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WO2025085891A1 - Dispositif de bioréacteur pour fabriquer de multiples feuilles de cellules dans un réseau vertical et procédés associés - Google Patents

Dispositif de bioréacteur pour fabriquer de multiples feuilles de cellules dans un réseau vertical et procédés associés Download PDF

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Publication number
WO2025085891A1
WO2025085891A1 PCT/US2024/052203 US2024052203W WO2025085891A1 WO 2025085891 A1 WO2025085891 A1 WO 2025085891A1 US 2024052203 W US2024052203 W US 2024052203W WO 2025085891 A1 WO2025085891 A1 WO 2025085891A1
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WO
WIPO (PCT)
Prior art keywords
bioreactor
culture medium
coupled
racks
cavity
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/US2024/052203
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English (en)
Inventor
Feng Zhao
Eric Wang
Brandon Bohan ZHAO
Roland Kaunas
Dhavan Divyanshu SHARMA
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.)
Texas A&M University System
Texas A&M University
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Texas A&M University System
Texas A&M University
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Publication of WO2025085891A1 publication Critical patent/WO2025085891A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F27/00—Mixers with rotary stirring devices in fixed receptacles; Kneaders
    • B01F27/05—Stirrers
    • B01F27/11—Stirrers characterised by the configuration of the stirrers
    • B01F27/19—Stirrers with two or more mixing elements mounted in sequence on the same axis
    • B01F27/192—Stirrers with two or more mixing elements mounted in sequence on the same axis with dissimilar elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F27/00—Mixers with rotary stirring devices in fixed receptacles; Kneaders
    • B01F27/80—Mixers with rotary stirring devices in fixed receptacles; Kneaders with stirrers rotating about a substantially vertical axis
    • B01F27/90—Mixers with rotary stirring devices in fixed receptacles; Kneaders with stirrers rotating about a substantially vertical axis with paddles or arms 
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F27/00—Mixers with rotary stirring devices in fixed receptacles; Kneaders
    • B01F27/80—Mixers with rotary stirring devices in fixed receptacles; Kneaders with stirrers rotating about a substantially vertical axis
    • B01F27/91—Mixers with rotary stirring devices in fixed receptacles; Kneaders with stirrers rotating about a substantially vertical axis with propellers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F35/20—Measuring; Control or regulation
    • B01F35/21—Measuring
    • B01F35/213—Measuring of the properties of the mixtures, e.g. temperature, density or colour
    • 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
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • 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
    • C12M23/48—Holding appliances; Racks; Supports
    • 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
    • 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
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

Definitions

  • BIOREACTOR DEVICE FOR FABRICATING MULTIPLE CELL-SHEETS IN A VERTICAL ARRAY AND RELATED METHODS Related Application [0001] This application is based upon prior filed copending Application No.63/591,547 filed October 19, 2023, the entire subject matter of which is incorporated herein by reference in its entirety.
  • the present disclosure relates to the field of bioreactors, and, more particularly, to a bioreactor for cell sheet and cell-derived extracellular matrix (ECM) generation and related methods.
  • ECM extracellular matrix
  • a bioreactor device may include a housing assembly comprising a body defining a bioreactor cavity therein with an opening, a lid to be received by the opening and comprising a plurality of rack openings, and at least one port coupled to the bioreactor cavity.
  • the bioreactor device may also include a plurality of racks respectively received by the plurality of rack openings.
  • Each of the plurality of racks may include a vertical arm, a plurality of vertically spaced supports coupled to the vertical arm, and a plurality of substrates respectively carried by the plurality of vertically spaced supports, each substrate having culture media thereon.
  • the bioreactor device may also include a control unit coupled to the at least one port and configured to selectively supply culture medium to the bioreactor cavity based upon at least one characteristic within the bioreactor cavity. 6230TEES23 (0137616_PCT) [0005]
  • the control unit may comprise a controller, and a pressure source coupled to the controller and the at least one port.
  • the bioreactor device may also include a supply of culture medium coupled to the at least one port via the pressure source.
  • the controller may be configured to cause the pressure source to move the culture medium from the supply of culture medium to the bioreactor cavity based upon the at least one characteristic within the bioreactor cavity.
  • the at least one characteristic may include a plurality thereof, the plurality of characteristics comprising a pH level, a dissolved oxygen (DO) level, a glucose level, and a lactate level, for example.
  • the bioreactor device may also include a stirrer carried by the housing assembly and extending within the bioreactor cavity.
  • the plurality of racks may be arranged in pairs, and the stirrer may be between each pair.
  • the stirrer may be configured to drive the culture medium over each substrate in the plurality of racks.
  • the bioreactor device may also include a heater carried by the housing assembly and coupled to the control unit.
  • the control unit may be configured to cause the heater to maintain the culture medium at a set temperature.
  • Each substrate may comprise a flat or patterned surface, where cells will grow and receive the culture media, and each rack may define a recess to receive a respective substrate.
  • the body may be cylinder- shaped.
  • the culture media may comprise components to support the growth of different cell types or stimulate extracellular matrix (ECM) production.
  • Each substrate may comprise a polymer material, for instance.
  • the bioreactor device may comprise a housing assembly comprising a body defining a bioreactor cavity therein with an opening, a lid to be received by the opening and comprising a plurality of rack openings, and at least one port coupled to the bioreactor cavity, and a plurality of racks respectively received by the plurality of rack openings.
  • Each of the plurality of racks may comprise a vertical arm, a plurality of vertically spaced supports coupled to the vertical arm, and a plurality of substrates respectively carried by the plurality of vertically spaced supports.
  • Each substrate may have culture media thereon.
  • the method may include operating a control unit coupled to the at least one port to selectively supply culture medium to the bioreactor cavity based upon at least one characteristic within the bioreactor cavity..
  • the method 6230TEES23 may also include coupling a stirrer to be carried by the housing assembly and extending within the bioreactor cavity.
  • the control unit is configured to cause the stirrer to adjust a flowrate through each substrate.
  • Yet another aspect is directed to a method for making a bioreactor device.
  • the method may also include forming a housing assembly comprising a body defining a bioreactor cavity therein with an opening, a lid to be received by the opening and comprising a plurality of rack openings, and at least one port coupled to the bioreactor cavity.
  • the method may also include positioning a plurality of racks respectively in the plurality of rack openings.
  • FIG.1 is a schematic diagram of a bioreactor device, according to a first example embodiment of the present disclosure.
  • FIG.2 is a partially exploded view of the bioreactor device of FIG.1.
  • FIG.3A is a perspective view of a rack from the bioreactor device of FIG.1.
  • FIG.3B is a perspective view of a stirrer from the bioreactor device of FIG.1.
  • FIG.3C is a schematic top plan view of the stirrer and racks from the bioreactor device of FIG.1.
  • FIG.3D is a diagram of fluid flow from the bioreactor device of FIG.1.
  • FIG.4 is a perspective view of a bioreactor device, according to a second example embodiment of the present disclosure.
  • FIG.5 is a partially exploded view of the bioreactor device of FIG.4.
  • FIG.6A is a perspective view of a bioreactor device, according to a third example embodiment of the present disclosure.
  • FIG.6B is another perspective view of a bioreactor device of FIG.6A. 6230TEES23 (0137616_PCT)
  • FIGS.7A & 7B are top plan views of the supports from the bioreactor device of FIG.6A.
  • FIG.7C is a perspective view of the removable lid from the bioreactor device of FIG.6A.
  • FIG.7D is a perspective view of the first base and the lid from the bioreactor device of FIG.6A.
  • FIG.7E is a top plan view of the bioreactor device of FIG.6A.
  • FIG.7F is a top plan view of the bioreactor device of FIG.6A with the first base removed.
  • FIG.8 is a perspective view of the rack and substrates from the bioreactor device of FIG.6A.
  • FIGS.9 is a perspective view of the tray from the rack from the bioreactor device of FIG.6A.
  • FIGS.10A & 10B are perspective views of the supports from the bioreactor device, according to a fourth example embodiment of the present disclosure.
  • FIG.11A is an image of a bioreactor device, according to a fifth example embodiment of the present disclosure.
  • FIG.11B is an image of a rack and tray system within the bioreactor device of FIG.11A.
  • FIGS.12A & 12B are images of a bioreactor device, according to a sixth example embodiment of the present disclosure.
  • FIG.12C is an image of a bioreactor device, according to a seventh example embodiment of the present disclosure.
  • FIG.13A is a perspective view of a rack from a bioreactor device, according to an eighth example embodiment of the present disclosure.
  • FIG.13B is a schematic side view of the bioreactor device of FIG.13A.
  • a bioreactor device 100, 200, 300 may facilitate the expansion of cells and fabrication of a large number of cell sheets or cell-derived extracellular matrices in a vertical array system with improved reproducibility, minimal batch-to-batch variation, and reduced risk of contamination in comparison with static cultures.
  • the bioreactor device 100, 200, 300 is capable of gentle yet thorough mixing of gases and culture media, and monitoring of gas flow and its concentration.
  • Cell cultures require a precise amount of DO to be released into the bioreactor cavity 103 to facilitate optimum cell growth.
  • the bioreactor device 100, 200, 300 can control nitrogen gas and carbon dioxide while maintaining optimum pH. Adequate mixing of the nutrient is essential to maintain homogenous culture media inside the bioreactor cavity 103; however, harsh mixing can damage cell sheets.
  • the mixing apparatus (stirrer 114) allows for low power output and steady mixing control.
  • the bioreactor parts are autoclavable or sterilized using other methods, such as ethylene oxide or radiation, thereby ensuring absolute sterility.
  • the bioreactor device 100, 200, 300 incorporates essential sensors to analyze media pH and dissolved oxygen concentration, allowing the user to continuously monitor cell growth and health. These parameters can be used to calculate additional factors that can be used to automatically correct the bioreactor system to changing inner conditions.
  • the bioreactor device 100, 200, 300 has been designed to facilitate fabrication of large numbers of cell sheets or extracellular matrices (e.g., 13-52 sheets or more, each with an area of 25-40 cm 2 ) stacked in a vertical array.
  • a bioreactor device 300 may improve reproducibility with minimal batch-to-batch variation, and minimum risk of contamination.
  • the bioreactor device 100, 200, 300 has been 6230TEES23 (0137616_PCT) designed that is capable of gentle yet thorough mixing of gases and culture media, and monitoring of gas flow and its concentration during the cell sheet production.
  • a bioreactor device 300 comprises first and second supports (e.g., polytetrafluoroethylene (PTFE)), a tubular housing between the first and second supports and having bioreactor fluid (e.g., sterile solution) therein, and a plurality of racks within the tubular housing, each rack carrying a substrate with culture media thereon.
  • PTFE polytetrafluoroethylene
  • each of the first and second supports may define at least one port.
  • Each rack may define a recess to receive a respective substrate.
  • the substrate may carry the culture media, for example, engineered stem cells, a skin graft, or an ECM.
  • the bioreactor device may also include a mixing device coupled to at least one of the first and second supports.
  • the top-down view in FIGS.7A-7B show the 3 ports 308a-308c on the cap of the bioreactor device 300. Each port can be utilized to monitor oxygen and gas exchange ports.
  • FIGS.6A-6B show the entire bioreactor device 300 assembled without culture media filled inside.
  • the bioreactor tube is also made from autoclavable polycarbonate material in addition to single-use filters for sterilizing input and output gases.
  • FIGS.7A-7B show the base of the bioreactor device 300 made from polytetrafluoroethylene (PTFE). The four threaded holes on the corners allow for securing the cap and base together to create an air-tight seal. The circular groove in the middle leaves space for the O-ring to sit and slot the polycarbonate tube into it.
  • PTFE polytetrafluoroethylene
  • FIG.7B shows the cap of the bioreactor, allowing 3 ports for gas/liquid exchange and 4 corner holes for the rod to pass through. A nut will be used to secure and clamp the base and lid together.
  • the cap of the bioreactor also includes a square cutout for the lid to be inserted into. The circular groove in the middle of the cap also allows space for the O-ring to sit into and slot the polycarbonate tube together to create an air-tight seal.
  • FIGS.7C-7D show the lid 305 of the bioreactor device 300 made from autoclavable polycarbonate material.
  • FIGS.7E-7F show the top plan view of the bioreactor device 300.
  • the rack slots into the two holes at the bottom of the base, securing the rack and substrates inside the bioreactor.
  • FIG.7E shows the cap of the bioreactor, allowing 3 ports for gas/liquid exchange and 4 corner holes for the rod to pass through.
  • FIGS.8-9 showcase the geometry of the racks 307 that allow for clipping the plates into the rod system.
  • This rack provides a space above the bottom of the bioreactor device 300 for the stir bar to sit on.
  • Each rack can hold 8 trays of the substrate.
  • These racks have an additional bar at the top to provide ease of carrying and removal.
  • the PDMS is modeled as 60 mm x 60 mm and will allow cells to adhere and 6230TEES23 (0137616_PCT) grow on it.
  • FIG.9 highlights a single tray (support 311) that composes the rack as a single part. Each tray has a bezel for the PDMS substrate to rest securely on the plate. It also shows the adjustable horizontal bar on each tray, which holds the PDMS substrate in place gently, preventing floating or movement.
  • FIGS.10A-10B show a potential container cap with the mixing apparatus (stirrer 414) attached. The prior embodiments utilize a supplemental stir bar placed at the bottom of the system. For larger bioreactor systems, a built-in stirring mechanism as shown in the figure is critical.
  • the mixing apparatus shown herein allows for low power output and steady mixing control. Again, the four ports on the top are available for gas control and sensors.
  • the initial prototype is shown in the images 1000, 1005 of FIGS.11A-11B with the bioreactor system consisting of the top and bottom supports clamping the polycarbonate tube in the middle. Nuts and bolts are used to secure the system and create a seal along with the O-rings. The rack system is secured into the holes at the bottom of the support.
  • FIG.11B shows the rack system with one tray clipped on and remaining spots for more trays.
  • the concept prototype is shown in the images 1010, 1015 of FIGS.12A-12B with a spinner flask apparatus attached.
  • the stoppers with glass tubes demonstrate the ports that will allow for media and gas exchange within the sterile bioreactor.
  • the spinner apparatus will ensure adequate mixing of homogenous culture media inside the bioreactor.
  • the model rack above holds two PDMS substrates and can be scaled to fit more.
  • the engineered cell sheet may be attractive among different engineered tissue forms. Although cell sheet engineering has brought a new avenue to fabricate 3D completely biological tissues, the cultures to generate cell sheets normally takes a few weeks, necessitating frequent manual media changes.
  • a closed, semi-automated bioreactor device 100, 200, 6230TEES23 (0137616_PCT) 300 that maintains a 3D culture environment that can house several tens of cell sheets.
  • Such a system may significantly increase culture efficiency, decrease contamination risk, and reduce batch-to-batch variation.
  • the decellularization process can be performed in the same bioreactor system upon the completion of the cell sheet culture. This facilitates the generation of cell-derived ECM scaffolds with desired structures for various tissue engineering applications.
  • the bioreactor devices 100, 200, 300 disclosed herein can accommodate several tens of substrates for cell sheet culture and subsequent decellularization (FIGS.6A-6B).
  • the chamber will be custom-made from autoclavable polycarbonate and will have clear walls for easy visual inspection.
  • the chamber will have shelf inserts secured to the chamber wall that can stack individual PDMS substrates, with the desired dimensions and surface nano or micro patterns.
  • medium inlet and outlet ports will be created at the top of the chamber.
  • a rotating shaft with rotor blades will be installed in the center of the chamber to mix the medium at a controlled speed during the culture process.
  • a custom-made header plate will allow media and mixed gases to flow in and out of the vessel, and probes will be used to monitor pH and DO levels. Mixed gases will be automatically placed into the culture medium to maintain optimal conditions for cell growth.
  • cell sheets grown from human cells or animal cells and ECM scaffolds derived from decellularizing tissues mimic the complex composition and architecture of native tissues. They have been widely used in regenerative medicine to regenerate or repair human/animal tissues while addressing issues such as donor scarcity, mismatched mechanical properties, pathogen transfer, and undesired host immune responses.
  • Patient-specific ECM scaffolds can be created by decellularizing cell sheets cultured from their own cells in pathogen-free conditions, thereby avoiding these problems.
  • Cultured cell sheets can be engineered in several ways to enhance their therapeutic efficacy. For example, the alignment of cell sheet ECM fibers can be controlled via nanogrooves in the growth surface to guide and increase blood vessel growth after implantation.
  • the major feature of the bioreactor device 100, 200, 300 is that it can accommodate several tens of substrates for cell sheet culture and subsequent decellularization. It is a closed, semi-automated bioreactor system that maintains a 3D culture environment that can house several tens of cell sheets. Such a system will significantly increase culture efficiency, decrease contamination risk, and reduce batch- to-batch variation. Moreover, the decellularization process can be performed in the same bioreactor system upon the completion of the cell sheet culture. This facilitates the generation of cell-derived ECM scaffolds with desired structures for various tissue engineering applications.
  • the bioreactor device 100 illustratively includes a housing assembly 101 comprising a body 102 defining a bioreactor cavity 103 therein with an opening 104.
  • the body 102 is illustratively cylinder-shaped, but other shapes are possible in other embodiments, such as a rectangle-shaped box.
  • the housing assembly 101 also includes a lid 105 to be received by the opening 104 and comprising a plurality of rack openings 106a-106d, and a plurality of ports 108a-108d (i.e., both gas ports 108c-108d and media exchange/fluid ports 108a-108b) carried by the lid 105 and fluidly coupled to the bioreactor cavity 103.
  • the plurality of ports 108a- 108d may be carried by the body 102.
  • the body 102 may comprise one or more rigid autoclavable materials, for example, glass, polycarbonate, aluminum, and steel.
  • the body 102 illustratively comprises first and second windows 109a-109b for providing viewing ports for culture observation.
  • the body 102 illustratively includes an outer body/jacket 118a, and an inner vessel 118b received by the outer body/jacket.
  • the lid 105 is coupled to the inner vessel 118b via a plurality of fasteners, and optionally an O-ring therebetween.
  • the body 102 may alternatively comprise one or more bioprocess bags.
  • flexible sterile sleeves respectively cover the respective racks 107a-107d for transport between a sterile environment used for cell seeding and the bioreactor device 100.
  • the body 102 may comprise disposable material components, permitting easy turnover to additional cycles.
  • the bioreactor device 100 illustratively includes a plurality of racks 107a-107d respectively received by the plurality of rack openings 106a-106d.
  • Each of the plurality of racks 107a-107d illustratively includes a handle on an uppermost portion thereof for providing easy grip for a user to remove racks when necessary.
  • Each of the plurality of racks 107a-107d illustratively comprises a vertical arm 110, a plurality of vertically spaced supports 111a-111m coupled to the vertical arm, and a plurality of substrates 112a-112m respectively carried by the plurality of vertically spaced supports.
  • each vertically spaced support 111a-111m defines a recess to receive a respective substrate 112a-112m.
  • Each substrate 112a-112m has culture media thereon.
  • the culture media may comprise components to stimulate ECM culture media, but in other embodiments, the culture media may comprise artificial stem cells or a skin graph, for example.
  • each substrate 112a-112m comprises a flat surface.
  • each substrate 112a-112m comprises a patterned surface to receive the culture media (i.e., the culture grows on the flat or patterned surface).
  • each substrate 112a-112m may be perfused by culture media or immersed in culture media.
  • the patterned surface may comprise either a micropatterned surface or a nanopatterned surface.
  • the culture media may comprise components to support the growth of different cell types or stimulate ECM production.
  • Each substrate 112a-112m may comprise a polymer or other material, for example, PDMS.
  • the bioreactor device 100 illustratively comprises a heater 113 carried by the outer body/jacket 118a of the housing assembly 101.
  • the heater 113 may comprise a resistive heater.
  • the bioreactor device 100 further comprises a stirrer 114 (i.e., a mixer or impeller device) carried by the lid 105 of the housing assembly 101 and extending within the bioreactor cavity 103.
  • the uppermost portion of the bioreactor cavity 103 may define a “headspace”.
  • the headspace may be defined as the volume defined between the lid 105 and an uppermost vertically spaced support 111a.
  • each of the plurality of racks 107a-107d has a sloped top for increasing gas exchange between the headspace and the culture medium via a falling film flow.
  • the bioreactor device 100 illustratively comprises a gas supply and associated mass flow controller 6230TEES23 (0137616_PCT) 119 (one shown, but other embodiments may include a plurality thereof) coupled to the gas ports 108e-108f.
  • the bioreactor device 100 illustratively includes a control unit 115 configured to control conditions within the bioreactor cavity 103 to encourage growth of the culture media.
  • the control unit 115 illustratively includes a controller 116, and a pressure source 117 (i.e., one or both of a positive pressure source and a negative pressure source) coupled to the controller and fluidly coupled to the plurality of ports 108a-108d.
  • the pressure source 117 comprises an electrical pump.
  • the controller 116 is configured to cooperate with the pressure source 117 to selectively supply culture medium to the bioreactor cavity 103 based upon a plurality of characteristics within the bioreactor cavity.
  • the plurality of characteristics illustratively includes a pH level, a DO level, a glucose level, and a lactate level.
  • the controller 116 is configured to cooperate with the heater 113 to maintain the culture medium at a set temperature.
  • the set temperature may comprise 98.6oF/ 37oC, for example.
  • the bioreactor device 100 illustratively comprises a supply of culture medium 120, a spent culture medium reservoir 121, and a supply of cell culture supplement (e.g., human platelet lysate (HPL), or fetal bovine serum (FBS)) 122, each being fluidly coupled to one or more of the plurality of ports 108a-108d via the pressure source 117, one or more tubes, and one or more tube connectors.
  • cell culture supplement e.g., human platelet lysate (HPL), or fetal bovine serum (FBS)
  • the controller 116 is configured to cause the pressure source 117 to: move the culture medium from the supply of culture medium 120 to the bioreactor cavity 103 based upon the plurality of characteristics within the bioreactor cavity; move the cell culture supplement fluid from the supply of cell culture supplement 122 to the bioreactor cavity 103 based upon the plurality of characteristics within the bioreactor cavity; and move the culture medium from the bioreactor cavity 103 to the spent culture medium reservoir 121 based upon the plurality of characteristics within the bioreactor cavity.
  • this may automate maintenance and monitoring of the culture growth in the bioreactor device 100.
  • the controller 116 is configured to control the culture growth process. 6230TEES23 (0137616_PCT) [0058] Further, the controller 116 is configured to cooperate with the stirrer 114 to circulate fluids within the bioreactor cavity 103.
  • the stirrer 114 illustratively includes an electric motor 123 coupled to the controller 116, a rod 124 coupled to the electrical motor, a first set of fins 125a-125f coupled to the rod, and a second set of fins 126a-126b coupled to the rod.
  • the electric motor 123 is configured to rotate the rod 124.
  • the plurality of racks 107a-107d is arranged in opposing pairs, and the stirrer 114 is between each opposing pair.
  • the first and second pairs of racks (107a/107c, 107b/107d) are radially spaced by substantially 90o (i.e., ⁇ 5o of 90o), and the stirrer 114 is between respective racks 107a-107d for each pair.
  • the stirrer 114 is configured to drive the culture medium over each substrate 112a-112m in the plurality of racks 107a-107d.
  • the controller 116 is configured to cooperate with the gas supply and associated mass flow controller 119 to dry the plurality of substrates 112a-112m by directing gas into upper side ports 108e-108f in the body 103.
  • Another aspect is directed to a method of operating a bioreactor device 100 comprising a housing assembly 101 comprising a body 102 defining a bioreactor cavity 103 therein with an opening 104, a lid 105 to be received by the opening and comprising a plurality of rack openings 106a-106d, and a plurality of ports 108a-108d coupled to the bioreactor cavity, and a plurality of racks 107a-107d respectively received by the plurality of rack openings.
  • Each of the plurality of racks 107a-107d comprises a vertical arm 110, a plurality of vertically spaced supports 111a-111m coupled to the vertical arm, and a plurality of substrates 112a-112m respectively carried by the plurality of vertically spaced supports.
  • Each substrate 112a-112m has culture media thereon.
  • the method includes operating a control unit 115 coupled to the plurality of ports 108a-108d to selectively supply culture medium to the bioreactor cavity 103 based upon a plurality of characteristics within the bioreactor cavity. [0060] In some embodiments, the method may comprise operating the control unit to cause the stirrer 114 to regulate the flow of different gases from individual supplies that are mixed and flowed through the headspace above the media.
  • mass 6230TEES23 (0137616_PCT) flow controllers to measure and regulate the flow rates of the gases.
  • the operation of the mass flow controllers may be based on feedback information from pH and DO sensors located within the culture media. Further, additional sensors may be located within the headspace to measure the composition of the gases within the headspace.
  • Yet another aspect is directed to a method for making a bioreactor device 100.
  • the method also includes forming a housing assembly 101 comprising a body 102 defining a bioreactor cavity 103 therein with an opening 104, a lid 105 to be received by the opening and comprising a plurality of rack openings 106a-106d, and a plurality of ports 108a-108d coupled to the bioreactor cavity, and positioning a plurality of racks 107a-107d respectively in the plurality of rack openings.
  • Each of the plurality of racks 107a-107d comprises a vertical arm 110, a plurality of vertically spaced supports 111a- 111m coupled to the vertical arm, and a plurality of substrates 112a-112m respectively carried by the plurality of vertically spaced supports, each substrate having culture media thereon.
  • the method also includes configuring a control unit 115 coupled to the plurality of ports 108a-108d to selectively supply culture medium to the bioreactor cavity 103 based upon a plurality of characteristics within the bioreactor cavity.
  • a control unit 115 coupled to the plurality of ports 108a-108d to selectively supply culture medium to the bioreactor cavity 103 based upon a plurality of characteristics within the bioreactor cavity.
  • FIGS.6A-6B, 7A-7F, 8-9 another embodiment of the bioreactor device 300 is now described.
  • this embodiment of the bioreactor device 300 differs from the previous embodiment in that this bioreactor device 300 illustratively includes a single rack 307, and three ports 308a-308c. As will be appreciated, this embodiment is for reduced scale applications.
  • This bioreactor device 300 illustratively comprises first and second bases 330a- 330b (e.g., PTFE), a tubular housing 331 between the first and second bases and having bioreactor fluid (e.g., sterile solution) therein, a plurality of vertical supports 332a-332d extending outside the tubular housing and between the first and second bases, and a rack 307 within the tubular housing.
  • the bioreactor device 300 illustratively includes a lid 305 which will fit into the cutout on the first support 330a, allowing for easy removal of rack and access to the trays.
  • the rack 307 illustratively includes first and second vertical arms 310a-310b, and a plurality of vertically spaced supports 311a- 311h coupled to the first and second arms.
  • Each support 311a-311h illustratively includes a recess for carrying a substrate 312a-312h with culture media thereon, a retention arm 333 for securing a respective substate 312a-312h, and first and second coupling arms 334a-334b respectively coupled to the first and second vertical arms 310a-310b.
  • FIGS.10A-10B another embodiment of the first base 430 is now described.
  • this embodiment of the first base 430 differs from the previous embodiment in that this first base 430 illustratively includes a stirrer 414.
  • this first base 430 illustratively includes a stirrer 414.
  • FIGS.13A-13B another embodiment of the rack 507 is now described. In this embodiment of the rack 507, those elements already discussed above with respect to FIGS.1-2 & 3A-3D are incremented by 400 and most require no further discussion herein.
  • this embodiment differs from the previous embodiment in that this bioreactor rack 507 illustratively includes a shelf insert 527 with a sloped top for draping falling film flow.
  • the lid 505 illustratively includes an expandable plug 528 extending along a periphery thereof.
  • Media will be circulated with a central impeller shaft and a falling film gas transfer strategy will be employed to avoid issues associated with gas sparging.
  • experimental and computational fluid dynamic (CFD) studies are performed to characterize media flow and oxygenation as bioreactor dimensions and operating conditions are optimized.
  • 6230TEES23 (0137616_PCT) [0068]
  • the four shelf inserts 527 are used to reduce media volume and confine flow to the sheet channels.
  • the flow within the bioreactor cavity 503 is shown with the arrows, and the flow exits the sheet channels reconverge in the side channels, which direct flow to the bottom center of the bioreactor cavity 503.
  • the impellers then propelled the media vertically through the core channel to again be distributed to the individual sheet channels and also spill over the tops of the racks to create a flowing surface film that increases the oxygen transfer rate well above that of a simple free surface, thus negating the need for gas sparging typically required for stirred bioreactors of this size.
  • Pitch-blade impellers are located at the vessel bottom to provide vertical (axial) flow while additional vertical impellers (e.g., Rushton) will be used to promote axial mixing.
  • an example embodiment of the bioreactor device 100, 200, 300 may include a custom-made chamber constructed from autoclavable glass for visual inspection of the cultures and media convection using a magnetic stir bar.
  • the chamber includes shelf inserts that can accommodate 10 individual PDMS substrates.
  • the bioreactor device can successfully culture 10 interwoven hDF cell sheets (6 x 6 cm2) over 6 weeks. Cell number analyses using DNA assays indicated faster proliferation of cells in this system relative to static dish cultures. No significant difference was found in the DNA amount of three cell sheets harvested from different positions, indicating a uniform culture environment in the bioreactor.
  • the bioreactor device 100 may accommodate four racks 107a-107d of sheets for cell sheet culture and subsequent decellularization.
  • the bioreactor device 100 illustratively includes a large diameter glass cylindrical vessel (body 102) with a custom-made head plate (lid 105) designed with ports (rack openings 106a-106d) for the fixation of 4 rack inserts (rack openings 106a- 106d).
  • Each rack insert contains 13 individual shelves (vertically spaced supports 111a- 111m) that hold PDMS substrates 112a-112m, featuring the desired dimensions and surface nano or micro patterns.
  • the impeller (stirrer 114) will drive media flow radially 6230TEES23 (0137616_PCT) between sheets and return the fluid vertically along the vessel wall to recirculate back to the impeller.
  • a heating jacket (heater 113) is used to maintain the glass vessel at the desired temperature, typically 37°C.
  • the multiple components can be easily assembled in an aseptic environment, such as a biosafety hood, to ensure sterile conditions.
  • a flow cell system with integrated DO, pH, glucose and lactate sensors is utilized for feedback control of O2 and pH in the bioreactor device 100. Glucose and lactate levels are monitored to signal when a medium change is required. [0074] In the bioreactor device 100, it may be helpful to characterize fluid flow using CFD simulations.
  • the circulation flow is shown in FIG.3C-3D, with media in the core of the bioreactor device 100 distributing uniformly to each sheet channel.
  • Four solid insets are used to ensure that the fluid exiting the channels reconverges in peripheral collector channels, which then return the flow to bottom center of the vessel. From there, fluid is propelled vertically through the core and diverges into the individual sheet channels.
  • Pitch-blade impellers second set of fins 126a-126b
  • first set of fins 125a-125f e.g., Rushton
  • bioreactor device 100 may include restrictions to flow through varying the channel heights (narrower channels towards the bottom).
  • the optimal size, number and distribution of impellers and channel geometries will be determined using CFD.
  • To assess the flow rate in each channel it is helpful to visualize the flow exiting the channels in the peripheral collector channel. Since this flow is primarily directed axially along the outer wall of the glass vessel, particle image velocimetry is employed using neutrally buoyant tracer particles travelling in the peripheral channels with a laser sheet aligned tangential to the vessel face. The velocity field and known cross-sectional areas will be converted to local flow rates that allow back calculation of flow exiting individual channels.
  • a feedback control system may be used, which may employ optical chemosensor flow cells for DO and pH sensing integrated with software to regulate flow rates to a mixing 6230TEES23 (0137616_PCT) gas manifold for use with stirred mammalian cell bioreactor cultures.
  • the flow cells involve a flow loop that continuously passes media from the bioreactor through the flow cells and returns the flow back to the bioreactor device 100.
  • This system will be employed for feedback control of DO and pH within the bioreactor device 100. While the single-rack prototype bioreactor performed well without gas sparging, the larger bioreactor device 100 will need sparging to meet the metabolic needs of more cells.
  • e-beam-sterilized glucose and lactate sensors for providing alerts for media exchanges.
  • a tube extending to the bottom of the vessel will be employed for media exchange.
  • Individual sheets will have a small lip designed to retain sufficient media to maintain culture viability while emptying and refilling the vessel.
  • Sterilization, operation, and xeno-free cell sheet production with the bioreactor device 100 is now described.
  • the glass vessel, PDMS substrates, head plate and tubing are autoclavable while the sensors can be sterilized via e-beam.
  • cell-seeded PDMS substrates are loaded into the glass container and sealed.
  • a xeno-free culture medium containing 5% hPL, rather than fetal bovine serum (FBS), is used to promote cell growth and ECM deposition.
  • Adipose stem cells cultured as sheets in 5% hPL produce thicker cell sheets than 10% FBS, with significantly more ECM deposition, including collagen and fibronectin4. After the designated culture period, the medium will be removed, and the cell sheets will be collected for characterization. [0077] Xeno-free ECM scaffold production and characterization with the bioreactor device 100 is now described. Once the uniformity of the cell sheets and batch-to-batch variation are confirmed, a new batch of cell sheets will be cultured. The glass vessel will then be rinsed a few times with PBS to wash the cell sheets.
  • the vessel will be filled with decellularization solutions, as outlined in Aim II.
  • the ECM scaffolds will be washed and removed for usage characterization. After washing the decellularization buffer, sterile and dry air will be purged through the bioreactor to result in dried decellularized ECM scaffolds.

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Abstract

Dispositif de bioréacteur pouvant comprendre un ensemble boîtier ayant un corps définissant une cavité de bioréacteur avec une ouverture, un couvercle devant être reçu par l'ouverture et comprenant des ouvertures de crémaillère et un orifice couplé à la cavité de bioréacteur. Le dispositif de bioréacteur peut également comprendre des crémaillères respectivement reçues par les ouvertures de crémaillère. Chacune des crémaillères peut comprendre un bras vertical, des supports espacés verticalement couplés au bras vertical, ainsi que des substrats portés respectivement par les supports espacés verticalement, chaque substrat ayant un milieu de culture sur celui-ci. Le dispositif de bioréacteur peut également comprendre une unité de commande couplée à l'orifice et conçue pour fournir sélectivement un milieu de culture à la cavité de bioréacteur sur la base d'une caractéristique à l'intérieur de la cavité de bioréacteur.
PCT/US2024/052203 2023-10-19 2024-10-21 Dispositif de bioréacteur pour fabriquer de multiples feuilles de cellules dans un réseau vertical et procédés associés Pending WO2025085891A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060286664A1 (en) * 1999-11-22 2006-12-21 Cytograft Tissue Engineering, Inc. Bioreactor for the Manufacture of Tissue Engineered Blood Vessels
US20130210130A1 (en) * 2010-04-21 2013-08-15 Octane Biotech, Inc. Automated cell culture system
US20180187139A1 (en) * 2016-03-14 2018-07-05 Ravindrakumar Dhirubhai Patel A bioreactor system and method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060286664A1 (en) * 1999-11-22 2006-12-21 Cytograft Tissue Engineering, Inc. Bioreactor for the Manufacture of Tissue Engineered Blood Vessels
US20130210130A1 (en) * 2010-04-21 2013-08-15 Octane Biotech, Inc. Automated cell culture system
US20180187139A1 (en) * 2016-03-14 2018-07-05 Ravindrakumar Dhirubhai Patel A bioreactor system and method thereof

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