Cell culture and tissue engineering system with controlled environmental zone

Abstract

An automated cell culture and tissue engineering system including a defined and separated environmental zone provides increased control and maintenance of the internal environment of the system such that the temperature, gas flow, and gas around a bioreactor module form a zone that is maintained separately from a second zone formed around a reagent fluid reservoir. The system further comprises means for eliminating and / or managing condensation within the second zone of the system.

Description

[0001] This application is a divisional application with application number 201980086836.9, application date December 20, 2019, and invention name “Cell culture and tissue engineering system with controlled environment zone”. Technical Field

[0002] The present invention relates to a tissue engineering system and method for automated cell culture and tissue engineering, wherein the tissue engineering system comprises a unified operating environment area to provide a more consistent biological process. Such systems and methods can be used in various clinical and laboratory environments. Background Art

[0003] Cell culture automation is a desired trend that provides scalability for large-scale production, reduced culture variability, reduced risk of culture contamination, and many economic cost and time-frame benefits associated with producing cell- or tissue-based implants for clinical therapies and cell-based assay systems for diagnostic assessments.

[0004] However, automated cell culture protocols for more complex biological processes (e.g., autologous patient treatments) can be more complex and require more precise operational control. For autologous patient treatments, successful biological culture is critical, and therefore each operational aspect must strictly adhere to specific protocols. For example, while the initial culture medium can be programmed to be delivered to the cell culture at an appropriate temperature (such as 37°C), maintaining strict adherence to this temperature throughout the cell culture process, which can be up to several days, proves to be more difficult. Many currently used automated systems that provide cell incubation capabilities experience thermal challenges such as: trying to achieve and maintain overall thermal uniformity; air temperature changes due to external operator access; maintaining refrigerated reagent storage; and the development of condensation within the enclosure that can lead to potential microbial contamination.

[0005] Refrigerated storage of reagents is desirable to avoid spoilage, however, refrigerated reagent storage can negatively impact the thermal performance and stability of the cell bioreactor, making it more difficult to maintain the desired high temperatures required for cell culture. In addition, lowering the temperature of the storage environment for process reagents to refrigeration temperatures inevitably results in condensation. Condensation forms when air cools below the dew point, causing water vapor in the air to condense into liquid form, especially on surfaces that are cooler relative to the surrounding environment. When the system is opened to load reagents, the warm, humid air may come into contact with the even cooler surfaces of the storage environment. This condensation can be problematic if the volume of condensate causes handling issues or contamination problems within the storage environment.

[0006] When gases such as CO2 are used to adjust the pH of active cultures, the gas concentration may also significantly affect biological performance. If the culture system experiences limited access to the buffering effect of the delivered CO2, pH control may not be uniform across the culture area. Therefore, gas concentration uniformity is also desired in cell culture and tissue engineering systems.

[0007] Therefore, there remains a need for continuous improvement in various aspects of automated cell culture and tissue engineering systems, such as providing higher fidelity environmental control within the system.

[0008] The background discussion herein is included to explain the context of the invention described herein. It is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge before the priority date of any claim. Summary of the invention

[0009] Described herein are cell culture and tissue engineering systems and methods that include a unified operating environment to provide more consistent control during a bioprocess. A unified operating environment related to airflow, temperature, gas control, and condensation control is integrated within the systems and methods described herein.

[0010] The automated cell culture and tissue engineering system as described herein is configured to create, adjust and maintain separate controlled environmental zones to properly operate the cell culture boxes and therefore the ongoing biological processes. Temperature, humidity levels and gas concentrations are controlled. Temperature fluctuations and humidity changes are minimized.

[0011] Surprisingly, two different temperature zones are created and maintained in the box storage area of ​​the system for installing and operating cell culture boxes. During system operation, the bioreactor module assembly of the box remains in the warm zone, while the reagent fluid reservoir assembly remains in the cold zone. The warm zone includes a recirculating warm high airflow path around the bioreactor module, and the recirculating warm high airflow path is produced by an adjacent separate heating assembly. The warm circulating air also penetrates into the culture aspect of the box through ventilation slots. This is conceptually similar to the groove in the reservoir. The cold zone includes a circulating cold conduit airflow path, which surrounds and partially penetrates (flows through) a part of the reagent fluid reservoir. The cold airflow path is produced by adjacent hot and cold assemblies. The cold zone also contains a condensation control device for controlling and removing the condensation in the cold zone.

[0012] Both the warm zone and the cold zone further include a separate independently controlled gas environment. In the warm zone, this helps to provide a specific gas concentration that affects dissolved gases present in the cell culture medium, such as oxygen and carbon dioxide, through fluid surface gas exchange. The adjustment and / or maintenance of dissolved gases have an impact on biological performance (including aspects such as oxygen delivery and maintenance of target pH of cell culture). The control of dissolved gases in the culture medium is achieved by recirculating the culture medium across a gas exchange membrane (such as a silicone tube), whereby the concentration of dissolved gases in the culture fluid responds to the concentration gradient between the fluid and the surrounding gas environment. By adjusting the environment, the level of dissolved gases in the culture fluid is adjusted simultaneously.

[0013] The automated cell culture and tissue engineering system is configured with a removable thermal barrier assembly that is used to lock the cassette into the system operating interface during installation of the cell culture cassette and in doing so forms a warm zone and a cold zone and keeps the two zones thermally and physically separated. An insulation mechanism is provided to ensure that the warm zone and the cold zone are insulated from each other and do not affect the properties of the cell culture or the stored reagents. The removable thermal barrier assembly creates and defines a portion of the boundary of each of the warm zone and the cold zone.

[0014] System heating and cooling assembly and operation robot are contained / positioned to be separated from heating and cooling assembly and operation robot of the box storage area of ​​system, which is conducive to not interfering with warm air flow path or cold air flow path and its function. Further, separate heating assembly to be separated and insulated from cooling assembly. System heating assembly is configured to produce and adjust the temperature of continuous warm high air flow path as required for several days, and further can be opened and any temperature drop that may occur due to colder room temperature air entering during inspection system is quickly adjusted. The configuration and shape of system help to set warm air flow path, and described warm air flow path is only for bioreactor module and continuously circulates around described bioreactor module and circulates through it. Hot and cold assembly is configured to remove heat continuously as the air flow in cold zone is continuously sucked through hot and cold assembly and air temperature is reduced. Cold zone is configured to have guided air flow, that is to say, cold air flow path meets the structural features comprising air flow channel, air flow baffle and air flow vent, to help carry colder air through the cold zone around and partially through the reagent fluid reservoir and further through the optional adjacent cold reservoir outside the reagent fluid reservoir. These structural features help prevent and minimize any obstruction of return air traveling toward the hot and cold assembly (through the filled fluid bag).

[0015] Providing separate warm zones and separate cold zones minimizes the amount and location of condensation that may form within the system, as the system is only subject to condensation in the cold zones, which have mechanisms to effectively prevent, control and remove condensation.

[0016] The present invention includes automated cell culture and tissue engineering systems in some aspects, and the automated cell culture and tissue engineering systems include closed automated cell culture boxes for one or more of the following: cell source separation, cell proliferation / amplification, cell differentiation, cell separation, cell labeling, cell purification, cell washing and cell seeding on a support for tissue formation (product formation). In some aspects, the cell is a mammalian cell. In other aspects, the cell is a human cell. The type of cell or tissue is not restrictive. In a non-limiting example, pluripotent stem cells (such as embryonic stem cells) and induced pluripotent stem (iPS) cells can be cultured and amplified for cell replacement therapy.

[0017] In some aspects, the automated cell culture box is a closed, single-use disposable box that includes one or more sterile bioreactor modules that are fluidically connected to a reagent fluid reservoir. The sterile bioreactor is loaded with desired cells and / or tissues and is connected to a reagent fluid reservoir that is preloaded to accommodate required fluid reagents. The sterility of the box is maintained throughout.

[0018] The automated cell culture cassette and dedicated software program are operatively employed within an automated cell culture and tissue engineering system to deliver and track desired one or more processes. Suitable non-limiting automated cell culture and tissue engineering systems are described in U.S. 8,492,140; U.S. 9,701,932; U.S. 9,534,195; U.S. 9,499,780; and U.S. 9,783,768 (the contents of each of these U.S. patents are incorporated by reference in their entirety).

[0019] Embedded sensors within the cell culture cassette provide real-time biofeedback and enable automatic adjustment of the bioprocess to accommodate natural variations in cell source behavior. The entire bioprocess is contained within the disposable cassette to maximize patient and operator safety and simplify logistics. Further, to successfully support multiple biological steps in the cell process sequence, one or more cassette bioreactors are integrated in combination with biosensor feedback within one or more interconnected bioreactors to provide a highly intuitive system with precise control at each cell and tissue stage. This comprehensive level of automation enables technically feasible and economically scalable, patient-scale cell manufacturing capabilities and allows for simplified production of cell therapies under good manufacturing practice (GMP) conditions, thereby meeting the unique challenges of different autologous and allogeneic clinical applications of cell and tissue therapies.

[0020] Advantageously, the cell culture box is installed in the cell culture and tissue engineering system housing and is operatively maintained in the housing in a manner that the bioreactor module is only located in different warm zones and the reagent fluid reservoir is only located in different cold zones. The cell culture box is installed in the system by actuating and locking a removable thermal barrier assembly, which separates the first hot zone from the second hot zone environmentally in terms of temperature, gas and humidity. The cell culture box comprises a bioreactor module and an attached reagent fluid reservoir. Although this combination in a single cell culture box is more user-friendly, it has proposed more challenges in creating separate and different environmental zones for bioreactor modules and reagent fluid reservoirs compared to the simpler design based on physically separated bioreactor modules and reservoirs that can be positioned in a separate environmental zone.

[0021] The present invention provides dedicated gas path paths within warm and cold zones, thereby ensuring controlled temperature / gas distribution around bioreactor modules containing one or more cell cultures and reagent fluid reservoirs in a manner that eliminates uniformity distortion points in each zone.

[0022] In some aspects, the present invention is a cell culture and tissue engineering system comprising two different independent thermal gas flows, namely a first gas flow comprising a high-speed warm gas flow for directing at and around a bioreactor module of a cell culture cassette, and a second gas flow comprising a cold gas flow for circulating around and through a reagent fluid reservoir operatively connected to the bioreactor module,

[0023] Wherein the first gas stream and the second gas stream are substantially separate (in embodiments these zones are not hermetically sealed relative to each other) and cannot intermix.

[0024] In some aspects, the first airflow and the second airflow are contained within a cell culture cartridge receiving area of ​​the system.

[0025] In some aspects, the cell culture cassette storage area is separate from the operating, heating and cooling assemblies of the system.

[0026] In some aspects, the cell culture cassette storage area defines a warm zone comprising a high velocity warm air flow.

[0027] In some aspects, the warm zone comprises a substantially uniform temperature within the warm zone.

[0028] In some aspects, the cell culture cassette storage area defines a cold zone including the cold air flow, the cold zone being positioned below the warm zone.

[0029] In some aspects, the cold zone includes means for reducing or eliminating condensation.

[0030] In some aspects, a thermal platform separates the warm zone from the cold zone. In some aspects, the thermal platform contains a seal.

[0031] In some aspects, the invention is a method for maintaining a controlled thermal environment for a bioprocess within a bioreactor module of a cell culture cassette, the method comprising:

[0032] directing a first gas flow comprising a high velocity warm gas flow at and around the bioreactor module and simultaneously circulating a cool gas flow around and through a reagent fluid reservoir operatively connected to the bioreactor module,

[0033] The first airflow and the second airflow are separated and cannot be mixed.

[0034] According to one aspect of the present invention is a cell culture and tissue engineering system comprising a thermal zone architecture for providing a more consistent and controlled environment to promote biological processes, wherein the system comprises:

[0035] a distinct warm zone compartment that holds a bioreactor module while continuously circulating a high velocity warm gas flow path around the bioreactor module and directing the high velocity warm gas flow path at and around the bioreactor module; and

[0036] A distinct cold zone compartment holds a reagent fluid reservoir functionally connected to the bioreactor module while having a cold gas flow path continuously circulate around and through the reagent fluid reservoir.

[0037] In some aspects, the distinct cold zone compartments further comprise means for reducing or eliminating condensation.

[0038] In some aspects, the warm zone compartment further comprises a gas environment that is different from the gas environment of the cold zone compartment.

[0039] According to another aspect of the invention is an automated system for cell culture and tissue engineering, the automated system maintaining a cell culture box in two different temperature and gas controlled environments, wherein the bioreactor component of the box is operatively maintained in a substantially uniform warm air flow zone with controlled gas concentration, and wherein the reagent fluid reservoir component of the box resides in a substantially uniform cold air flow zone, wherein the warm air flow zone is clearly separated from the cold air flow zone, and wherein the cold air flow zone further includes a device for preventing or eliminating undesirable moisture accumulation therein.

[0040] According to another aspect of the present invention is a cell culture and tissue engineering system for housing and operationally supporting an automated cell culture cassette for a bioprocess in a more uniform controlled environment, the cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the system comprising:

[0041] a warm zone configured to circulate a warm air flow path around the bioreactor module;

[0042] a cold zone configured to circulate a cold gas flow path around the reagent fluid reservoir; and

[0043] A removable thermal barrier assembly for thermally isolating the warm zone from the cold zone when the cell culture cassette is installed and for securing the bioreactor module solely within the warm zone and the reagent fluid reservoir solely within the cold zone.

[0044] According to another aspect of the present invention, there is a cell culture box, comprising:

[0045] - a bioreactor module having a bottom portion to which a reagent fluid reservoir is attached;

[0046] - A reagent fluid reservoir comprising a fluid bag container having open air ducts on the front and rear walls of the reservoir.

[0047] In some aspects, the fluid bag container includes a top panel and a bottom panel, the top panel including a downwardly extending baffle.

[0048] In some aspects, the cartridge further comprises an insulation layer between a bottom of the bioreactor module and a top plate of the fluid bag container, the insulation layer insulating against heat migration from the bioreactor module.

[0049] In some aspects, the reagent fluid reservoir is attached via a port connection positioned on a top plate of the fluid bag container and unobstructed by the insulation layer.

[0050] In some aspects, the reagent fluid reservoir further comprises a snap tab for attachment to the bioreactor module.

[0051] According to a further aspect of the invention is a reagent fluid reservoir for connection to a bioreactor module, the reagent fluid reservoir comprising a fluid bag container having open air conduits on the front and rear walls of the reservoir.

[0052] In some aspects, the fluid bag container includes a top panel and a bottom panel, the top panel including a downwardly extending baffle.

[0053] According to one aspect of the present invention, an automated cell culture and tissue engineering system is provided for housing and operationally supporting an automated cell culture cassette for a bioprocess in a more uniform controlled environment, the cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the system comprising:

[0054] a warm zone configured to circulate a tangential warm gas flow path around the bioreactor module;

[0055] a cold zone configured to circulate a tangential cold gas flow path around the reagent fluid reservoir; and

[0056] A removable thermal barrier assembly for thermally isolating the warm zone from the cold zone when the cell culture cassette is installed and for securing the bioreactor module solely within the warm zone and the reagent fluid reservoir solely within the cold zone.

[0057] In any of the foregoing aspects, the systems and methods may include one or more controllers and associated software, sensors, and user interfaces.

[0058] Non-limiting aspects are as follows:

[0059] 1A. A cell culture and tissue engineering system comprising two distinct independent thermal gas streams, a first gas stream comprising a high velocity warm gas stream for directing at and around a bioreactor module of a cell culture cassette, and a second gas stream comprising a cold gas stream for circulating around and through a reagent fluid reservoir operatively connected to the bioreactor module,

[0060] The first airflow and the second airflow are separated and cannot be mixed.

[0061] 1B. The system of aspect 1A, wherein the first airflow and the second airflow are contained within a cell culture cassette receiving area of ​​the system.

[0062] 1C. The system of aspect 1B, wherein the cell culture cassette storage area defines a warm zone comprising a high velocity warm air flow.

[0063] 1D. The system of aspect 1C, wherein the warm zone comprises a substantially uniform temperature within the warm zone.

[0064] 1E. The system of aspects 1B, 1C or 1D, wherein the cell culture cassette storage area defines a cold zone including the cold air flow, the cold zone being positioned below the warm zone.

[0065] 1F. The system of aspect 1E, wherein the cold zone comprises means for reducing or eliminating condensation.

[0066] 1G. The system of aspect 1F, wherein a thermal platform separates the warm zone from the cold zone.

[0067] 1H. A method for maintaining a controlled thermal environment for a bioprocess within a bioreactor module of a cell culture cassette, the method comprising:

[0068] directing a first gas flow comprising a high velocity warm gas flow at and around the bioreactor module and simultaneously circulating a cool gas flow around and through a reagent fluid reservoir operatively connected to the bioreactor module,

[0069] The first airflow and the second airflow are separated and cannot be mixed.

[0070] 1J. A method according to aspect 1H, using a system according to any one of aspects 1A to 1H.

[0071] 2A. A cell culture and tissue engineering system comprising a thermal zone architecture for providing a more consistent and controlled environment to promote biological processes, wherein the system comprises:

[0072] a distinct warm zone compartment that holds a bioreactor module while continuously circulating a high velocity warm gas flow path around the bioreactor module and directing the high velocity warm gas flow path at and around the bioreactor module; and

[0073] A distinct cold zone compartment holds a reagent fluid reservoir functionally connected to the bioreactor module while having a cold gas flow path continuously circulate around and through the reagent fluid reservoir.

[0074] 2B. The system of aspect 2A, wherein the distinct cold zone compartments further comprise means for reducing or eliminating condensation.

[0075] 2C. The system of aspect 2A or 2B, wherein the warm zone compartment further comprises a gas environment that is different from the gas environment of the cold zone compartment.

[0076] 2D. A method for maintaining a controlled environment for a bioprocess within a bioreactor and for maintaining fluids required by the bioreactor at low temperatures to achieve stability, the method comprising using a system according to any one of aspects 2A to 2C.

[0077] 3A. An automated system for cell culture and tissue engineering, the automated system maintaining a cell culture box in two different temperature and gas controlled environments, wherein the bioreactor component of the box is operatively maintained in a substantially uniform warm air flow zone with controlled gas concentration, and wherein the reagent fluid reservoir component of the box resides in a substantially uniform cold air flow zone, wherein the warm air flow zone is clearly separated from the cold air flow zone, and wherein the cold air flow zone further includes a device for preventing or eliminating undesirable moisture accumulation therein.

[0078] 3B. A method for maintaining a controlled environment for a bioprocess within a bioreactor and for maintaining fluids required by the bioreactor at low temperatures to achieve stability, the method comprising using the system according to aspect 3A.

[0079] 1. A cell culture and tissue engineering system for housing and operationally supporting an automated cell culture cassette for a bioprocess in a more consistent controlled environment, the cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the system comprising:

[0080] a warm zone configured to circulate a warm air flow path around the bioreactor module;

[0081] a cold zone configured to circulate a cold gas flow path around the reagent fluid reservoir; and

[0082] A removable thermal barrier assembly for thermally isolating the warm zone from the cold zone when the cell culture cassette is installed and for securing the bioreactor module solely within the warm zone and the reagent fluid reservoir solely within the cold zone.

[0083] 2. The cell culture and tissue engineering system according to aspect 1, wherein the cold zone further comprises a condensation control device for minimizing and eliminating undesirable moisture accumulation therein.

[0084] 3. The cell culture and tissue engineering system of aspect 1 or 2, wherein the warm zone and the cold zone each comprise substantially separate gas environments.

[0085] 4. The cell culture and tissue engineering system according to aspects 1, 2 or 3, wherein the system has a housing comprising an outer shell cover and an inner shell, wherein the outer shell cover encloses a front opening of the inner shell in an encapsulated manner when the system is closed.

[0086] 5. A cell culture and tissue engineering system according to aspect 5, wherein the outer shell cover is connected to the inner shell to rotate while the inner shell remains fixed, the outer shell cover rotates along the outer arc of the inner shell to expose the front opening of the inner shell to enter the inner shell and stops rotating when the outer shell cover is nested in the inner shell.

[0087] 6. A cell culture and tissue engineering system according to aspect 4 or 5, wherein the front opening of the inner shell includes a perimeter having an expandable sealing device to seal with the inner surface of the outer shell cover when the system is closed.

[0088] 7. The cell culture and tissue engineering system according to aspect 6, wherein the perimeter comprises a U-shaped channel for retaining the expandable sealing device.

[0089] 8. A cell culture and tissue engineering system according to aspect 7, wherein the sealing device includes an elastic tube assembled in the U-shaped channel, the elastic tube is substantially flat when the system is opened, and the sealing device is activated when the system is closed to introduce expansion pressure into the cavity of the inflatable seal, thereby shifting the seal to produce a reliable seal between the outer shell cover and the front opening of the inner shell.

[0090] 10. The cell culture and tissue engineering system of aspect 9, wherein the outer shell cover is configured as an arc-shaped body that helps guide a circulating warm air flow path around the bioreactor module and also helps guide a circulating cold air flow path around the reagent fluid reservoir.

[0091] 11. The cell culture and tissue engineering system according to aspect 10, wherein the arc-shaped body comprises a plurality of thermal units to act as an external thermal barrier.

[0092] 12. The cell culture and tissue engineering system of any one of aspects 4 to 11, wherein the movable thermal barrier assembly is disposed on a manipulation robotic interface positioned within the front opening of the inner housing.

[0093] 13. A cell culture and tissue engineering system according to aspect 12, wherein the operating robot interface is connected to an associated internal robot and includes valve actuators, peristaltic pumps and related control systems to cooperate with corresponding connections on the cell culture box.

[0094] 14. The cell culture and tissue engineering system according to any one of aspects 4 to 11, wherein the movable thermal barrier assembly comprises:

[0095] - A pair of spaced apart lever arms internally connected at either side of the manipulation robot interface, the pair of spaced apart lever arms supporting a central thermal platform with associated upper handrails.

[0096] 15. A cell culture and tissue engineering system according to aspect 14, wherein the movable thermal barrier assembly can be moved from a first raised position for mounting the cell culture box to a second lowered position, the second lowered position locking and holding the cell culture box against the operating robot interface for accurate alignment and retention thereto and simultaneously separating the warm zone from the cold zone.

[0097] 16. The cell culture and tissue engineering system of aspect 15, wherein the upper armrest is sized to closely conform to the dimensions of the cell culture cassette and provides a gripping device to lock the cassette into position for operating or raising the thermal platform.

[0098] 17. A cell culture and tissue engineering system according to aspects 14, 15 or 16, wherein a recess is provided on the operating robotic interface adjacent to the lever arm to provide an operator with controlled access to open the assembly.

[0099] 18. The cell culture and tissue engineering system according to any one of aspects 14 to 17, wherein the thermal platform extends forward and radiates from around the cell culture box to the outer housing cover.

[0100] 19. A cell culture and tissue engineering system according to aspect 18, wherein the thermal platform includes a flexible outer seal and a flexible inner seal, the flexible outer seal being at its front outer boundary to accommodate relative movement between the thermal platform and the outer shell cover, and the flexible inner seal being directly adjacent to the cell culture box.

[0101] 20. The cell culture and tissue engineering system of aspect 19, wherein the flexible inner seal accommodates installation tolerances of the cell culture cassette.

[0102] 21. The cell culture and tissue engineering system of aspect 19 or 20, wherein the flexible outer seal and the flexible inner seal are elastomeric and vapor impermeable.

[0103] 22. The cell culture and tissue engineering system of aspect 21, wherein the flexible outer seal and the flexible inner seal are scraper seals.

[0104] 23. A cell culture and tissue engineering system according to any one of aspects 14 to 22, wherein when in the locked position, the thermal platform includes a portion of a lower surface boundary of the warm zone and a portion of an upper surface boundary of the cold zone.

[0105] 24. The cell culture and tissue engineering system of any one of aspects 14 to 18, wherein the inner housing comprises a floor supporting a tray having a forward cantilevered shelf supporting a portion of the bottom surface of the cell culture box.

[0106] 24a. The cell culture and tissue engineering system of aspect 24, wherein an air flow conduit is defined by the top plate and the tray.

[0107] 25. The cell culture and tissue engineering system of any one of aspects 4 to 24, wherein a heating assembly is disposed in an upper section of the inner housing, generating hot air directed at the bioreactor module.

[0108] 26. The cell culture and tissue engineering system of aspect 25, wherein the heating arrangement comprises a high-capacity long-blade fan operably connected to the heated air flow director to generate and direct a high-velocity warm air flow path.

[0109] 27. The cell culture and tissue engineering system of aspect 26, wherein a highly tangential warm air flow path around the bioreactor module promotes temperature uniformity of the warm zone and, thereby, promotes temperature uniformity of the bioprocess within the bioreactor.

[0110] 28. A cell culture and tissue engineering system according to aspect 27, wherein the high tangential velocity warm air flow path surrounding the bioreactor module helps maintain the internal temperature of the bioreactor at about 35°C, at about 36°C, at about 37°C, at about 38°C or at about 39°C.

[0111] 28a. The cell culture and tissue engineering system of aspect 28, wherein gas is selectively introduced into the warm zone.

[0112] 28b. The cell culture and tissue engineering system of aspect 28a, wherein the gas comprises one or more of oxygen, carbon dioxide and nitrogen.

[0113] 28c. The cell culture and tissue engineering system of aspect 28a or 28b, wherein the warm zone comprises a sensor in the warm gas flow path to monitor the gas.

[0114] 28d. The cell culture and tissue engineering system according to aspect 28c, wherein the sensor is operatively connected to a PID (proportional integral derivative) controller providing gas feedback control.

[0115] 29. A cell culture and tissue engineering system according to any one of aspects 4 to 28, wherein the hot and cold assembly forms the rear portion of the internal housing and generates and controls a cold zone airflow path around the reagent fluid reservoir to enhance reagent stability.

[0116] 30. The cell culture and tissue engineering system of aspect 29, wherein the hot and cold assembly comprises a cold channel array comprising a plurality of cold channels compressed to a hot channel using a peltier device to transfer heat to the hot channel.

[0117] 31. A cell culture and tissue engineering system according to aspect 30, wherein the compression is spring compression using a Peltier solid state device, the Peltier solid state device comprising an array of spring compression bolts, each spring compression bolt comprising a coil spring, and the heat slot acts as a thermally conductive path for heat removal.

[0118] 32. The cell culture and tissue engineering system of aspect 31, wherein the Peltier solid state device is used to pump heat from the cold tank to the hot tank for subsequent transfer of heat to the surrounding environment.

[0119] 33. The cell culture and tissue engineering system of any one of aspects 30 to 32, wherein each cold channel array is divided into functional units comprising a cold channel and an associated axial flow fan.

[0120] 34. The cell culture and tissue engineering system of aspect 33, wherein the axial flow fan has an axial flow configuration.

[0121] 35. The cell culture and tissue engineering system of aspect 33 or 34, wherein the cold channel comprises a vertical fin structure.

[0122] 36. The cell culture and tissue engineering system according to any one of aspects 29 to 35, further comprising a thermal insulator for preventing heat from returning from the hot tank to the cold tank.

[0123] 37. A cell culture and tissue engineering system according to any one of aspects 2 to 36, wherein the condensation control device includes a moisture transport material enclosed in a conduit connected to the hot tank, the moisture transport material collecting and moving any condensate that may form in the hot tank and travels along the cold tank until it is evaporated.

[0124] 38. The cell culture and tissue engineering system of aspect 37, wherein the condensation control device minimizes and eliminates unwanted microbial contamination due to moisture accumulation.

[0125] 39. The cell culture and tissue engineering system of any one of aspects 29 to 38, wherein the hot and cold assembly can be unhinged from the inner housing bottom to be suspended in an open configuration for cleaning.

[0126] 40. The cell culture and tissue engineering system of aspect 39, wherein the cold channel array and associated axial flow fans can be decoupled for cleaning.

[0127] 41. The cell culture and tissue engineering system of aspect 40, wherein the heat sink fan and associated shroud can be unlatched from the upper portion of the internal housing and raised into an open configuration for cleaning.

[0128] 42. A cell culture and tissue engineering system according to any one of aspects 1 to 41, wherein the reagent fluid reservoir is connected to the bioreactor module via a top mounted port connection to mate with a port on the underside of the bioreactor module.

[0129] 43. The cell culture and tissue engineering system of aspect 42, wherein the reagent fluid reservoir further comprises a snap tab for reversibly attaching to the bioreactor module.

[0130] 44. The cell culture and tissue engineering system of aspect 43, wherein the reagent fluid reservoir contains reagents required for a biological process and contains the retention of waste products eliminated from the bioreactor module.

[0131] 45. The cell culture and tissue engineering system of aspect 44, wherein the reagent fluid reservoir comprises a fluid bag container for storing a plurality of individual reagents and for storing the waste product.

[0132] 46. ​​The cell culture and tissue engineering system of aspect 45, wherein the plurality of separate reagents are stored in reagent bags.

[0133] 47. The cell culture and tissue engineering system of aspect 46, wherein the waste products are stored in a reagent bag.

[0134] 48. The cell culture and tissue engineering system according to aspect 46 or 47, wherein the reagent bag is a flexible reagent bag.

[0135] 49. A cell culture and tissue engineering system according to any one of aspects 44 to 48, wherein the reagent fluid reservoir further includes an open air duct located at the top of the front wall and the rear wall, so that the open air duct is located above the reagent bag, thereby providing a cool air flow directly above the reagent bag.

[0136] 50. The cell culture and tissue engineering system of aspect 49, further wherein the reagent fluid reservoir comprises a top plate having a downwardly extending baffle to help direct the flow of cool air over and across the reagent bag.

[0137] 51. The cell culture and tissue engineering system of aspect 50, wherein the cell culture cassette further comprises an insulation layer between the bioreactor module and the reagent fluid reservoir, the insulation layer insulating any migration of heat from the bioreactor module.

[0138] 52. A cell culture box comprising:

[0139] - a bioreactor module having a bottom portion to which a reagent fluid reservoir is attached;

[0140] - A reagent fluid reservoir comprising a fluid bag container having open air ducts on the front and rear walls of the reservoir.

[0141] 53. The cell culture cassette of aspect 52, wherein the fluid bag container comprises a top plate and a bottom plate, the top plate comprising a downwardly extending baffle.

[0142] 54. The cell culture cassette of aspect 52 or 53, wherein the cassette further comprises an insulation layer between the bottom of the bioreactor module and the top plate of the fluid bag container, the insulation layer insulating heat migration from the bioreactor module.

[0143] 55. The cell culture cassette of any one of aspects 53 or 54, wherein the reagent fluid reservoir is attached via a port connection positioned on a top plate of the fluid bag container and unobstructed by the insulation layer.

[0144] 56. The cell culture cassette of aspect 55, wherein the reagent fluid reservoir further comprises a snap tab for attachment to the bioreactor module.

[0145] 57. An automated cell and tissue culture engineering system comprising the cell culture cassette according to any one of aspects 52 to 56.

[0146] 57a. The automated system of aspect 57, wherein the cartridge comprises one or more sensors connected to a logic device.

[0147] 58. A reagent fluid reservoir for connection to a bioreactor module, the reagent fluid reservoir comprising a fluid bag container having open air conduits located on the front and rear walls of the reservoir.

[0148] 59. The reagent fluid reservoir of aspect 57, further wherein the fluid bag container comprises a top plate and a bottom plate, the top plate comprising a downwardly extending baffle.

[0149] 60. A cell culture cassette comprising a bioreactor module according to aspect 58 or 59 and a reagent fluid reservoir.

[0150] 61. An automated cell culture and tissue engineering system for housing and operationally supporting an automated cell culture cassette for a bioprocess in a more consistent controlled environment, the cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the system comprising:

[0151] a warm zone configured to circulate a tangential warm gas flow path around the bioreactor module;

[0152] a cold zone configured to circulate a tangential cold gas flow path around the reagent fluid reservoir; and

[0153] A removable thermal barrier assembly for thermally isolating the warm zone from the cold zone when the cell culture cassette is installed and for securing the bioreactor module solely within the warm zone and the reagent fluid reservoir solely within the cold zone.

[0154] 62. A method for maintaining a controlled thermal environment for a bioprocess within a bioreactor module of a cell culture cassette, the method comprising:

[0155] generating and maintaining different high-velocity warm gas streams at and around the bioreactor module and simultaneously generating and circulating different cold gas streams around and through a reagent fluid reservoir operatively connected to the bioreactor module,

[0156] The first airflow and the second airflow are separated and cannot be mixed. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] The following description of typical aspects described herein will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the current typical aspects are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangement and means of the aspects shown in the accompanying drawings. It should be noted that in the different embodiments as shown in the drawings and mentioned in the description, like reference numerals refer to like elements.

[0158] The description herein will be more fully understood in view of the following drawings:

[0159] Figure 1 A cross-section of one embodiment of the cell culture and tissue engineering system of the present invention is presented, showing a cell culture cassette engaged with a manipulation robot and a thermal barrier assembly in a locked position;

[0160] Figure 2 shows a cross section of the system in an open configuration exposing the cell culture cassette shown with the thermal barrier assembly in an unlocked position;

[0161] Figure 3A A front elevation view of the system is shown showing the operating robotic interface and the thermal barrier assembly in an upright position for mounting a cell culture cassette;

[0162] Figure 3B Shown Figure 3A A front elevation view of the system and the thermal barrier assembly in a locked position;

[0163] Figure 3C A close-up front elevation view of an external cold reservoir forming part of the cold zone and positioned adjacent to the mounted reagent fluid reservoirs is shown, the external cold reservoir having baffles to ensure that air returning to the cold tank is not obstructed. These baffles are similar to those in the Figure 3B The underlying structure of the thermal barrier assembly as seen in FIG.

[0164] Figure 4 Features of the warm zone airflow path and heating assembly are shown;

[0165] Figure 5 The cold zone airflow paths and the characteristics of the hot and cold assemblies are shown;

[0166] Figure 6 A close-up perspective view showing the reagent fluid reservoir structure;

[0167] Figure 7 A close-up cross-sectional view of the cold slot array is shown;

[0168] Fig. 8A The positioning of the condensation control structure adjacent to the cold slot array is shown;

[0169] Figure 8B An enlarged cross-sectional view of the condensation control structure is shown;

[0170] Fig. 9 The hot and cold assemblies are shown unhinged from the system;

[0171] Fig.10 A hot and cold assembly is shown with a detached cooling fan; and

[0172] Fig.11 Shown is the heat sink fan and associated shroud released from the rear of the system. DETAILED DESCRIPTION

[0173] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The publications and applications discussed herein are provided only for disclosure prior to the filing date of the present application. Anything herein should not be construed as admitting that the present invention has no right to precede such publications due to existing inventions. In addition, these materials, methods and examples are illustrative only and are not intended to be limiting.

[0174] In case of conflict, the present specification, including definitions, will control.

[0175] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the subject matter herein belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0176] As used herein, the article "a / an" before an element or component is intended to be non-restrictive with respect to the number of instances (i.e., occurrences) of the element or component. Therefore, "a / an" should be understood to include one or at least one, and the singular form of an element or component also includes the plural unless the number is obviously intended to be singular.

[0177] As used herein, the term "invention" or "present invention" is a non-limiting term and is not intended to refer to any single aspect of the particular invention, but encompasses all possible aspects described in the specification and claims.

[0178] As used herein, the terms "comprises," "comprising," "includes," "including," "having," and variations and conjugations thereof mean "including, but not limited to," and should be understood to be open-ended, for example, meaning including but not limited to.

[0179] As used herein, the term "about" refers to a change in the numerical quantity. On the one hand, the term "about" means within 10% of the reported numerical value. On the other hand, the term "about" means within 5% of the reported numerical value. However, on the other hand, the term "about" means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the reported numerical value.

[0180] It will be understood that any component defined herein may be expressly excluded from the claimed invention by way of a proviso or negative limitation.

[0181] As may be used herein, the term "substantially" (or its synonyms) means, relative to the context, a measure or extent or amount or degree that covers a large portion or majority of the referenced entity, or to a degree that is at least moderately or much greater or larger or more effective or more important relative to the referenced entity or to the referenced subject matter.

[0182] As used herein, the term "may" means options or actions that are included or not included and / or used and / or implemented and / or occur, but the options constitute at least part of some embodiments of the present invention or their results, without limiting the scope of the present invention.

[0183] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, e.g., the elements are present in conjunction in some cases and separately in other cases. In addition to the elements specifically identified with the "and / or" phrase, other elements may optionally be present, whether related or unrelated to those elements specifically identified, unless explicitly stated to the contrary.

[0184] As used herein, with respect to the engagement of components of the automation device of the present invention, "supported," "mounted," "attached," "connected," "engaged," "coupled," and "linked" may be used interchangeably. Further, any of these terms may be used with the term "reversibly."

[0185] As used herein, "thermal zone" is understood to be a separate area with a defined consistent temperature in terms of warmth or cold. Further, a zone with a defined temperature or a defined temperature range is maintained at the defined temperature or within the defined temperature range so that the zone is stable, uniform, unbiased or homogeneous with respect to the defined temperature or defined temperature range.

[0186] As used herein, a "warm zone" is understood to be a separate, enclosed area with a precise temperature above room temperature (i.e., above about 23°C). Typically, the precise temperature of mammalian cells is about 37°C. However, depending on the special needs of a particular cell culture, temperatures above and below 37°C can still be selected as precise temperatures and completed as a "warm zone". For example, if there are no differentiation factors, stem cells will proliferate at 37°C without differentiation. On the contrary, stem cells grown at temperatures above and below 37°C will differentiate in the absence of differentiation factors. In addition, different "warm zone" temperatures may be required to apply temperature stress to a given culture. A "warm zone" will contain a high-speed warm air flow path. Those skilled in the art will understand the meaning of "high speed" compared to conventional air flow velocities. The "warm zone" accommodates bioreactor modules and has a unique gas regulating device.

[0187] As used herein, a "cold zone" is understood to be a separate enclosed area with a temperature range of about 2°C to 8°C. The exact temperature of the "cold zone" need not be precise but rather the overall temperature is reduced, such as about 2°C to 8°C. The "cold zone" contains a cold air stream. The "cold zone" houses the fluid reagent reservoir and any additional fluid reagent bags. The "cold zone" houses the condensation control device.

[0188] The "warm zone" is separated from the "cold zone" so that the temperature in one zone does not migrate to the other zone. High-speed warm air currents do not mix with cold air currents.

[0189] The cell culture cassettes reside in the warm zone.

[0190] The reagent fluid reservoir resides in the cold zone.

[0191] The outer (additional) fluid bag resides in the cold zone.

[0192] As used herein, "resident" with respect to a "warm zone" or a "cold zone" means that the referenced structural element is only subjected to the atmosphere in that particular zone during closed operation of the culture system.

[0193] The Invention and Practice A general non-limiting summary of the invention is presented below. The summary summarizes exemplary practices of embodiments / aspects of the invention, thereby providing a constructive basis for variations and / or alternatives and / or different aspects / embodiments, some of which are described subsequently.

[0194] Figure 1 and Figure 2 An embodiment of the system of the present invention is shown in a closed configuration and an open configuration, respectively. Figure 1An embodiment of a cell culture and tissue engineering system 1 of the present invention is shown, in which a disposable cell culture cassette 2 has been installed and operated under automated conditions. The cell culture cassette 2 includes a bioreactor module 4 and an attached reagent fluid reservoir 6. The system 1 is similar to a "cocoon" and includes an inner shell 8 and an outer shell cover 10 covering an opening 12 of the inner shell. The outer shell cover 10 encloses the front opening 12 of the inner shell and is connected to the inner shell by rotatable connections (not shown) at both sides of the inner shell 8, so that the outer shell cover 10 can be rotated to open along the outer arc shape of the inner shell from a first closed position to an open position rotated around the connection point until the outer shell cover is nested in the inner shell (at Figure 2 ). The front opening 12 of the inner housing 8 includes a perimeter 14 having a U-shaped channel 16, the U-shaped channel including an expandable seal 17, so that when the system is closed, the expandable seal is actuated to expand and engage with the inner surface of the outer shell cover to seal the system in an airtight manner. The outer shell cover 10 includes a thermal unit 18 that acts as an external thermal barrier.

[0195] The cell culture box 2 is mounted against an operating robot interface 20 positioned in the opening 12 of the inner shell 8. The operating robot interface 20 further includes an associated robot and electronic device 22 in the inner shell. The cell culture box 2 is operationally constrained against the operating robot interface 20 and is locked in this position by a removable thermal barrier assembly 24 extending from the cell culture box 2 to the outer shell cover 10. The upper warm zone 26 and the lower cold zone 28 are generated by locking the cell culture box 2 to the operating robot interface 20 by the removable thermal barrier assembly 24. The bioreactor module 4 is fixed in the warm zone 26. The reagent fluid reservoir is fixed in the cold zone 28. An additional external cold reservoir 29 is positioned in the cold zone and adjacent to the reagent fluid reservoir of the installed cell culture box. This external cold reservoir 29 may contain one or more additional storage bags for collecting fluid waste and / or providing one or more culture solutions and one or more reagents required in addition. Thus, the cell culture system provides both a warm incubation environment suitable for bioprocesses (eg, about 37°C + / - 5°C) and a separate cool environment (eg, over 0°C to about 10°C) for enhancing reagent stability during reagent storage periods.

[0196] exist Figure 2In the embodiment of the present invention, the system 1 is in an open configuration exposing the cell culture box 2 for inspection and / or removal, and further shows a removable thermal barrier assembly 24 in an open raised position. The bioreactor module 4 of the cell culture box 2 is fluidly connected to a reagent fluid reservoir 6 by a matching port connection (not shown). The bioreactor module 4 supports the operational requirements of a bioprocess (such as cell culture or tissue engineering) and includes one or more bioreactors that can be operably connected in series. The reagent fluid reservoir 6 basically accommodates and stores the reagents required for the bioprocess that occurs in one or more bioreactors of the bioreactor module. The reagent fluid reservoir 6 includes a plurality of flexible reagent bags (not shown), each of which contains one or more of a culture medium, a growth factor, a medicament, a cell marker, and a waste product eliminated from the bioreactor module.

[0197] In operation, the outer housing cover 10 is rotated to an open position to enable access to the cassette 2 to install or remove a cell culture cassette, which is restrained against the operating robotic interface 20 by the structural configuration of the movable barrier assembly 24. When it is necessary to install / remove a cell culture cassette, the movable thermal barrier assembly 24 can be raised away from the cassette by an internal linkage mechanism that allows full access to the cell culture cassette. After the cassette 2 is installed, the thermal barrier assembly 24 is moved to the position as shown in FIG. Figure 1 The engaged (lowered) position is shown, thereby ensuring accurate alignment with the robot 7 and associated interface connections and forming separate warm and cold zones. When the cell culture cassette is installed and the removable thermal barrier is locked in place, the thermal barrier effectively forms a portion of the lower surface surrounding the warm zone of the cassette and the upper surface surrounding the cold zone of the cassette.

[0198] Figure 3A and 3B A front elevation view of an operating robotic interface 20 and a removable thermal barrier assembly 24 in an open raised position is shown. The operating robotic interface 20 includes a number of valve actuators and peristaltic pump connections with which the cell culture cassette is aligned and connected. The removable thermal barrier assembly 24 includes spaced apart lever arms 30 connected at either side of the operating robotic interface 20 that support a thermal platform 32 shown in a raised position. The underside of the thermal platform is shown as having a channel 33. Figure 3B, a thermal platform 32 and its associated upper armrest 34 are shown that closely conform to the dimensions of the mounted and locked cell culture cassette. A recess 36 is provided adjacent to the lever arm 30 that provides an operator with controlled access to open and extend the removable thermal barrier assembly 24. The upper armrest 34 provides a gripping device for the user to lock the cell culture cassette in place for operation or to unlock and raise the thermal platform 32. The thermal platform 32 is shown extending forward and radiating around the cell culture cassette to the outer housing cover (at Figure 1 The thermal platform has both a flexible outer seal at its front outer boundary to accommodate relative movement between the thermal platform and the outer housing cover (not shown) and a flexible inner seal directly adjacent to the cell culture cassette (not shown) to accommodate installation tolerances of the cell culture cassette. Both seals are elastomeric and impermeable to moisture and vapor.

[0199] Figure 3C The interior of an external cold reservoir 38 is shown, and the external cold reservoir has a baffle 40 to help prevent obstruction of air returning to the hot and cold assembly shown at the back of the external cold reservoir. The external cold reservoir 38 is positioned adjacent to the reagent fluid reservoir and both are located within the cold zone 28. The refrigerated space can contain a separate one or more external storage bags outside the reagent fluid reservoir (not shown). The baffle 40 is used to prevent any storage bag contained in the external cold reservoir from being isolated from the return air back to the cold tank. When the thermal platform is in a lowered locked position around the cell culture box, the lower side channel 33 is aligned with the baffle 40 to create a continuous structure for unimpeded cold air flow.

[0200] Figure 4 The temperature-controlled warm zone 26 and cold zone 28 of the cell culture system 1 are shown. The system 1 is shown in a closed position. The warm zone has a gas environment that is substantially separated from the gas environment of the cold zone. The warm zone includes a gas control system for gases such as oxygen, carbon dioxide and nitrogen. Nitrogen makes the concentration of oxygen and / or carbon dioxide lower than the ambient concentration. The warm zone 26 is generated by a heating assembly 41 positioned in the upper section of the inner shell. The heating assembly includes a large-capacity long-blade fan 42, which is operably connected to a heating air flow guide 44 to generate and guide a high-warm air flow path 45 (shown by arrows) so as to circulate around the bioreactor module 4. The large-capacity long-blade fan provides high-speed airflow to minimize spatial and temporal temperature non-uniformity in the warm zone. Due to the combination of laminar flow, minimum pressure drop and minimum airflow direction change, the long-blade fan 42 has a flow configuration that provides a high-speed airflow rate. Due to high convective heat transfer, high airflow speed promotes temperature uniformity, which inherently minimizes the surface temperature difference caused by competing heat sources in the warm zone.

[0201] Further, the continuous circulation of the high-speed warm air flow is assisted by the arc shape of the inner wall of the outer shell cover, and helps the circulation path of the high-speed warm air flow to be consistent and uniform. The warm zone is shown to be completely thermally separated from the cold zone. Competitive heat loads are placed on the warm zone through heat transfer with other areas within the cell culture system and through heat transfer to the surrounding ambient environment. The cold zone and typically the surrounding environment tend to operate at temperatures below the warm zone temperature set point, and therefore these factors represent heat losses in the warm zone. In contrast, specific electronic components within the robot architecture can operate at temperatures above the warm zone temperature, and therefore such components represent heat gains in the warm zone. The configuration and operation of the warm zone avoids the problem of heat transfer conditions that inherently drive temperature non-uniformity, so that the uniform warm zone maintains more consistent operating conditions for the ongoing biological process within the bioreactor module.

[0202] A consistent heating temperature can be selected for the warm zone as required by a particular biological process. The controlled temperature can be selected from 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C or higher.

[0203] Figure 5 Shown are the major functional components of the cold zone 28. The inner housing has a floor that supports a tray 46 having a forward cantilevered shelf 48 for supporting the bottom surface of a cell culture cassette, thereby forming a ducted airflow path beneath the tray to create a cold zone airflow path 49 (shown by arrows).

[0204] A hot-cold assembly 50 is included at the rear of the inner housing to generate and control a cold zone airflow path around the reagent fluid reservoir to enhance reagent stability. The hot-cold assembly 50 includes a cold slot array 52, each cold slot array including a plurality of fin-shaped cold slots 54 (see Figure 7 ), the fin-shaped cold slot receives heat from the cold zone airflow path through the cold slot fan 56. Peltier solid-state thermoelectric device (see Figure 7 ) transfers heat from the cold tank 54 to the adjacent hot tank 58. The hot tank fan 60 then exhausts the heat from the hot tank into the surrounding ambient air 62.

[0205] The cold tank fan 56 has an axial flow configuration that provides a high convective heat transfer coefficient due to turbulence at the hot tank fins 58, thereby producing a minimum temperature difference between the airflow in the cold storage zone and the cold tank. The reagents in the cold storage zone are cooled by being surrounded by the cold air flow path. The temperature in the cold zone may not be as uniform as in the warm zone because the key criterion for enhancing reagent stability is overall temperature reduction rather than precise temperature accuracy.

[0206] Figure 6Shown is a reagent fluid reservoir 6 in which multiple reagents are stored in separate fluid containers such as flexible reagent bags (not shown). The reagent bag is constrained in a fluid bag container 64. The reagent fluid reservoir 6 is connected to the bioreactor module by helping to maintain a port connection 67 and a snap tab 66 attached to the bioreactor module. The reagent bag is isolated by a box thermal insulation 68 to prevent heat from migrating from a warm zone. The refrigerated temperature of the reagent bag is maintained by circulating cold air around and inside the reagent fluid reservoir, which comprises an air flow above the reagent bag through the reagent fluid reservoir open air conduit 70 on the front wall and rear wall of the top portion of the adjacent reagent fluid reservoir. The top (top plate portion) of the reagent fluid reservoir has a baffle (not shown) extending downwardly into the reservoir, which helps to guide cold air flow above the reagent bag and across the reagent bag, and allows air flow to move through an external cold reservoir guided by baffle 40 therein to flow back to the cold tank array unhindered.

[0207] The cold zone is structured so as to create a minimally obstructed ducted / guided airflow therein so as to provide a continuous flow of cold air that will circulate throughout the cold zone, providing a cold airflow under and around the reagent fluid reservoirs and having the cold airflow penetrate the top portion of the reagent fluid reservoirs through vents for cold air to flow through the reservoirs and directly across the fluid bags and exit through further vents into an external cold reservoir where it is directed through baffles back to the cold tank of the hot and cold assembly to dissipate heat. The arrangement of channels, baffles, vents, insulation, and seals of the hot platform and underside channels together ensure that the cold air is maintained in the cold zone with minimal obstruction to the flow of the cold air so as to remove heat and circulate the cold air.

[0208] Figure 7 The structure of a Peltier solid-state thermoelectric device is shown, which pumps heat from the cold tank to the hot tank for subsequent transfer of heat to the surrounding environment. Compared with other traditional refrigeration methods such as vapor compression refrigeration, the Peltier solid-state device is preferred because the solid-state device is compact and has no moving parts that are prone to failure. A thermal insulator is provided to prevent heat from returning from the hot tank to the cold tank, because such heat return will impair thermal efficiency. The cold tank has an adjacent monument-shaped object (monument) incorporated therein, thereby forming an extension of the cold tank and representing a conductive path for heat to travel from the cold tank to the Peltier solid-state device. The subsequent heat delivered from the Peltier solid-state device to the hot tank is the sum of the heat pumped from the cold tank and the power consumed by the Peltier solid-state device. In this way, the accumulated heat delivered to the hot tank is significantly greater than the heat removed from the cold tank.

[0209] In contrast to hot sinks, it is advantageous to incorporate the monument into a cold sink because less heat transfer is then required through the monument. Thus, the temperature differential across the monument is significantly less than that present when the monument is located on a hot sink, thereby reducing heat losses. For Peltier solid-state devices, the coefficient of performance (the ratio of heat pumped to power consumed) increases as the temperature differential decreases. Thus, positioning the monument on a cold sink provides a significant gain in coefficient of performance relative to the alternative of incorporating the monument as part of a hot sink.

[0210] The cold slot array 52 comprises a plurality of individual cold slots 54 relative to a hot slot 58 which is integral. To ensure close thermal contact between the monument of the cold slot 54, the Peltier solid state device and the hot slot 58, the cold slot is divided into functional units (cold slot plus axial flow fan), whereby each functional cold slot unit is in close contact with the integral hot slot by using an array of spring compression bolts 70 (through the Peltier solid state device). The spring compression is achieved by using a coil spring 72. The key advantage of the spring compression of the cold slot to the integral hot slot is that thermal and / or production distortions are self-correcting because each cold slot assembly can be independently aligned and achieve a uniform compressive load against the associated Peltier solid state device and forward against the integral hot slot. This self-compensating compressive load maximizes the effectiveness of heat transfer from the individual cold slots to the integral hot slot.

[0211] When the cell culture system is turned on, the cold zone inevitably exchanges air with the surrounding environment. Therefore, when the cell culture system is subsequently turned off, air from the surrounding environment is entrained within the cold zone. If the humidity of the incoming ambient air causes the dew point to be higher than the final temperature of the cold zone, moisture from the ambient air will condense. The resulting inevitable condensation may create undesirable areas of moisture accumulation within the cold zone, as such accumulation may create potential sites for microbial contamination. Condensation naturally begins and continues on the cold tank, as the surfaces of this component are the coldest surfaces within the refrigerated zone. In order to automatically manage and thereby remove the complications of condensation, the cell culture system employs a condensation control mechanism 73 within the cold zone, Fig. 8A The positioning of the condensation control mechanism is shown in FIG. Figure 8B The condensation control mechanism 73 shown more closely in FIG. 7 includes a moisture transport material 74. The moisture transport material 74 forms condensation on the cold tank and the condensation collects condensate as it travels along the fins of the cold tank through the effects of gravity and airflow down through the cold tank fins. The moisture transport material protrudes from the cold tank to the hot tank through a dedicated conduit 76, with the transfer of moisture through the heat of the hot tank followed by subsequent and continuous evaporation into the surrounding environment. This condensation management strategy does not require moving parts or additional electricity.

[0212] Good manufacturing practices require maintenance and cleaning of cell culture systems. Fig. 9The hot and cold assembly 50 is shown to be unlatched and supported on hinges 51 to hang open to enable servicing and cleaning. The hot and cold assembly is released from the normal operating position by activation of latch 78 and subsequent downward rotation about the hinge point. When the hot and cold assembly is returned to the normally closed operating configuration, the refrigerated power connector 82 provides a reliable electrical connection for data and power.

[0213] Fig.10 The cold sink fan 56 is further illustrated as including an assembly 84 that can be decoupled from and removed from the cold sink to enable further careful cleaning of the underlying cold sink.

[0214] Fig.11 It is further shown how the heat sink fan 60 and associated shroud may be hingedly withdrawn upward and away from the heat sink, thereby enabling further careful cleaning of the heat sink below.

[0215] In embodiments, the use of a hollow shaft enables the interior of a cell culture system to be connected to the exterior of the system, allowing for the creation of a unique third control zone within the shaft to enable processes to be run at temperatures other than culture or refrigeration temperatures. Such embodiments may be used for process steps that may benefit from an intermediate temperature and may be controlled in this transition zone.

[0216] In additional embodiments, thermal windows may be included in the warm and / or cold zones, comprising dual liquid crystal (LCD) windows (or functional equivalent) incorporated into the housing, the LCD windows allowing: viewing of internal box action when the LCD is transparent; not conducting harmful light degradation of the reagent when the LCD is activated and opaque; and a thermal barrier created by the dual LCD walls forming an entrained air space.

[0217] In an embodiment, the individual internal air flows may be connected to a central air flow management system capable of controlling multiple production units.

[0218] In other embodiments, air filtration may be incorporated into the air circulation path, and such filters may be disposable after each treatment or other reasonable period of time.

[0219] It will be understood by those skilled in the art that materials used to manufacture components of the systems described herein are selected to maximize thermal insulation properties, where feasible, without compromising the primary function of the components in terms of biocompatibility (e.g., non-toxic, USP Class VI compliant) or structural properties (e.g., strength, stiffness, toughness, and weight). While the systems are shown as being generally configured in a cocoon shape, this can vary, and the dimensions can vary, as long as the shape maintains warm and cold airflow paths therein.

[0220] In addition, the cell culture and tissue engineering system of the present invention includes various sensors associated with cold zones, hot zones, cell culture boxes, heating assemblies, hot and cold assemblies and / or positioned therein and associated with operating robotic interfaces and associated internal robotics and electronic devices and further associated with computer devices.

[0221] It should be understood that various changes and modifications to the currently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the subject matter of the present invention and without reducing its expected advantages. Therefore, the appended claims are intended to cover such changes and modifications.

Claims

1. A method for preparing a controlled thermal environment for a bioprocess within an automated cell culture cassette, the automated cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the method comprising: raising the movable thermal barrier assembly to an unlocked, raised position; installing the automated cell culture cassette into a housing having an outer housing cover and an inner shell and abutting against an operator robot interface positioned within the inner shell; lowering the movable thermal barrier assembly to a locked lowered position to constrain the automated cell culture cassette against the manipulation robotic interface and to form a portion of a lower surface boundary of a warm zone and a portion of an upper surface boundary of a cold zone; and A warm gas flow path around the bioreactor module and a cold gas flow path around the reagent fluid reservoir are circulated. 2 . The method of claim 1 , wherein the circulating comprises circulating the warm airflow path through a heating assembly in an upper section of the inner housing.

3. The method of claim 2, wherein the heating assembly includes a high-volume long-blade fan operably connected to a heated airflow director, and the circulating produces a consistent and uniform airflow through the high-volume long-blade fan and the heated airflow director.

4. The method of claim 2 further comprising maintaining a substantially uniform temperature throughout the warm zone by heating the assembly.

5. The method of claim 1 further comprising creating the cold gas flow path around the reagent fluid reservoir by a hot and cold assembly at a rear portion of the inner housing.

6. The method of claim 5, wherein the hot and cold assembly creates the cold airflow path via a cold slot array comprising a plurality of cold slots compressed to a hot slot using a Peltier device to transfer heat to the hot slot.

7. A cell culture system for housing and operationally supporting an automated cell culture cassette for use in a bioprocess, the automated cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the system comprising: shell; Operate the robot interface; as well as a movable thermal barrier assembly having an unlocked raised position for mounting the automated cell culture cassette and a locked lowered position for thermally isolating a warm zone from a cold zone, the movable thermal barrier assembly operatively constraining the automated cell culture cassette against the operating robotic interface; and Wherein when in the locked lowered position, the movable thermal barrier assembly forms a portion of a lower surface boundary of the warm zone and a portion of an upper surface boundary of the cold zone.

8. The cell culture system of claim 7, wherein the warm zone is configured to circulate a warm air flow path around the bioreactor module. 9 . The cell culture system of claim 8 , wherein a heating assembly is disposed in an upper section of the housing, and the heating assembly generates the warm air flow path directed at the bioreactor module.

10. The cell culture system of claim 7, wherein the cold zone is configured to circulate a cold gas flow path around the reagent fluid reservoir.

11. The cell culture system of claim 7, wherein the operating robotic interface comprises valve actuators, peristaltic pump connectors and associated control systems to mate with corresponding connections on the automated cell culture cassette.

12. The cell culture system of claim 7, wherein the movable thermal barrier assembly comprises a pair of spaced-apart lever arms internally connected at either side of the manipulation robot interface, the pair of spaced-apart lever arms supporting a central thermal platform with associated upper handrests.

13. The cell culture system of claim 7, wherein the heating assembly creates a warm air flow path directed at the bioreactor module.

14. The cell culture system of claim 7, wherein the hot and cold assembly generates and controls a cold gas flow path around the reagent fluid reservoir.

15. The cell culture system of claim 7, wherein the bioreactor module is fluidly connected to the reagent fluid reservoir.

16. The cell culture system of claim 7, wherein the cold zone further comprises a condensation control.

17. The cell culture system of claim 7, wherein the cold zone comprises a cold reservoir positioned adjacent to the reagent fluid reservoir.

18. The cell culture system of claim 17, wherein the cold reservoir comprises at least one baffle.

19. The cell culture system of claim 18, wherein the movable thermal barrier assembly comprises at least one channel, and when the movable thermal barrier assembly is in the locked lowered position, the at least one baffle of the cold reservoir is aligned with the at least one channel of the movable thermal barrier assembly.

20. A cell culture system for housing and operationally supporting an automated cell culture cassette for use in a bioprocess, the automated cell culture cassette comprising a bioreactor module and a reagent fluid reservoir, the system comprising a thermoelectric device for pumping heat from a cold tank to a hot tank for subsequent transfer of the heat to the surrounding environment; a thermal insulator configured to prevent heat from returning from the hot tank to the cold tank; an incorporated monument adjacent to the cold sink and further forming an extension of the cold sink, the incorporated monument providing a conductive path for heat to travel from the cold sink to the thermoelectric device; The heat pumped from the thermoelectric device is the sum of the heat pumped from the cold sink and the electricity consumed by the thermoelectric device.

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