Cell culture and tissue engineering system with controlled environmental zones

Abstract

To provide tissue engineering systems for automated cell culture and tissue engineering that include uniform operational environmental zones to provide more consistent biological processes.SOLUTION: An automated cell culture and tissue engineering system 1 comprising defined and separate environmental zones improves control and maintenance of an internal environment of the system such that a temperature, an air flow and gases surrounding a bioreactor module 4 form one zone that is maintained separately from a second zone formed surrounding a reagent fluid reservoir 6. The system further comprises means for elimination and / or management of condensation within the second zone of the system.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to tissue engineering systems and methods for automated cell culture and tissue engineering, and the system includes a uniform operating environment zone for providing a more consistent biological process. Such systems and methods are used in various clinical and laboratory environments.

Background Art

[0002] Automation of cell culture is a desirable trend in order to provide scalability for mass production, reduction of variability in culture, reduction of the risk of culture contamination, and many economic cost and time frame advantages related to the generation of cell or tissue-based implants for clinical treatment and cell-based assay systems for diagnostic evaluation.

[0003] However, in more complex biological processes, such as automated cell culture protocols used for autologous patient treatment, more complex and accurate operation control may be required. For autologous patient treatment, since it is important to succeed in biological culture, each operation mode needs to strictly follow a specific protocol. For example, the initial culture medium can be programmed to be supplied to the cell culture at a suitable temperature such as 37°C, but it is more difficult to strictly maintain this temperature throughout the entire cell culture process that can last for several days. Many automated systems that currently provide cell incubation capabilities have thermal problems such as attempts to achieve and maintain overall thermal uniformity, changes in air temperature due to external operator access, maintenance of refrigerated reagent storage, and the occurrence of condensation inside the enclosure that can lead to potential microbial contamination.

[0004] While refrigerated storage is desirable to avoid reagent degradation, it can negatively impact the thermal performance and stability of cell bioreactors, potentially making it more difficult to maintain the desired high temperatures required for cell culture. Furthermore, reducing the storage environment temperature for process reagents to refrigerated levels inevitably leads to condensation. Condensation forms when air is cooled below its dew point. In particular, water vapor in the air condenses into liquid on surfaces that are colder than their surroundings. When the system is opened to fill reagents, warm, humid air may come into contact with colder surfaces in the storage environment. Depending on the amount of condensation, this can pose problems in handling or contamination issues in the storage environment.

[0005] Furthermore, since gases such as CO2 are used to regulate the pH of active cultures, gas concentrations can significantly affect biological performance. In culture systems, if access to the buffering role of supplied CO2 is limited, non-uniform pH control may occur throughout the culture zone. Therefore, uniformity of gas concentration is also necessary in cell culture and tissue engineering systems.

[0006] Therefore, there is a need for continuous improvement of the characteristics of automated cell culture and tissue engineering systems, such as providing higher fidelity in environmental control within the system.

[0007] The background discussions in this specification are included to explain the context of the inventions described herein. It should not be construed that any of the referenced materials were published, known, or part of the general knowledge as of the priority date of any of the claims. [Overview of the project]

[0008] This specification describes cell culture and tissue engineering systems and methods that include a uniform operating environment to provide more consistent control over biological processes. A uniform operating environment with respect to airflow, temperature, gas control, and condensation control is integrated into the systems and methods described herein.

[0009] The automated cell culture and tissue engineering systems described herein are configured to generate, regulate, and maintain controlled and isolated environmental zones for the proper operation of cell culture cassettes and, consequently, ongoing biological processes. Temperature, humidity levels, and gas concentrations are controlled. Temperature and humidity fluctuations are minimized.

[0010] Notably, within the cassette receiving area of ​​the system in which the cell culture cassette is installed and operated, two separate temperature zones are created and maintained. During system operation, the components of the reagent fluid reservoir are kept in the cold zone, while the components of the cassette's bioreactor module are kept in the warm zone. The warm zone includes a recirculating warm high-airflow path surrounding the bioreactor module, generated by an adjacent separate heating assembly. The warm circulating air also permeates the culture surface of the cassette through ventilation slots, a similar concept to the slots in the reservoir. The cold zone includes a circulating cold airflow path with ducts that surrounds and partially permeates (flows through) a portion of the reagent fluid reservoir. The cold airflow path is generated by an adjacent cold thermal assembly. The cold zone also includes condensation control means for controlling and removing condensation within the cold zone.

[0011] Both the warm and cold zones further include independently controlled gas environments. In the warm zone, this helps to provide specific gas concentrations. Gas concentrations affect dissolved gases present in the cell culture medium, such as oxygen and carbon dioxide, through gas exchange at the fluid surface. The adjustment and / or maintenance of dissolved gases has implications from a bioperformance standpoint, including aspects such as oxygen supply and maintenance of a target pH for cell culture. Since the control of dissolved gases in the culture medium is achieved through the recirculation of the culture medium across the gas exchange membrane (e.g., silicone tubing), the concentration of dissolved gases in the culture fluid responds to the concentration gradient between the fluid and the surrounding gaseous environment. By adjusting the environment, the levels of dissolved gases in the culture fluid are simultaneously adjusted.

[0012] The automated cell culture and tissue engineering system consists of a movable thermal barrier assembly, which, during the installation of the cell culture cassette, locks the cassette within the system's operating interface, thereby forming a warm zone and a cold zone and maintaining the thermal and physical separation of these two zones. An insulating mechanism is provided so that the warm and cold zones are insulated from each other and do not affect the properties of either the cell culture or the stored reagents. The movable thermal barrier assembly creates and defines a portion of the boundary of each of the warm and cold zones.

[0013] The system's heating and cooling assemblies, as well as the operating robot, are contained / positioned independently of the system's cassette receiving area, which is beneficial as it does not interfere with the warm airflow path or the cold airflow path and its function. Furthermore, the heating assembly is isolated to be separated and insulated from the cooling assembly. The system's heating assembly is configured to continuously generate and regulate the temperature of the warm, high-temperature airflow path for several days as needed, and can more quickly adjust any temperature drops that may occur due to the influx of cooler room-temperature air during system opening and inspection. The system's configuration and geometry help to provide a warm airflow path that is directed only to the bioreactor module and continuously circulates around and through it. The cold thermal assembly is configured to continuously draw airflow in the cold zone through the cold thermal assembly, removing heat continuously as the air temperature decreases. The cold zone is configured to have channeled airflow. In other words, the cryogenic airflow path, following structural features including airflow channels, airflow shutoffs, and airflow vents, helps to deliver cooled air through the cryogenic zone to and partially through the reagent fluid reservoir, and further helps to pass it through an optional adjacent cold reservoir outside the reagent fluid reservoir. These structural features help to prevent and minimize any blockage of return air (by the filled fluid bag) moving toward the cold thermal assembly.

[0014] By providing separate warm and cold zones, the system is exposed to condensation only within the cold zone, which has mechanisms to effectively prevent, control, and remove condensation, thus minimizing the amount and location of condensation that may form within the system.

[0015] Aspects of the present invention include automated cell culture and tissue engineering systems. The system includes a closed automated cell culture cassette for one or more of the following: cell source isolation, cell proliferation / expansion, cell differentiation, cell isolation, cell labeling, cell purification, cell washing, and cell seeding onto a scaffold for tissue formation (product formation). In some aspects, the cells are mammalian cells. In further aspects, human cells. The type of cell or tissue is not limited. In one non-limiting example, pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, may be cultured and expanded for cell replacement therapy.

[0016] The automated cell culture cassette is in the form of a closed, single-use, disposable cassette, which includes one or more sterile bioreactor modules fluidly connected to a reagent fluid reservoir. The sterile bioreactor is filled with the desired cells and / or tissues, and the sterile bioreactor is connected to a reagent fluid reservoir that is pre-filled to contain the required fluid reagents. Sterility of the cassette is maintained throughout.

[0017] Automated cell culture cassettes are employed to operate within automated cell culture and tissue engineering systems, along with dedicated software programs for supplying and tracking the desired process(s). Preferred and non-limiting automated cell culture and tissue engineering systems are described in U.S. Patents No. 8,492,140, ​​No. 9,701,932, No. 9,534,195, No. 9,499,780, and No. 9,783,768 (the content of each of these U.S. patents is incorporated in whole by reference).

[0018] Sensors embedded within the cell culture cassette provide real-time biofeedback, enabling automated adjustment of bioprocessing to respond to natural changes in the behavior of the cell source. The entire bioprocess is contained within a disposable cassette, ensuring maximum safety for patients and operators and streamlining logistics. Furthermore, to successfully support multiple biological steps within the cell process sequence, the cassette bioreactor(s) are integrated in combination with biosensor feedback within one or more interconnected bioreactors, providing a highly intuitive system with precise control at each cell and tissue stage. This comprehensive level of automation enables technically feasible and economically scalable cell production capabilities at patient scale, allowing for streamlined production of cell therapies under good Good Manufacturing Practice (GMP) conditions, thus addressing the challenges inherent in various autologous and allogeneic clinical applications of cell and tissue therapies.

[0019] Advantageously, the cell culture cassette is installed and operationally held within the cell culture and tissue engineering system housing such that the bioreactor module resides only in a separate warm zone and the reagent fluid reservoir resides only in a separate cold zone. Installation of the cell culture cassette into the system is performed via the activation and locking of a movable thermal barrier assembly, which environmentally separates the first thermal zone from the second thermal zone with respect to temperature, gas, and humidity. The cell culture cassette includes a bioreactor module with an attached reagent fluid reservoir. While this combination with a single cell culture cassette is more user-friendly, it presents more challenges compared to a simpler design based on physically separate bioreactor modules and reservoirs that can reside in separate environmental zones, as it involves creating separate and distinct environmental zones for the bioreactor module and reagent fluid reservoir.

[0020] In the present invention, dedicated airflow paths are provided within the warm zone and the cold zone, and a controlled temperature / gas distribution is ensured around the bioreactor module and the reagent fluid reservoir that contain the cell culture(s) so as to eliminate the distortion of uniformity in each zone.

[0021] In an aspect of the present invention which is a cell culture and tissue engineering system, the system includes two separate and independent hot airflows, a first airflow including a high-speed warm airflow for guiding around the bioreactor module of the cell culture cassette and its surroundings, and a second airflow including a cold airflow for circulating around and through the reagent fluid reservoir operably connected to the bioreactor module. The first airflow and the second airflow are substantially isolated (in an embodiment, these zones are not airtight sealed from each other) and cannot be mixed.

[0022] In an aspect, the first airflow and the second airflow are included within the cell culture cassette receiving region of the system.

[0023] In an aspect, the cell culture cassette receiving region is isolated from the heating assembly and the cooling assembly operable in the system.

[0024] In an aspect, the cell culture cassette receiving region defines a warm zone including a high-speed warm airflow.

[0025] In an aspect, the warm zone includes a substantially uniform temperature within the warm zone.

[0026] In an aspect, the cell culture cassette receiving region defines a cold zone including a cold airflow, and the cold zone is positioned below the warm zone.

[0027] In an aspect, the cold zone includes means for reducing or eliminating condensation.

[0028] In an aspect, the thermal platform separates the warm zone from the cold zone. In an aspect, the thermal platform includes a seal.

[0029] In an aspect of the present invention which is a method for maintaining control of the thermal environment for a biological process within a bioreactor module of a cell culture cassette, the method includes inducing a first air flow including a high-speed warm air flow around the bioreactor module and its surroundings, and simultaneously circulating a low-temperature air flow around and through a reagent fluid reservoir operably connected to the bioreactor module, where the first air flow and the second air flow are isolated and cannot mix.

[0030] According to an aspect of the present invention which is a cell culture and tissue engineering system including a thermal zone architecture for providing a more consistent controlled environment for promoting a biological process, the system includes a separate warm zone compartment that holds the bioreactor module while continuously circulating a high-speed warm air flow path induced around and around the bioreactor module, and a separate cold zone compartment that holds a reagent fluid reservoir functionally connected to the bioreactor module while continuously circulating a low-temperature air flow path around and through the reagent fluid reservoir.

[0031] In an aspect, the separate cold zone compartment further includes means for reducing or eliminating condensation.

[0032] In an aspect, the warm zone compartment further includes a gas environment different from that of the cold zone compartment.

[0033] According to a further aspect of the present invention, which is an automated system for cell culture and tissue engineering that holds a cell culture cassette in two different temperature and gas-controlled environments, the bioreactor components of the cassette are operably held in a substantially homogeneous warm airflow zone having a controlled gas concentration, and the reagent fluid reservoir components of the cassette are located in a substantially homogeneous cold airflow zone, the warm airflow zone being isolated separately from the cold airflow zone, and the cold airflow zone further includes means for preventing or eliminating undesirable moisture accumulation within the cold airflow zone.

[0034] According to a further aspect of the present invention, which is a cell culture and tissue engineering system for receiving and operationally supporting an automated cell culture cassette in a more consistently controlled environment for biological processes, the cell culture cassette comprises a bioreactor module and a reagent fluid reservoir, and the system is A warm zone configured to circulate warm airflow paths surrounding the bioreactor module, A low-temperature zone configured to circulate a low-temperature airflow path surrounding the reagent fluid reservoir, The system includes a movable thermal barrier assembly for thermally separating the warm zone from the cold zone when installing the cell culture cassette, fixing the bioreactor module only within the warm zone, and fixing the reagent fluid reservoir only within the cold zone.

[0035] According to a further aspect of the present invention, which is a cell culture cassette, the cell culture cassette is - A bioreactor module with a reagent fluid reservoir attached to the bottom, - A reagent fluid reservoir including a fluid bag container having open air ducts located on the front and rear walls of the reservoir.

[0036] In one embodiment, the fluid bag container includes a roof section and a floor section, the roof section including a baffle extending downward.

[0037] In one embodiment, the cassette further includes a thermal insulating layer positioned between the bottom of the bioreactor module and the roof of the fluid bag container, the thermal insulating layer insulating against heat transfer from the bioreactor module.

[0038] In one embodiment, the reagent fluid reservoir is attached via a port connection located on the roof of the fluid bag container, which is not blocked by the thermal insulation layer.

[0039] In one embodiment, the reagent fluid reservoir further includes a snap tab for attachment to a bioreactor module.

[0040] According to a further embodiment of the present invention, which is a reagent fluid reservoir for connection to a bioreactor module, the reagent fluid reservoir includes a fluid bag container having open air ducts located on the front and rear walls of the reservoir.

[0041] In one embodiment, the fluid bag container includes a roof section and a floor section, the roof section including a baffle extending downward.

[0042] According to one aspect of the present invention, an automated cell culture and tissue engineering system for receiving and operationally supporting an automated cell culture cassette in a more consistently controlled environment for biological processes, the cell culture cassette comprises a bioreactor module and a reagent fluid reservoir, and the system is A warm zone is configured to circulate tangential warm airflow paths surrounding the bioreactor module, A low-temperature zone configured to circulate a tangential low-temperature airflow path surrounding the reagent fluid reservoir, The system includes a movable thermal barrier assembly for thermally separating the warm zone from the cold zone when installing the cell culture cassette, fixing the bioreactor module only within the warm zone, and fixing the reagent fluid reservoir only within the cold zone.

[0043] In any of the embodiments described above, the system and method may include one or more controllers, as well as associated software, sensors, and user interfaces.

[0044] The following are non-restrictive examples. 1A. A cell culture and tissue engineering system comprising two separate and independent hot airflows, a first airflow including a fast warm airflow for guiding a cell culture cassette in and around the bioreactor module, and a second airflow including a cold airflow for circulating around and around a reagent fluid reservoir operably connected to the bioreactor module, A system in which the first airflow and the second airflow are isolated and cannot be mixed. 1B. The system according to claim 1A, wherein the first airflow and the second airflow are contained within the cell culture cassette receiving region of the system. 1C. The system according to claim 1B, wherein the cell culture cassette receiving region defines a warm zone including a high-speed warm airflow. 1D. The system according to claim 1C, wherein the warm zone includes a substantially uniform temperature within the warm zone. 1E. The system according to claim 1B, 1C, or 1D, wherein the cell culture cassette receiving region defines a low-temperature zone including a low-temperature airflow, and the low-temperature zone is positioned below a warm zone. 1F. The system according to claim 1E, wherein the low-temperature zone includes means for reducing or eliminating condensation. 1G. The system according to claim 1F, wherein the thermal platform separates a warm zone from a cold zone. 1H. A method for maintaining a controlled thermal environment for a biological process within a bioreactor module of a cell culture cassette, wherein the method is This includes inducing a first airflow containing a high-speed warm airflow around the bioreactor module and its surroundings, and simultaneously circulating a low-temperature airflow around a reagent fluid reservoir operably connected to the bioreactor module and the low-temperature airflow passing through it, A method in which the first airflow and the second airflow are isolated and cannot be mixed. 1J. The method according to claim 1H, using the system described in any one of claims 1A to 1H. 2A. A cell culture and tissue engineering system comprising a thermal zone architecture for providing a more consistent and controlled environment to facilitate biological processes, wherein the system While continuously circulating the bioreactor module and the high-speed warm airflow path induced around it, a separate warm zone section containing the bioreactor module is provided. A system comprising a separate cryogenic zone compartment that continuously circulates a cryogenic airflow path around and through a reagent fluid reservoir, while holding a reagent fluid reservoir functionally connected to a bioreactor module. 2B. The system according to claim 2A, wherein a separate low-temperature zone compartment further includes means for reducing or eliminating condensation. 2C. The system according to claim 2A or 2B, wherein the warm zone section further includes a gas environment different from that of the cold zone section. 2D. A method for maintaining a controlled environment for a biological process within a bioreactor and for maintaining the fluids required for the bioreactor at a cooling temperature for stability, comprising the use of a system according to any one of claims 2A to 2C. 3A. An automated system for cell culture and tissue engineering that holds a cell culture cassette in two different temperature and gas-controlled environments, wherein the bioreactor components of the cassette are operably held in a substantially homogeneous warm airflow zone having a controlled gas concentration, and the reagent fluid reservoir components of the cassette are located in a substantially homogeneous cold airflow zone, the warm airflow zone being isolated separately from the cold airflow zone, and the cold airflow zone further includes means for preventing or eliminating undesirable moisture accumulation within the cold airflow zone. 3B. A method for maintaining a controlled environment for a biological process within a bioreactor and for maintaining the fluids required for the bioreactor at a cooling temperature for stability, comprising the use of the system described in claim 3A. 1. A cell culture and tissue engineering system for receiving and operationally supporting an automated cell culture cassette in a more consistently controlled environment for biological processes, wherein the cell culture cassette comprises a bioreactor module and a reagent fluid reservoir, and the system A warm zone configured to circulate warm airflow paths surrounding the bioreactor module, A low-temperature zone configured to circulate a low-temperature airflow path surrounding the reagent fluid reservoir, A cell culture and tissue engineering system comprising: a movable thermal barrier assembly for thermally separating the warm zone from the cold zone when a cell culture cassette is installed, fixing the bioreactor module only within the warm zone, and fixing the reagent fluid reservoir only within the cold zone. 2. The cell culture and tissue engineering system according to claim 1, further comprising condensation control means for minimizing and eliminating undesirable moisture accumulation within the low-temperature zone. 3. The cell culture and / or tissue engineering system according to claim 1 or 2, wherein the warm zone and the cold zone each include a substantially isolated gas environment. 4. The cell culture and tissue engineering system according to claim 1, 2, or 3, wherein the system has a housing comprising an outer shell cover and an inner shell body, and the outer shell cover encloses the front opening of the inner shell body when the system is closed. 5. The cell culture and tissue engineering system according to claim 5, wherein the outer shell cover is connected to the inner shell body for rotation while the inner shell body remains stationary, the outer shell cover rotates along the outer arc of the inner shell body to expose the front opening of the inner shell body for access to the inner shell body, and stops rotating when the outer shell cover retracts the inner shell body. 6. The cell culture and tissue engineering system according to claim 4 or 5, wherein the front opening of the inner shell body includes a periphery having an expandable sealing means for seal engagement with the inner surface of the outer shell body when the system is closed. 7. The cell culture and tissue engineering system according to claim 6, wherein the peripheral portion includes a U-shaped channel for holding the expandable sealing means. 8. The cell culture and tissue engineering system according to claim 7, wherein the sealing means includes an elastomer tube fitted into a U-shaped channel, the elastomer tube being substantially flat when the system is open, and when the system is closed, the sealing means acts to cause a displacement of the seal, introducing an expansion thickness pressure into an inflatable seal cavity that provides a secure seal between the outer shell cover and the front opening of the inner shell body. 10. The cell culture and tissue engineering system according to claim 9, wherein the outer shell cover is configured as an arc-shaped body that helps guide a circulating warm airflow path surrounding the bioreactor module and a circulating cold airflow path surrounding the reagent fluid reservoir. 11. The cell culture and tissue engineering system according to claim 10, wherein the arc-shaped body includes a plurality of thermal cells that function as an external heat barrier. 12. The cell culture and tissue engineering system according to any one of claims 4 to 11, wherein a movable thermal barrier assembly is positioned on a robotic interface located within a forward opening of the inner shell body. 13. The cell culture and tissue engineering system according to claim 12, wherein the operating robot interface is connected to an associated internal robot and includes a valve actuator, a peristaltic pump, and an associated control system for mating with a corresponding connection on a cell culture cassette. 14. The movable heat barrier assembly - A cell culture and tissue engineering system according to any one of claims 4 to 11, comprising a pair of spaced-apart lever arms internally connected at either side of a working robot interface supporting a central thermal platform with associated upper handrails. 15. The cell culture and tissue engineering system according to claim 14, wherein the movable thermal barrier assembly is movable from a first raised position for installing a cell culture cassette to a second lowered position for locking and holding the cell culture cassette against a robotic interface for precise alignment and retention, and simultaneously isolating the warm zone from the cold zone. 16. The cell culture and tissue engineering system according to claim 15, wherein the upper handrail portion is sized to closely conform to the dimensions of a cell culture cassette and to provide a gripping means for locking the cassette in place for operation or for raising a thermal platform. 17. The cell culture and tissue engineering system according to claim 14, 15, or 16, wherein a recess is provided on the operating robot interface adjacent to the lever arm to provide operator control access for releasing the assembly. 18. The cell culture and tissue engineering system according to any one of claims 14 to 17, wherein the thermal platform extends forward and radiates from around the cell culture cassette to the outer shell cover. 19. The cell culture and tissue engineering system according to claim 18, wherein the thermal platform comprises a flexible outer seal at its forward outer boundary to accommodate relative motion between the thermal platform and the outer shell cover, and a flexible inner seal directly adjacent to the cell culture cassette. 20. The cell culture and tissue engineering system according to claim 19, wherein a flexible internal seal accommodates the mounting tolerance of the cell culture cassette. 21. The cell culture and tissue engineering system according to claim 19 or 20, wherein the flexible outer seal and the flexible inner seal are elastic and impermeable to vapor. 22. The cell culture and tissue engineering system according to claim 21, wherein the flexible outer seal and the flexible inner seal are wiper-type seals. 23. The cell culture and tissue engineering system according to any one of claims 14 to 22, wherein, when in a locked position, the thermal platform includes a portion of the lower surface boundary of the warm zone and a portion of the upper surface boundary of the cold zone. 24. The cell culture and tissue engineering system according to any one of claims 14 to 18, wherein the inner shell body includes a floor portion that supports a tray having a forward cantilever-shaped shelf portion that supports a portion of the bottom surface of a cell culture cassette. 24a. The cell culture and tissue engineering system according to claim 24, wherein the airflow duct is defined by a floor section and a tray. 25. A cell culture and tissue engineering system according to any one of claims 4 to 24, wherein a heating assembly is provided in the upper section of the inner shell body and generates warm air that is directed to the bioreactor module. 26. The cell culture and tissue engineering system according to claim 25, wherein the heating device includes a high-capacity linear fan operably connected to a heated airflow induction device for generating and inducing a high-speed warm airflow path. 27. The cell culture and tissue engineering system according to claim 26, wherein a high tangential warm airflow path surrounding the bioreactor module promotes temperature uniformity in the warm zone and, consequently, uniformity of biological processes within the bioreactor. 28. The cell culture and tissue engineering system according to claim 27, wherein a tangential high-speed warm airflow path surrounding the bioreactor module helps maintain the internal temperature of the bioreactor at approximately 35°C, 36°C, 37°C, 38°C, or 39°C. 28a. The cell culture and tissue engineering system according to claim 28, wherein the gas is selectively introduced into a warm zone. 28b. The cell culture and tissue engineering system according to claim 28a, wherein the gas comprises one or more of oxygen, carbon dioxide, and nitrogen. 28c. The cell culture and tissue engineering system according to claim 28a or 28b, wherein the warm zone includes sensors in the warm airflow path for monitoring gases. 28d. The cell culture and tissue engineering system according to claim 28c, wherein the sensor is operably connected to a PID (proportional-integral-derivative) controller that provides gas feedback control. 29. A cell culture and tissue engineering system according to any one of claims 4 to 28, wherein a cold thermal assembly forms the back surface of the inner shell body and generates and controls a low-temperature zone airflow path surrounding the reagent fluid reservoir to improve reagent stability. 30. The cell culture and tissue engineering system according to claim 29, wherein the cold thermal assembly includes a cold sink array comprising a plurality of cold sinks pressurized together with a Peltier device to a hot sink in order to transfer heat to the hot sink. 31. The cell culture and tissue engineering system according to claim 30, wherein the compression is spring compression utilizing a Peltier solid device comprising an array of spring compression bolts, each comprising a coil spring, and a hot sink functions as a heat conduction path for heat removal. 32. The cell culture and tissue engineering system according to claim 31, wherein the Peltier solid device functions to transfer heat from a cold sink to a hot sink for subsequent heat transfer to the surrounding environment. 33. The cell culture and tissue engineering system according to any one of claims 30 to 32, wherein each cold sink array is segmented into functional units including a cold sink and associated axial fans. 34. The cell culture and tissue engineering system according to claim 33, wherein the axial fan has an axial flow configuration. 35. The cell culture and tissue engineering system according to claim 33 or 34, wherein the cold sink includes a vertical fin structure. 36. The cell culture and tissue engineering system according to any one of claims 29 to 35, further comprising a thermal insulator that inhibits the return of heat from the hot sink to the cold sink. 37. The cell culture and tissue engineering system according to any one of claims 2 to 36, wherein the condensation control means includes a moisture transport material sealed in a duct connected to a hot sink, the moisture transport material collects and moves any condensates that may be formed, which move down a cold sink and to a hot sink where they evaporate. 38. The cell culture and tissue engineering system according to claim 37, wherein the condensation control means minimizes and eliminates undesirable microbial contamination from moisture accumulation. 39. The cell culture and tissue engineering system according to any one of claims 29 to 38, wherein the cold thermal assembly can be suspended in an open configuration for cleaning by detaching the hinge from the bottom of the inner shell body. 40. The cell culture and tissue engineering system according to claim 39, wherein the cold sink array and associated axial fan can be uncoupled for cleaning. 41. The cell culture and tissue engineering system according to claim 40, wherein the hot sink fan and associated cowling can be removed from the top of the inner shell body and raised to an open configuration for cleaning. 42. The cell culture and tissue engineering system according to any one of claims 1 to 41, wherein the reagent fluid reservoir is fluidly connected to the bioreactor module via an upper-mounted port connector for mating with a lower port of the bioreactor module. 43. The cell culture and tissue engineering system according to claim 42, wherein the reagent fluid reservoir further comprises a snap tab for reversible attachment to a bioreactor module. 44. The cell culture and tissue engineering system according to claim 43, wherein a reagent fluid reservoir contains reagents required for a biological process and is responsible for holding waste products removed from the bioreactor module. 45. The cell culture and tissue engineering system according to claim 44, wherein the reagent fluid reservoir includes a fluid bag container for storing a plurality of separate reagents and for storing waste. 46. ​​The cell culture and tissue engineering system according to claim 45, wherein the plurality of separate reagents are stored in a reagent bag. 47. The cell culture and tissue engineering system according to claim 46, wherein the waste is stored in a reagent bag. 48. The cell culture and tissue engineering system according to claim 46 or 47, wherein the reagent bag is a flexible reagent bag. 49. The cell culture and tissue engineering system according to any one of claims 44 to 48, wherein the reagent fluid reservoir further includes open air ducts located at the top of the front and rear walls, so that the open air ducts are positioned above the reagent bag, providing a low-temperature airflow directly above the reagent bag. 50. The cell culture and tissue engineering system according to claim 49, wherein the reagent fluid reservoir further comprises a roof having a downward-extending baffle that helps to guide a low-temperature airflow above and across the reagent bag. 51. The cell culture and tissue engineering system according to claim 50, wherein the cell culture cassette further comprises a thermal insulating layer positioned between the bioreactor module and the reagent fluid reservoir, the thermal insulating layer insulating against any heat transfer from the bioreactor module. 52. A cell culture cassette, - A bioreactor module with a reagent fluid reservoir attached to the bottom, A cell culture cassette comprising a reagent fluid reservoir including a fluid bag container having open air ducts located on the front and rear walls of the reservoir. 53. The cell culture cassette according to claim 52, wherein the fluid bag container includes a roof and a floor, and the roof includes a baffle extending downward. 54. The cell culture cassette according to claim 52 or 53, further comprising a thermal insulating layer positioned between the bottom of a bioreactor module and the roof of a fluid bag container, the thermal insulating layer insulating against heat transfer from the bioreactor module. 55. The cell culture cassette according to claim 53 or 54, wherein the reagent fluid reservoir is attached via a port connection located on the roof of the fluid bag container, which is not blocked by the thermal insulating layer. 56. The cell culture cassette according to claim 55, wherein the reagent fluid reservoir further comprises a snap tab for attachment to a bioreactor module. 57. An automated cell and tissue culture engineering system comprising a cell culture cassette according to any one of claims 52 to 56. 57a. The automated system according to claim 57, wherein the cassette includes one or more sensors connected to a logic means. 58. A reagent fluid reservoir for connection to a bioreactor module, comprising a fluid bag container having open air ducts located on the front and rear walls of the reservoir. 59. The reagent fluid reservoir according to claim 57, wherein the fluid bag container further comprises a roof portion and a floor portion, the roof portion comprising a baffle extending downward. 60. A cell culture cassette comprising a bioreactor module and a reagent fluid reservoir according to claim 58 or 59. 61. An automated cell culture and tissue engineering system for receiving and operationally supporting an automated cell culture cassette in a more consistently controlled environment for biological processes, wherein the cell culture cassette comprises a bioreactor module and a reagent fluid reservoir, and the system is A warm zone is configured to circulate tangential warm airflow paths surrounding the bioreactor module, A low-temperature zone configured to circulate a tangential low-temperature airflow path surrounding the reagent fluid reservoir, An automated cell culture and tissue engineering system comprising: a movable thermal barrier assembly for thermally separating the warm zone from the cold zone when installing a cell culture cassette, fixing the bioreactor module only within the warm zone, and fixing the reagent fluid reservoir only within the cold zone. 62. A method for maintaining control of the environment for biological processes within a bioreactor module of a cell culture cassette, wherein the method is This includes creating and maintaining a separate high-speed warm airflow around the bioreactor module and, at the same time, creating and circulating a separate low-temperature airflow around and through a reagent fluid reservoir operably connected to the bioreactor module. A method in which the first airflow and the second airflow are separated and cannot be mixed. [Brief explanation of the drawing]

[0045] The following description of typical embodiments described herein will be better understood in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show currently typical embodiments. However, it should be understood that the present invention is not limited to the exact configuration and fixtures of the embodiments shown in the drawings. It should be noted that similar reference numerals refer to similar elements across different embodiments, as shown in the drawings and referenced in the description. The description in this specification will be better understood in consideration of the following drawings. [Figure 1] A cross-sectional view illustrating one embodiment of the cell culture and tissue engineering system of the present invention is shown, illustrating a cell culture cassette engaged with a robotic arm, with the thermal barrier assembly in a locked position. [Figure 2] This diagram illustrates a cross-section of an open-configuration system, showing the thermal barrier assembly in the unlocked position and the cell culture cassette exposed. [Figure 3a] An example of a system front view is shown, illustrating the operational robot interface with the thermal barrier assembly in an upright position for installing the cell culture cassette. [Figure 3b] Figure 3a illustrates a front view of the system with the thermal barrier assembly in the locked position. [Figure 3c] An enlarged front view illustrating an external cold reservoir, which forms part of the low-temperature zone and is located adjacent to the installed reagent fluid reservoir, is shown. The external cold reservoir has baffles to prevent obstruction of air return to the cold sink. These baffles are aligned with the underlying structure of the thermal barrier assembly shown in Figure 3b. [Figure 4] The characteristics of the warm zone airflow path and heating assembly are illustrated. [Figure 5] This illustrates the characteristics of the low-temperature zone airflow path and the cold thermal assembly. [Figure 6] An enlarged perspective view of the reagent fluid reservoir structure is shown as an example. [Figure 7] An enlarged cross-sectional view of a cold sink array is shown as an example. [Figure 8a] The location of the condensation control structure adjacent to the cold sink array is illustrated as an example. [Figure 8b] An enlarged cross-sectional view of the condensation control structure is shown as an example. [Figure 9] This example shows a cold thermal assembly with the hinge removed from the system. [Figure 10] An example of a cold thermal assembly with an unattached cold fan is provided. [Figure 11] This example illustrates the hotsink fan and associated cowling that vent from the rear of the system. [Modes for carrying out the invention]

[0046] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. The publications and applications discussed herein were made available exclusively for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention has no prior rights to such publications by prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to limit the scope of the invention.

[0047] In the event of any conflict, including definitions, this specification shall prevail.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in the field to which the subject matter of this specification pertains. The following definitions are provided for the convenience of understanding the invention when used herein:

[0049] As used herein, the articles “a” and “an” preceding an element or component are intended to be non-restrictive with respect to the number of instances (i.e., occurrences) of the element or component. Thus, “a” or “an” should be read as including one or at least one, and the singular form of an element or component also includes plurals unless the number clearly implies singular.

[0050] As used herein, the terms “invention” or “this invention” are non-limiting and are not intended to refer to any single aspect of a particular invention, but rather encompass all possible aspects described herein and in the claims.

[0051] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” and “having,” as well as their inflections and conjugations, indicate that “including” is not limited to these, and should be understood to be open-ended, for example, meaning that “including” is not limited to these.

[0052] As used herein, the term “approximately” refers to variation in a numerical quantity. In one aspect, “approximately” means within 10% of the reported value. In another aspect, “approximately” means within 5% of the reported value. In yet another aspect, “approximately” means within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reported value.

[0053] Any component defined as included herein may be expressly excluded from the claimed invention by means of a proviso or negative limitation.

[0054] As may be used herein, the term “substantially” (or its synonyms) means, with respect to the context, a measure or range, or a quantity or degree, encompassing a large part or most of the entity being referenced, or a range that is at least moderate or very large, or more effective or significant, with respect to the entity being referenced or the subject being referenced.

[0055] As used herein, the term “may” indicates options or effects that constitute, but are included in, or not included in, and / or used in, and / or implemented in, and / or result of, some embodiments or results of the present invention, without limiting the scope of the present invention.

[0056] When used in this specification and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, for example, elements that exist concomitantly in some cases and separately in others. Other elements other than those specifically identified by the phrase “and / or” may exist optionally, whether related to or unrelated to those specifically identified elements, unless otherwise explicitly indicated.

[0057] As used herein, “supported,” “mounted,” “attached,” “connected,” “joined,” “bonded,” and “linked” may be used interchangeably with respect to the engagement of components of the automated device of the present invention. Furthermore, any of these terms may be used in conjunction with the term “reversibly.”

[0058] As used herein, “thermal zone” should be understood as an isolated area having a consistent temperature, defined in terms of being warm or cold. Furthermore, a zone having a defined temperature or defined temperature range is maintained within that defined temperature or defined temperature range, and as a result, the zone is stable, uniform, invariant, or homogeneous with respect to the defined temperature or defined temperature range.

[0059] As used herein, the “warm zone” should be understood as an isolated, restricted area having a precise temperature above room temperature (i.e., above approximately 23°C). Generally, the precise temperature for mammalian cells is approximately 37°C. However, depending on the specific needs of a particular cell culture, temperatures above and below 37°C can still be selected as precise temperatures and realized as a “warm zone.” For example, in the absence of differentiation factors, stem cells will proliferate at 37°C even without differentiation. Conversely, stem cells growing at temperatures above and below 37°C will differentiate even in the absence of differentiation factors. Furthermore, different “warm zone” temperatures may be required to apply temperature stress to a given culture. The “warm zone” may include a high-speed warm airflow pathway. Those skilled in the art will understand the meaning of “high-speed” in comparison to normal airflow velocity. The “warm zone” may house a bioreactor module and have separate gas conditioning means.

[0060] As used herein, the “low temperature zone” should be understood as an isolated, restricted area having a temperature range of approximately 2°C to 8°C. The exact temperature of the “low temperature zone” does not need to be precise, but rather needs to be an overall temperature reduction such as approximately 2°C to 8°C. The “low temperature zone” contains a low-temperature airflow. The “low temperature zone” houses a fluid reagent reservoir and any further fluid reagent bags. The “low temperature zone” houses condensation control means.

[0061] The "warm zone" is isolated from the "cold zone," so temperatures from one zone do not transfer to the other. Fast warm air currents do not mix with cold air currents.

[0062] The cell culture cassettes are located in the warm zone.

[0063] The reagent fluid reservoir is located within the low-temperature zone.

[0064] The external (additional) fluid bag is located within the low-temperature zone.

[0065] As used herein, "present" with respect to a "warm zone" or "cold zone" means that the structural element in question is exposed only to the atmosphere within that particular zone during the closure operation of the culture system.

[0066] A general and non-limiting overview of the present invention and its implementation are presented below. The overview outlines exemplary implementations of embodiments / models of the present invention and provides a constructive basis for variations and / or alternative and / or different embodiments / models, some of which will be described later.

[0067] Figures 1 and 2 illustrate embodiments of the system of the present invention in a closed and open configuration, respectively. Figure 1 illustrates an embodiment of the cell culture and tissue engineering system 1 of the present invention, in which a disposable cell culture cassette 2 is installed and which operates under automated conditions. The cell culture cassette 2 includes a bioreactor module 4 and an adhesive reagent fluid reservoir 6. The system 1 is “cocoon-like” and includes an inner shell body 8 and an outer shell cover 10 that covers the opening 12 of the inner shell body. The outer shell cover 10 encloses the front opening 12 of the inner shell body and is connected to the inner shell body 8 via rotatable connectors (not shown) on both sides of the inner shell body, so that the outer shell cover 10 can be opened by rotating along the outer arc shape of the inner shell body from a first closed position to an open position rotating around the connector until the outer shell cover occupies the inner shell body (as shown in Figure 2). The front opening 12 of the inner shell body 8 includes a periphery 14 having a U-shaped channel 16 containing an inflatable seal 17. As a result, when the system is closed, an inflatable seal is activated and inflates, engaging with the inner surface of the outer shell body to airtightly seal the system. The outer shell cover 10 includes a thermal cell 18 that functions as an external thermal barrier.

[0068] The cell culture cassette 2 is installed against a robotic interface 20 positioned within an opening 12 of the inner shell body 8. The robotic interface 20 further includes associated robots and electronic equipment 22 within the inner shell body. The cell culture cassette 2 is operationally constrained to the robotic interface 20 and locked in this position by a movable thermal barrier assembly 24 extending from the cell culture cassette 2 to the outer shell cover 10. By locking the cell culture cassette 2 to the robotic interface 20 with the movable thermal barrier assembly 24, an upper warm zone 26 and a lower cold zone 28 are created. The bioreactor module 4 is fixed within the warm zone 26. The reagent fluid reservoir is fixed within the cold zone 28. An additional external cold reservoir 29 is located within the cold zone and is adjacent to the reagent fluid reservoir of the installed cell culture cassette. This external cold reservoir 29 may include additional reservoir bags for collecting fluid drainage and / or provide additional necessary culture fluids and reagents. Thus, the cell culture system provides both a warm, incubated environment suitable for biological processes (e.g., about 37°C ± 5°C) and a separate cold environment for improving reagent stability during reagent storage (e.g., 0°C to about 10°C).

[0069] In Figure 2, System 1 is in an open configuration that exposes the cell culture cassette 2 for inspection and / or removal, and further shows a movable thermal barrier assembly 24 in an open raised position. The bioreactor module 4 of the cell culture cassette 2 is fluidly connected to a reagent fluid reservoir 6 via a mating port connection (not shown). The bioreactor module 4 supports the operating requirements of a biological process 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 substantially contains and stores the reagents required for the biological process occurring 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 the culture media, growth factors, drugs, cell labels, and waste products removed from the bioreactor module.

[0070] During operation, the outer shell cover 10 rotates to the open position, allowing access to the cassette 2 for installation or removal of the cell culture cassette, which is constrained to the operating robot interface 20 by the structural configuration of the movable barrier assembly 24. When installation / removal of the cell culture cassette is required, the movable thermal barrier assembly 24 can be raised away from the cassette via an internal coupling 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 engaged (lowered) position, as shown in Figure 1, thereby ensuring precise alignment with the robot 7 and associated interface connections, and forming separate warm and cold zones. Once the cell culture cassette is installed and the movable thermal barrier is locked in place, the thermal barrier effectively forms a portion of the lower surface of the warm zone around the cassette and an upper surface of the cold zone around the cassette.

[0071] Figures 3a and 3b show front elevation views of the operational robot interface 20 with the movable thermal barrier assembly 24 in the open, raised position. The operational robot interface 20 includes several valve actuators and peristaltic pump connectors to which the cell culture cassette is aligned and connected. The movable thermal barrier assembly 24 includes a lever separation arm 30, which is connected to either side of the operational robot interface 20 supporting the thermal platform 32 shown in the raised position. The underside of the thermal platform is shown to have a channel 33. In Figure 3b, the thermal platform 32 is shown with an associated upper handrail 34, which is set to precisely match the dimensions of the locked cell culture cassette. A recess 36 is provided adjacent to the lever arm 30, which provides operator control access for opening and extending the movable thermal barrier assembly 24. The upper handrail 34 provides a gripping means for the user to lock the cell culture cassette in the operational position or to release the lock of the thermal platform 32 and raise it. The thermal platform 32 extends forward around the cell culture cassette and is shown to radiate to the outer shell cover (shown in Figure 1). The thermal platform has both a flexible outer seal at its forward outer boundary to accommodate relative motion between the thermal platform and the outer shell cover (not shown), and a flexible inner seal directly adjacent to the cell culture cassette (not shown) to accommodate mounting tolerances of the cell culture cassette. Both seals are elastic and impermeable to moisture and vapor.

[0072] Figure 3c shows the interior of an external cold reservoir 38, which has a baffle 40 to prevent obstruction of air return to the cold thermal assembly shown on the rear of the external cold reservoir. The external cold reservoir 38 is positioned adjacent to the reagent fluid reservoir, both located within the cold zone 28. The cold space may contain another(yes) external reservoir bag outside the reagent fluid reservoir (not shown). The baffle 40 functions to prevent any reservoir bag contained within the external cold reservoir from obstructing the return air to the cold sink. When the thermal platform is in the lower locking position around the cell culture cassette, the lower channel 33, alongside the baffle 40, creates a continuous structure for unobstructed cold airflow.

[0073] Figure 4 shows the temperature-controlled warm zone 26 and cold zone 28 of cell culture system 1. System 1 is shown in a closed position. The warm zone has a gas environment substantially isolated 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 is used to lower the concentrations of oxygen and / or carbon dioxide below ambient levels. The warm zone 26 is created by a heating assembly 41 located in the upper section of the inner shell body. The heating assembly includes a high-capacity linear fan 42, which is operably connected to a heated airflow inductor 44 to generate and induce a high-temperature warm airflow path 45 (indicated by arrows) that circulates around the bioreactor module 4. The high-capacity linear fan provides a high-speed airflow, minimizing spatial and temporal temperature heterogeneity within the warm zone. The linear fan 42 has a flow configuration, which provides a high-speed airflow velocity through a combination of stacked flow rate, minimum pressure drop, and minimum airflow direction change. High airflow velocity promotes temperature uniformity through high convective heat transfer, which essentially minimizes surface temperature differences within the warm zone from competing heat sources.

[0074] Furthermore, the continuous circulation of the high-speed warm airflow is aided by the arc shape of the inner wall of the outer shell cover, which helps ensure that the circular path of the warm high-speed airflow remains consistently homogeneous. The warm zone has been shown to be completely insulated from the low-temperature zone. Competing heat loads are located on the warm zone by heat transfer with other areas within the cell culture system and by heat transfer to the surrounding environment. The low-temperature zone and the typical surrounding environment tend to operate at temperatures below the warm zone's temperature setpoint, and these factors consequently represent the heat loss in the warm zone. In contrast, certain electronic components within the robotic architecture may operate at temperatures above the warm zone's temperature, and consequently, such components represent the thermal gain in the warm zone. The configuration and operation of the warm zone eliminate the problem of heat transfer conditions that inherently cause temperature non-uniformity, and as a result, a uniform warm zone maintains a more consistent operating state for the biological processes underway within the bioreactor module.

[0075] In the warm zone, a consistent heating temperature required for specific biological processes can be selected. The control temperature may be selected from 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, or higher.

[0076] Figure 5 illustrates the main functional components of the low-temperature zone 28. The inner shell body has a floor section that supports the tray 46, and the tray 46 is equipped with a forward cantilevered shelf section 48 for supporting the bottom surface of the cell culture cassette, which forms a ducted airflow path below the tray and generates the low-temperature zone airflow path 49 (indicated by the arrow).

[0077] The cold thermal assembly 50 is housed at the rear of the inner shell body and generates and controls a low-temperature zone airflow path surrounding the reagent fluid reservoir to improve reagent stability. The cold thermal assembly 50 includes a cold sink array 52, each containing a number of finned cold sinks 54 (see Figure 7) that receive heat from the low-temperature zone airflow path via a cold sink fan 56. Heat is transferred from the cold sinks 54 to an adjacent hot sink 58 by a Peltier solid thermoelectric device (see Figure 7). The heat is then released from the hot sinks to the surrounding ambient air 62 by a hot sink fan 60.

[0078] The cold sink fan 56 has an axial flow configuration that provides a high convective heat transfer coefficient due to turbulence in the hot sink fins 58, resulting in a minimum temperature difference between the airflow in the refrigerated zone and the cold sink. Reagents in the refrigerated zone are cooled by being surrounded by a low-temperature airflow path. Since the key criterion for improving reagent stability is the overall temperature reduction rather than the exact temperature, the temperature in the low-temperature zone may not be as uniform as in the warm zone.

[0079] Figure 6 illustrates a reagent fluid reservoir 6 that stores multiple reagents in separate fluid containers, such as flexible reagent bags (not shown). The reagent bags are confined within a fluid bag container 64. The reagent fluid reservoir 6 is connected to the bioreactor module via port connectors 67 and snap tabs 66, which help maintain attachment to the bioreactor module. The reagent bags are insulated against heat transfer from the warm zone by a cassette thermal insulator 68. The refrigerated temperature of the reagent bags is maintained by the circulation of cold air around and inside the reagent fluid reservoir, which includes airflow over the reagent bags via reagent fluid reservoir open air ducts 70 located on the front and rear walls adjacent to the top of the reagent fluid reservoir. The top of the reagent fluid reservoir (roof portion) has a downward-extending baffle that extends into the reservoir (not shown). The baffle helps guide a cold airflow above and across the reagent bag, allowing the airflow to move to an external cold reservoir guided by the internal baffle 40 and return to the cold sink array without obstruction.

[0080] The structure of the cryogenic zone creates a ducted / channeled airflow with minimal obstruction within the cryogenic zone, providing a continuous cryogenic airflow that circulates throughout the entire cryogenic zone, providing a cryogenic airflow below and around the reagent fluid reservoir, which then passes through vents to penetrate the top of the reagent fluid reservoir for the cryogenic air to flow through the reservoir, directly through the fluid bag, and further through vents to the external cold reservoir, where it returns to the cold sink of the cold thermal assembly via baffles to dissipate heat. By providing channels, baffles, vents, thermal insulators, and seals on the thermal platform, as well as lower channels, together to dissipate heat and circulate cryogenic air, obstruction to the cryogenic airflow is minimized, ensuring that the cryogenic air is maintained within the cryogenic zone.

[0081] Figure 7 shows the structure of a Peltier solid-state thermoelectric device, which transfers heat from a cold sink to a hot sink for subsequent heat transfer to the surrounding environment. Because the solid-state device is compact and has no easily failing moving parts, the Peltier solid-state device is preferred over other conventional refrigeration methods such as vapor compression refrigeration. A thermal insulator is provided to prevent heat from returning from the hot sink to the cold sink, as this would impair thermal efficiency. The cold sink has adjacent built-in monuments that form an extension of the cold sink, representing the heat conduction path from the cold sink to the Peltier solid-state device. The subsequent heat supplied from the Peltier solid-state device to the hot sink is the sum of the heat transferred from the cold sink and the power consumed by the Peltier solid-state device. Thus, the total heat supplied to the hot sink is significantly greater than the heat removed from the cold sink.

[0082] Integrating the monument into a cold sink rather than a hot sink is advantageous because it reduces heat transfer through the monument. Consequently, the overall temperature difference of the monument is significantly smaller than when the monument is located on a hot sink, thus reducing heat loss. In Peltier solid-state devices, the coefficient of performance (the ratio of heat discharged to power consumption) increases with decreasing temperature difference. Therefore, the location of the monument on a cold sink significantly improves the coefficient of performance compared to the alternative method of integrating the monument as part of a hot sink.

[0083] The cold sink array 52 consists of multiple individual cold sinks 54 relative to a monolithic hot sink 58. To ensure close thermal contact between the monument of the cold sinks 54, the Peltier solid device, and the hot sink 58, the cold sinks are segmented into functional units (cold sinks and axial fans), so that each functional cold sink unit makes close contact with the monolithic hot sink through the array (via the Peltier solid device) and by using spring compression bolts 70. Spring compression is achieved by using coil springs 72. The main advantage of spring compression of the cold sinks relative to the monolithic hot sink is that thermal and / or manufacturing distortions self-correct, and each cold sink assembly can be independently aligned forward relative to the associated Peltier solid device and relative to the monolithic hot sink, thereby achieving a homogeneous compressive load. Such self-correcting compressive loads maximize the efficiency of heat transfer from the individual cold sinks to the monolithic hot sink.

[0084] When the cell culture system is opened, the cold zone inevitably exchanges air with the surrounding environment. As a result, when the cell culture system is subsequently closed, air from the surrounding environment is trapped within the cold zone. If the humidity of the incoming ambient air causes the dew point to exceed the final temperature of the cold zone, moisture from the ambient air will condense. Consequently, condensation inevitably occurs, potentially creating an undesirable moisture accumulation zone within the cold zone. Such accumulation can become a potential site for microbial contamination. Since the surface of this component is the coldest surface in the cold zone, condensation will naturally begin and continue on the cold sink. To automatically manage and eliminate the annoyance of condensation, a condensation control mechanism 73 is employed within the cold zone of the cell culture system at the location shown in Figure 8a. As shown in more detail in Figure 8b, the condensation control mechanism 73 includes a moisture transport material 74. The moisture transport material 74 collects condensate when it forms on the cold sink and moves it downwards on the cold sink fins due to the effects of gravity and the downward airflow over the cold sink fins. The moisture transport material protrudes from the cold sink to the hot sink through a dedicated duct 76, and the heat from the hot sink then causes the transported moisture to continuously evaporate into the surrounding environment. This condensation control measure requires no moving parts or extra power.

[0085] Good Manufacturing Practices require servicing and cleaning of the cell culture system. Figure 9 shows that the cold thermal assembly 50 can be removed and supported on the hinge 51 to allow for servicing and cleaning. The cold thermal assembly is released from its normal operating position by the operation of the latch 78 and subsequent downward rotation around the hinge point. A refrigerated power connector 82 provides a reliable electrical connection for data and power when the cold thermal assembly is returned to its normal closed operating configuration.

[0086] Figure 10 further illustrates that the cold sink fan 56 includes an assembly 84. The assembly 84 can be detached from the cold sink and pulled out, allowing for more thorough cleaning of the cold sink below.

[0087] Figure 11 shows that the hot sink fan 60 and associated cowling can be pulled upward from the hot sink via a hinge, allowing for more detailed cleaning of the hot sink below.

[0088] In this embodiment, a hollow shaft can be used to connect the inside and outside of the cell culture system, and a third control zone can be provided within the shaft to run processes at temperatures other than the culture temperature or refrigeration temperature. Such an embodiment can be used for process steps that are potentially beneficial from intermediate temperatures and can be controlled in this transition zone.

[0089] In additional embodiments, the thermal window may comprise a warm zone and / or cold zone consisting of twin liquid crystal (LCD) windows (or functional equivalents) incorporated into the outer shell. The LCD windows enable the display of internal cassette operation when the LCD is transparent, opacity against photodegradation harmful to reagents when the LCD is activated and opaque, and a thermal barrier due to the twin LCD walls forming an enclosed air space.

[0090] In this embodiment, individual internal airflows can be connected to a centralized airflow management system capable of controlling multiple manufacturing units.

[0091] In additional embodiments, air filtration may be incorporated into the air circulation path, and such filters may be disposable after each treatment or other reasonable period.

[0092] Those skilled in the art will understand that, where possible, the materials for manufacturing the components of the systems described herein are selected to maximize thermal insulation properties without impairing the primary functions 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 generally shown to be cocoon-shaped, this may vary as well as in size, as long as the shape maintains warm and cold airflow paths within it.

[0093] Furthermore, the cell culture and tissue engineering system of the present invention includes various sensors, which are associated with and / or located within the low-temperature zone, hot zone, cell culture cassette, heating assembly, and cold thermal assembly, and are associated with the operating robot interface and associated internal robots and electronic equipment, and are further associated with computer means.

[0094] 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 this subject matter and without diminishing its intended merits. Accordingly, such changes and modifications are intended to be within the scope of the appended claims.

Claims

1. A method for preparing a controlled thermal environment for a biological process in an automated cell culture cassette, wherein the automated cell culture cassette comprises a bioreactor module and a reagent fluid reservoir, and the method is Raising the movable heat barrier assembly to the unlocked raised position, The automated cell culture cassette is installed within a housing having an outer shell cover and an inner shell body, with respect to the operating robot interface located within the inner shell body. Lowering the movable thermal barrier assembly to a locked lowered position restrains the automated cell culture cassette to the operating robot interface, forming a portion of the lower surface boundary of the warm zone and a portion of the upper surface boundary of the cold zone, The heating assembly generates and circulates a warm airflow path surrounding the bioreactor module within the warm zone, The cold thermal assembly generates and controls a low-temperature airflow path surrounding the reagent fluid reservoir within the low-temperature zone, wherein the cold thermal assembly comprises a cold sink array, the cold sink array has an integrated monument that forms an extension of the cold sink array, and the integrated monument provides a heat conduction path from the cold sink array to the thermoelectric device. To transfer heat to the subsequent surrounding environment, the thermoelectric device sends heat from the cold sink array to the hot sink, A method that includes this.

2. The method according to claim 1, wherein the heating assembly includes a high-capacity linear fan operably connected to a heating airflow induction device, and the circulation includes the high-capacity linear fan and the heating airflow induction device generating a consistent and homogeneous flow.

3. The method according to claim 1, further comprising maintaining a substantially uniform temperature throughout the warm zone by the heating assembly.

4. The method according to claim 1, wherein the cold sink array includes a plurality of cold sinks pressurized together with the thermoelectric device with respect to the hot sink in order to transfer the heat to the hot sink.

5. A cell culture system for receiving and operationally supporting an automated cell culture cassette for biological processes, The automated cell culture cassette comprises a bioreactor module and a reagent fluid reservoir, and the system is The casing and A robotic interface and A movable thermal barrier assembly has an unlocked raised position for installing the automated cell culture cassette and a locked lowered position for thermally insulating the warm zone from the cold zone, and operably restrains the automated cell culture cassette with respect to the operating robot interface, A cold thermal assembly configured to generate and control a cold airflow path surrounding the reagent fluid reservoir within the cold zone, comprising a cold sink array, wherein the cold sink array has an integrated monument that forms an extension of the cold sink array, and the integrated monument provides a heat conduction path for heat moving from the cold sink array, A heating assembly configured to generate and circulate a warm airflow path surrounding the bioreactor module within the warm zone, A thermoelectric device configured to transfer heat from the cold sink array to the hot sink, Equipped with, A cell culture system in which, in the locked lowered position, the movable thermal barrier assembly forms part of the lower surface boundary of the warm zone and part of the upper surface boundary of the cold zone.

6. The cell culture system according to claim 5, wherein the heating assembly is provided in the upper section of the housing.

7. The cell culture system according to claim 5, wherein the operating robot interface includes a valve actuator, a peristaltic pump connector, and an associated control system for mating with a corresponding connector of the automated cell culture cassette.

8. The cell culture system according to claim 5, wherein the movable thermal barrier assembly includes a pair of spaced arms that are internally connected to both sides of the operating robot interface and support a central thermal platform having associated upper handrails.

9. The cell culture system according to claim 5, wherein the bioreactor module is fluidly connected to the reagent fluid reservoir.

10. The cell culture system according to claim 5, wherein the low-temperature zone further includes a condensation control unit.

11. The cell culture system according to claim 5, wherein the low-temperature zone includes a cold reservoir located adjacent to the reagent fluid reservoir.

12. The cell culture system according to claim 11, wherein the cold reservoir includes at least one baffle.

13. The cell culture system according to claim 12, wherein the movable thermal barrier assembly includes 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.

14. The cell culture system according to claim 5, wherein the cold sink array includes a plurality of cold sinks pressurized together with the thermoelectric device relative to the hot sink.

15. The cell culture system according to claim 5, further comprising a cold sink fan comprising an assembly configured such that the cold thermal assembly is uncoupled from and withdrawn from the cold sink array.

16. The cell culture system according to claim 5, further comprising a condensation control mechanism having a moisture transport material for collecting condensation liquid when condensation forms on the cold sink array.

17. The cell culture system according to claim 16, wherein the moisture transport material protrudes from a dedicated duct to the hot sink, and in the hot sink, the condensation liquid evaporates due to the heat of the hot sink.

18. The cell culture system according to claim 5, further comprising a hot sink fan configured to release the heat from the hot sink to the surrounding environment.

19. The cell culture system according to claim 18, wherein the hot sink fan is configured to be pulled upward via a hinge so as to be separated from the hot sink.

Images