to denude COC cells, to gently transfer cells or embryos, individually or with multiple other co-cultured cells.
  • visualisation technologies such as Optical Coherence Tomography may be adopted and, optionally, incorporated into oocyte retrieval equipment in conjunction with low flow culture devices to support the timely and visible collection of oocytes.
  • Systems and/or devices described herein may comprise a precision incubator sufficiently smaller in size to suit the cells being incubated rather than the operator; providing a higher level of precision of temperature, humidity, and gas environment.
  • Methods for the use of low flow microfluidic devices described herein enable the delivery of controlled amounts of gas through integrated valves and pumps 102 to deliver a controlled flow at a microscale.
  • the devices and methods described herein may be adapted by persons skilled in the art for ART applications and may be used to make all forms of cell therapy and culturing more precise, efficient and repeatable. Such devices and methods may be adapted for cell models such as stem-cells, organoids, viruses and bacteria to automate production and improve quantity and quality of cellular output.
  • Methods for non-ART cell therapies may be adapted to incorporate the use of low flow microfluidic devices to provide media to cells under therapy or culture.
  • Each cradle may contain a cell, organoid or group of cells (e.g. for non-human non-ART).
  • Integrated valves and pumps 102, described herein may then deliver flow to an individual cradle at a desired flow rate such that the flow rate to each cradle within an array may be individually controlled.
  • Low flow microfluidic devices may be integrated into systems for the full automation of in vitro fertilisation procedures, to reduce the financial constraints and dependency on operator skill for successful procedures.
  • An end-to-end system may integrate devices described herein to automate the process of growing healthy and viable embryos.
  • devices and systems adapted for automated in vitro fertilisation may comprise an automated injector, fixed to image-controlled single micromanipulator, and may be used to align the Z-plane, identify the sperm, complete sperm pickup, identify the oocyte in the microwell, align the injector, and fertilise the oocyte with sperm.
  • first, second, third, etcetera may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, a first element, component, region, layer or section could be termed a second element, component, region, layer or section without departing from the teachings of the present disclosure.
  • Embodiments of the description are described herein with reference to diagrams and/or cross-section illustrations, for example, that are schematic illustrations of preferred embodiments (and intermediate structures) of the description. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. Thus, embodiments of the description should not be construed as limited to the particular shapes of components illustrated herein but are to include deviations in shapes that result, for example, from manufacturing.
  • any reference in this specification to "one embodiment,” “an embodiment,” “example embodiment,” etc. means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the description.
  • the appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment.
  • Embodiments are also intended to include or otherwise cover methods of using and methods of manufacturing any or all of the elements disclosed above.

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Abstract

La présente divulgation concerne de manière générale des dispositifs microfluidiques à faible débit, des systèmes et des procédés pour moduler l'écoulement de fluides dans une culture cellulaire. Les systèmes comprennent des dispositifs microfluidiques et des procédés comprenant l'utilisation de dispositifs microfluidiques qui, à leur tour, comprennent un support microfluidique sur ou à l'intérieur de celui-ci; un ou plusieurs réservoirs de fluide conçus pour contenir un milieu de culture, une ou plusieurs canalisations de fluides conçues pour diriger l'écoulement de fluide à travers celles-ci, du ou des réservoirs de fluide vers une ou plusieurs chambres de culture cellulaire, et la ou les canalisations de fluide comprenant une ou plusieurs vannes à l'intérieur de celles-ci pour moduler l'écoulement de fluide à travers les canalisations pour moduler l'écoulement de fluides à travers le système de culture cellulaire; en particulier pour fournir l'écoulement dynamique de milieux à des débits faibles ou très faibles.
PCT/AU2025/050086 2024-02-06 2025-02-06 Dispositif microfluidique à faible débit Pending WO2025166414A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010023497A1 (fr) * 2008-08-29 2010-03-04 Peking University Puce microfluidique pour culture de cellules contrôlable avec précision
US20120034695A1 (en) * 2010-06-30 2012-02-09 Palaniappan Sethu Tissue/cell culturing system and related methods
US20190093059A1 (en) * 2016-03-08 2019-03-28 National Institute Of Advanced Industrial Science And Technology Cell culture device and cell culture method
US20200216790A1 (en) * 2017-09-19 2020-07-09 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. Microfluidic device for cell culture experiments and uses thereof
US20200230597A1 (en) * 2019-01-18 2020-07-23 National Tsing Hua University Automatic Microfluidic System for Rapid Personalized Drug Screening and Testing Method for Personalized Antibiotic Susceptibility
WO2021108860A1 (fr) * 2019-12-03 2021-06-10 The University Of Adelaide Micro-dispositif de culture cellulaire
US20240002763A1 (en) * 2020-10-06 2024-01-04 Micronit Holding B.V. Microfluidic cell culture device

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010023497A1 (fr) * 2008-08-29 2010-03-04 Peking University Puce microfluidique pour culture de cellules contrôlable avec précision
US20120034695A1 (en) * 2010-06-30 2012-02-09 Palaniappan Sethu Tissue/cell culturing system and related methods
US20190093059A1 (en) * 2016-03-08 2019-03-28 National Institute Of Advanced Industrial Science And Technology Cell culture device and cell culture method
US20200216790A1 (en) * 2017-09-19 2020-07-09 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. Microfluidic device for cell culture experiments and uses thereof
US20200230597A1 (en) * 2019-01-18 2020-07-23 National Tsing Hua University Automatic Microfluidic System for Rapid Personalized Drug Screening and Testing Method for Personalized Antibiotic Susceptibility
WO2021108860A1 (fr) * 2019-12-03 2021-06-10 The University Of Adelaide Micro-dispositif de culture cellulaire
US20240002763A1 (en) * 2020-10-06 2024-01-04 Micronit Holding B.V. Microfluidic cell culture device

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