Modular configurable bioreactor system for manufacturing lines

Abstract

To provide a configurable system for repeatably performing processes related to tissue growth in a controlled environment, possibly part of an industrial production line.SOLUTION: A system (107) can accommodate various sizes and shapes of culture vessels (503), and can maintain cells at a desired temperature in the culture vessels, thus enabling a plug and play system that can produce consistent and repeatable results. The system includes gas management, fluid management, and control (111) of multiple processes simultaneously, and can be automatically operably coupled with a variety of supporting technologies that can enable tissue-related processes. The system can communicate with supporting technologies upstream and downstream on a manufacturing line, enabling fully automated process control, centralized data historization, and centralized control.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to configurable systems, and specifically to configurable manufacturing systems. In a configurable manufacturing system, the concept is to allow plug-and-play components to be added to and then removed from the system with little or no system reconfiguration, and enable robust communication with upstream and downstream equipment and technologies. Plug-and-play is a particular concern for industrial systems that accommodate the operable coupling of interchangeable components, where the footprint, characteristics, and thermal requirements of such components vary from one component type to another. Current systems can be improved by enabling upstream and downstream communication with other components of a manufacturing line. Examples of manufacturing line components for tissue processing can include, but are not limited to, cell lysis systems, bioreactor systems, and tissue maintenance systems. Standardized processes and communication between components along a manufacturing line allow for the addition and removal of components depending on a desired output. Accordingly, the manufacturing lines of the present teachings contemplate various components, including those listed herein and others, that operate simultaneously, sequentially, or in a dynamically determined and / or user-selected order determined by a recipe.

Background Art

[0002] When considering bioreactor systems for a manufacturing line, results can be improved, for example, by providing automatic medium exchange, sterilization, decellularization perfusion processes, integrated electrical and mechanical stimulation, and versatile bioreactor components. Other possibilities for improvement also exist. Examples of uses for a manufacturing line for tissue processing, including the system of the present teachings, include, but are not limited to, cell proliferation, decellularization, endothelial cell perfusion, recellularization, and maturation of recellularized tissue. This list does not limit the use of the system of the present disclosure or the present teachings in any way.

[0003] Cell proliferation is the intentional growth of cells to generate therapies for tissues or diseases. Human mesenchymal stromal cells (hMSCs) (adult stem cells) and human induced pluripotent stem cells (hiPCSs) (obtained by reprogramming somatic cells into human pluripotent stem cells (hPCSs)) are cells that can be effectively used in the cell proliferation process. These cell types can self-regenerate and differentiate into specific cell types depending on their efficacy. Bioreactors (culture vessels) can be used to enable cell proliferation, particularly because they can provide a 3D agitated, scalable, homogenized environment. Types of bioreactors that can be found in a production line can include agitation tanks, fixed beds, hollow fiber, rotating cell, rotating bed, and oscillating bioreactors, having disposable or durable components or combinations. Sensors can be associated with bioreactors and controllers to establish the operating range for temperature, acid / base level, aeration, agitation speed, and culture medium flow rate. To enable reproducibility in cell proliferation on the production line, consistent internal control of the bioreactor system and communication of the bioreactor system's status and other characteristics with other production line components are required.

[0004] Decellularization results in the removal of biological tissue from the extracellular matrix (ECM) scaffold, while retaining clues to cellular preservation and homeostasis in the structure of the ECM. The scaffold can be decellularized physically or chemically. Chemicals used in decellularization include surfactants such as sodium dodecyl sulfate (SDS) that lyse cells by disrupting the phospholipid cell membrane, acids such as peracetic acid and bases such as sodium hydroxide that solubilize the cell membrane. Physical decellularization can include methods such as freeze / thaw, high hydrostatic pressure, and supercritical carbon dioxide. All decellularization processes involve a washing process. To determine whether decellularization has been successful, aspects of the ECM that remain intact after decellularization can be examined to determine, for example, whether cells have been removed, genetic material has been eliminated, proteins in the matrix have been preserved, and any mechanical properties have been retained. More specifically, in some systems, the ECM after decellularization should not reach a pre-selected threshold for double-stranded DNA of pre-selected fragment lengths and should not contain any visible nuclear material. Mechanical properties, including elastic modulus and tensile strength, may be required to meet certain pre-selected criteria. The success of the decellularization method can be determined by the reduction of the tissue's immunogenicity, specifically, the reduction of genetic material and antigens. Insufficient reduction of immunogenicity can lead to in vivo rejection of the tissue. What is needed is a bioreactor system that includes a robust communication system, which includes internal controls to achieve the desired decellularization result and provides the status of decellularization and other properties to other components of the production line.

[0005] In recellularization, a scaffold is seeded with cells to form an organ. Complete organ regeneration requires that parenchyma, blood vessels, and supporting components must be re-established prior to cell seeding. Many cell types are considered for organ regeneration. For example, mesenchymal stem cells from bone marrow or adipose tissue, proliferated to a suitable number, can differentiate into various cell types, and scaffolds have been found to facilitate mesenchymal stem cell differentiation. Induced pluripotent stem cells can facilitate the use of patient-derived cells to provide a cell source for recellularization. Supporting cells, such as fibroblasts, can enhance the function of certain types of cells and thus enhance recellularization. The successful seeding route may be organ-dependent. For example, a renal scaffold can be re-seeded through the ureter or renal artery, and the renal artery route has been shown in some studies to yield higher cell distribution and retention than the ureteral route. Recellularization requires a bioreactor control system that manages properties such as temperature, gas, pH, and pressure. Successful recellularization yields the required number of specific types of cells to form an entire tissue or organ. What is required is a bioreactor system that includes internal controls to achieve the desired recellularization results and a robust communication system that provides the recellularization status and other characteristics to other components of the production line.

[0006] In systems designed to repeatedly control the growth of mammalian cells by supplying nutrients, removing waste, and controlling temperature and headspace gas mixing within the culture vessel, modular design can be beneficial to take advantage of technological advancements in the non-simultaneity of the system required to enable cell growth. Furthermore, such systems can offer greater efficiency by operating multiple cell culture vessel stations simultaneously. The main technological gap relates to the issue of the relative size of the vessels required for autologous and allogeneic processes involving small tissues. There is a need for vessels capable of producing varying amounts of cells without sacrificing the ability to monitor and control the growth process. Efficiently using certain commercial bioreactor systems requires a large minimum volume to accommodate current sensors. Harvesting cells of varying volumes is also difficult due to the dead volume present at the bottom of the growth vessel, which can range from tens to hundreds of milliliters in various types of culture vessels. New disposable vessels and sensors designed for smaller working volumes are needed to achieve the goal of efficiently automating the production of autologous and allogeneic tissue engineering components. Similarly, scaling up to larger vessels, for example, as large as 100 liters or larger, is required. Furthermore, a system may be required, which may include both disposable and durable components. Disposable components may be needed, such as tubing for supplying and recirculating culture media, disposing of waste, seeding cells, and moving fluids and cells from one production line component to the next, as well as culture media reservoirs and single-use sensors. Durable components may include, for example, system chassis, user interfaces, culture media refrigerators, waste containment systems, gas management systems, pneumatic control blocks, and other components or subcomponents on the production line. Components that may be durable or disposable include, but are not limited to, growth and maturation bioreactors.

[0007] Another technological gap relates to issues surrounding the use of pumps and valves. In current systems, pumps and valves are arranged in a fixed number, limiting the flexibility of the system, and requiring the purchase of different systems if more or fewer pumps and valves are needed for a particular application. Yet another technological gap involves the control of flexibly configured systems. For example, in a system with multiple bioreactors operating simultaneously, current systems assume that each bioreactor is performing the same operation, e.g., growing cells. Monitoring in these systems shows that cells may grow at different rates, but the same basic control functions are required for such parallel operation. What is needed is a system in which each bioreactor can be controlled separately while operating simultaneously. Such a system can enable the simultaneous preparation of cells and scaffolds to engineer complex organs and provide the results to other components on the production line. For example, a system with multiple bioreactors can simultaneously prepare multiple types of cells to decellularize and recellularize scaffolds. Each bioreactor and its associated pumps and valves can receive custom controlled flows and custom gas flow, guided by monitoring the conditions with the bioreactor contents. For each bioreactor, various different pumps, valves, and sensors can be activated in this embodiment to achieve tissue growth and scaffold preparation simultaneously.

[0008] The current system, Zoo Ning's Industrial-Scale Optical Bioreactor No. CN201045139Y, a utility model (Ning) granted on April 9, 2009, and expiring on September 19, 2014, transferred to Yantai Haishangchuanqi Biotechnology Co., LTD., describes a bioreactor constructed from simple glass for large-scale industrial production. Wang et al., "Development of Novel Bioreactor Control Systems Based on Smart Sensors and Actuators," Frontiers in Bioengineering and Biotechnology, 8:7, doi: 10.3389 / fbioe.2020.00007 (February 4, 2020) (Wang), describes the latest trends in bioreactor control technology, including hierarchical control systems, which are a form of networked control system in which a set of devices and control software are arranged in a hierarchical tree, and the links in the tree are implemented by a computer network. Wang describes an improvement to a flat-configuration control system for bioreactors, based on parallel distributed smart sensors and actuators, as a concise solution for process control in bioreactors. Laboratory configurations such as the Sartorius AMBR® 15 Cell Culture Generation 2 can be modified by the operator both at the start and during the process. The system includes single-use containers and an automated workstation, all housed within a biological safety cabinet. Multiple bioreactor cultures are monitored in parallel. The system includes an automated liquid handler that can dispense culture media, feed, and reagents into the containers and facilitate in-line or offline sampling. Other devices, such as Eppendorf's DASGIP® parallel bioreactor system, enable advanced bioprocess control and automation.The system provides precise parameter control, user-defined profiles, automated features, and configurable solutions to adapt to the requirements of microbial, phototrophic, mammalian, and human cell and stem cell applications. The system can control multiple durable or disposable bioreactors in parallel, maintain desired temperature profiles, and control agitation, pH, and DO, as well as TMFC gas mixtures of air, N2, O2, and CO2.

[0009] What is needed, but not currently available, is an industrial control system with at least one integrated programmable logic controller (PLC) in a closed-system bioreactor. Such a system could enable communication between the bioreactor PLC and other controllers along the production line. What is needed is a system with integrated sensors, i.e., a system that can adapt to changes in container size and shape and allow for fewer MFCs than the number of bioreactors. What is needed is a system that can adapt to the ability to grow one type of cell on one side of the bioreactor scaffold and another type on the other side, and to combine the two types of cells. [Overview of the Initiative] [Means for solving the problem]

[0010] The system described herein overcomes the technological gap outlined herein. The system efficiently automates the production of autologous and allogeneic tissues by adapting to processes involving cell culture vessels of various sizes and small tissues. The vessels described herein can produce varying quantities of cells without sacrificing the ability to monitor and control the growth process. The vessels include a head plate that can be adapted to the tubular needs of various organ types. The vessels described herein are configured to adapt to sensor placement so that the volume of cells covers the sensors. The vessels are disposable to allow for the harvesting of varying volumes of cells, thus reducing dead volume cells at the bottom of the vessel. The vessels can also be durable. The vessels can be scaled down to, for example, 0.1–3 liters, for small amounts of cells, or scaled up to, for example, up to 1,000 liters, for larger vessels. The system can also be adapted to a variable number of pumps and valves, which can be allocated based on specific applications. The system includes a variable number of valves and pumps, a variable number and size of vessels, and at least one controller configured to control fluids and gases associated with desired processes. Since the vessels can be composed of different types of cells, tissues, and / or scaffolds, the controller is configured to control various processes running in parallel within the vessels. The system is configured to control each culture vessel station independently while they are operating simultaneously. For example, a system with multiple culture vessel stations can simultaneously prepare multiple types of cells to decellularize and recellularize scaffolds. Each culture vessel and its associated pumps, valves, and gas systems can receive a custom controlled flow guided by monitoring the contents of the culture vessel. For each culture vessel, various pumps, valves, and sensors can be activated to achieve, for example, tissue growth and scaffold preparation simultaneously. The system of this instruction includes pressure-controlled circulating flow, along with flow feedback and monitoring. The flow paths can be modified as the system operates.Another feature of the system described herein is a gas control system configured to allow fewer mass fluid controllers (MFCs) than the number of culture vessels, as each culture vessel is given a time window for receiving gas within it. This feature allows for reduced gas consumption, less gas to be aerated, and less wasted gas compared to systems with a one-to-one correspondence between MFCs and culture vessels. Another feature is that the system can be adapted to grow one type of cell on one side of the culture vessel scaffold and another type on the other side, allowing for the combination of two types of cells. Uses of the system described herein, but are not limited to, cell culture, culture medium preparation (i.e., adjusting the temperature of the medium to a desired value and setting the pH and DO levels of the medium), decellularization and recellularization of scaffolds, generation of blood, blood components, viruses for gene therapy, recombinant proteins, pharmaceuticals, vaccines, allergens, genes, antibodies, fermentation, combinations, cosmetics, and food, and conversion of raw materials into useful by-products. Other applications are also envisioned by this instruction. The present invention provides, for example, the following: (Item 1) A system for repeatedly performing at least one type of organization-related process as part of a manufacturing line, wherein the system is At least one culture vessel station, the at least one culture vessel station comprising at least one variable-size culture vessel, a fluid handling system, and a culture vessel control system, wherein the at least one culture vessel station is configured to be adapted to perform at least one type of tissue-related process, A gas management system configured to supply at least one type of gas to the at least one culture vessel station, At least one controller, the at least one controller configured to control the gas management system and the at least one culture vessel station to carry out the at least one type of tissue-related process, and the at least one controller configured to communicate with components on the production line using a standard industrial communication protocol, and A system equipped with these features. (Item 2) The system according to item 1, wherein the at least one variable-size culture vessel comprises a disposable component. (Item 3) The system according to item 1, wherein the at least one variable-size culture vessel comprises a durable component. (Item 4) The aforementioned at least one variable-size culture vessel is A container sleeve surrounding at least one portion of the at least one variable-sized culture vessel, wherein the container sleeve is configured to transfer thermal energy to the at least one variable-sized culture vessel, A thermal sleeve operably coupled to the container sleeve, wherein the thermal sleeve controls the amount of thermal energy entering the container sleeve, A container clamp for stabilizing the at least one variable-sized culture vessel within the heat sleeve, The system described in item 1, comprising the features described in item 1. (Item 5) The fluid handling system is configured to move a fluid through at least one variable-sized culture vessel, as described in item 1. (Item 6) The system according to item 4, further comprising a sensor control system for controlling the amount of thermal energy entering the container sleeve. (Item 7) The gas management system according to item 1, configured to control the type and amount of gas entering the at least one variable-sized culture vessel. (Item 8) The container clamp is A stretchable device, wherein the stretchable device adapts to the height of at least one variable-sized culture vessel. The system described in item 4, which includes the features described in item 4. (Item 9) The system according to item 4, further comprising an insulating layer between the container sleeve and the environment surrounding the container sleeve. (Item 10) An electrical shutoff device that senses when the thermal sleeve reaches at least one pre-selected threshold temperature, wherein the electrical shutoff device is configured to disable the addition of further thermal energy to the container sleeve. The system described in item 4, which further includes the features described therein. (Item 11) The system according to item 4, further comprising at least one band clamp for securing the heat sleeve to the container sleeve. (Item 12) The aforementioned heat sleeve is The system described in item 4, comprising at least one expansion / contraction gap. (Item 13) The system according to item 4, further comprising a stabilizing pin configured to positionally fix the heat sleeve to the container sleeve. (Item 14) The system described in item 1, further comprising at least one temperature control element. (Item 15) The system according to item 4, further comprising a thermally conductive material for filling the space between the inner diameter of the container sleeve and the outer diameter of the at least one variable-sized culture vessel. (Item 16) The system according to item 4, further comprising a plurality of container sleeves that accommodate a plurality of sizes of the aforementioned at least one variable-size culture vessel. (Item 17) The system according to item 1, further comprising a sensor system configured to monitor a variable volume of cells in at least one variable-size culture vessel. (Item 18) The aforementioned fluid handling system is A variable number of at least one valve and at least one pump configured to move the fluid in and out of the at least one culture vessel station, wherein the at least one controller controls the at least one valve and at least one pump, The system described in item 5, which includes the features described in item 5. (Item 19) The aforementioned at least one controller is The system according to item 1, comprising instructions configured to control multiple culture vessel stations performing independent tasks simultaneously. (Item 20) The system according to item 19, wherein the first of the at least one culture vessel stations performs the first option of the at least one type of tissue-related process in parallel with the second of the at least one culture vessel station performing the second option of the at least one type of tissue-related process. (Item 21) The system according to item 20, wherein the first type of at least one option of the at least one type of organization-related process comprises the same options as the second option of the at least one type of organization-related process. (Item 22) The system according to item 20, wherein the first option of the at least one type of organizational process is a different selection from the second option of the at least one type of organizational process. (Item 23) The first type of the at least one type of tissue-related process is the system according to item 21, comprising decellularization. (Item 24) The first type of the above-mentioned at least one type of tissue-related process is the system according to item 21, comprising recellularization. (Item 25) The first type of the at least one type of tissue-related process is the system according to item 21, comprising cell maturation of recellularized tissue. (Item 26) The first type of the at least one type of tissue-related process is the system described in item 21, comprising perfusion of endothelial cells. (Item 27) The aforementioned at least one controller is The system described in item 1, which includes determining the fluid flow path, at least based on the recipe. (Item 28) The aforementioned at least one controller is The system described in item 1, which includes dynamically determining the fluid flow path. (Item 29) The aforementioned at least one controller is The system described in item 1, which includes determining a fluid flow path based at least on user input. (Item 30) The aforementioned at least one controller is The system described in item 1, which includes determining the fluid flow path based on at least a combination of a recipe, dynamically determined parameters, and user-provided parameters. (Item 31) The aforementioned at least one type of organization-related process is A system described in item 1, which includes a batch process. (Item 32) The system described in item 31, wherein the batch is produced according to at least one industrial standard process. (Item 33) The aforementioned at least one industry standard process is: The system described in item 32, which incorporates ANSI / ISA-88.01-1995. (Item 34) The aforementioned standard industrial communication protocol is: A system as described in item 32, featuring Ethernet® / Industrial Protocol. (Item 35) The aforementioned gas management system is At least one mass fluid controller configured to receive a gas source, wherein the amount of the gas is controlled by the at least one controller, A mixing manifold, wherein the mixing manifold is configured to blend multiple types of gases from a plurality of at least one mass flow controllers, and the amount and type of the multiple types of gases are controlled by the at least one controller, A distribution manifold, wherein the distribution manifold receives the compounded gases and distributes the compounded gases to the at least one culture vessel station according to a command from the at least one controller. The system described in item 1, comprising the features described in item 1. (Item 36) The system according to item 35, wherein the number of controllers of the at least one mass fluid controller is independent of the number of containers of the at least one culture vessel station. (Item 37) The system according to item 36, wherein a plurality of at least one mass fluid controllers are configured to provide the quantities and types of the plurality of gases to a plurality of at least one culture vessel station according to a periodic delivery function. (Item 38) The periodic delivery function is based on values ​​collected by a sensor associated with at least one culture vessel station, according to item 37. (Item 39) A cone feature attached to the shaft of a stirring device in at least one culture vessel, wherein the cone feature substantially prevents cells from settling on the stirring device. The system described in item 1, further comprising the features described above. (Item 40) A method for seeding multiple types of cells on multiple areas of a scaffold, wherein the method is The scaffold is operably coupled to a rotating means within a bioreactor, the bioreactor being configured to receive the plurality of types of cells through a plurality of ports within the bioreactor, When the cell density of the first cell type of the plurality of cell types in the selected culture vessel reaches a pre-selected threshold, or after a pre-selected waiting cycle, (a) Removing excess culture medium from the selected culture vessel, (b) Washing the first cells of the first cell type in the selected culture vessel, (c) Digesting the microcarriers / extracellularly attached proteins in the selected culture vessel using a digestion solution, (d) To suppress the digestive solution, (e) Adjusting the concentration of the culture medium, The first cells are pumped from the selected culture vessel to a first area of ​​the multiple areas of the scaffold in the bioreactor. When the first pre-selected time has elapsed, Processing a second cell type of the plurality of cell types according to steps (a)-(e), The second cell type is pumped from the second of the selected culture vessels to a second area of ​​the multiple areas of the scaffold in the bioreactor, wherein the second cell type is generated in the second of the culture vessels. Methods that include... (Item 41) After the first area has been seeded, the scaffold is rotated, the rotation of which positions the scaffold to receive a second cell type of the plurality of cell types on the second area. The method described in item 40, further including the method described in item 40. [Brief explanation of the drawing]

[0011] The aforementioned features of this disclosure will be more readily understood by referring to the following description, which will be considered with reference to the accompanying drawings.

[0012] [Figure 1A] Figure 1A is a schematic block diagram of a configurable industrial manufacturing system, including the system described in this teaching.

[0013] [Figure 1B]Figure 1B is a schematic block diagram of the system described in this instruction.

[0014] [Figure 1C] Figure 1C is a schematic block diagram of the implementation of the system shown in Figure 1.

[0015] [Figure 1D] Figure 1D-1G shows the components of the implementation in Figure 1C. [Figure 1E] Figure 1D-1G shows the components of the implementation in Figure 1C. [Figure 1F] Figure 1D-1G shows the components of the implementation in Figure 1C. [Figure 1G] Figure 1D-1G shows the components of the implementation in Figure 1C.

[0016] [Figure 2A] Figure 2A is a perspective view of the device implementation of the system described in this instruction.

[0017] [Figure 2B] Figures 2B and 2C are perspective views of the components of the implementation shown in Figure 2A. [Figure 2C] Figures 2B and 2C are perspective views of the components of the implementation shown in Figure 2A.

[0018] [Figure 3A] Figures 3A-3E are schematic diagrams of the implementation of this teaching, including the downstream recirculation system. [Figure 3B] Figures 3A-3E are schematic diagrams of the implementation of this teaching, including the downstream recirculation system. [Figure 3C] Figures 3A-3E are schematic diagrams of the implementation of this teaching, including the downstream recirculation system. [Figure 3D] Figures 3A-3E are schematic diagrams of the implementation of this teaching, including the downstream recirculation system. [Figure 3E] Figures 3A-3E are schematic diagrams of the implementation of this teaching, including the downstream recirculation system.

[0019] [Figure 3F] Figures 3F and 3G are perspective views of the impeller cone for this instruction. [Figure 3G] Figures 3F and 3G are perspective views of the impeller cone for this instruction.

[0020] [Figure 4] Figure 4 is a first-side perspective view of the assembly of this instruction, including a commercial culture vessel.

[0021] [Figure 5] Figure 5 is a perspective view of the assembly of this instruction, including a commercial culture vessel, from the second side.

[0022] [Figure 6] Figure 6 includes bottom and top perspective views of the assembly of this instruction, including a commercial culture vessel.

[0023] [Figure 7] Figure 7 is an exploded perspective view of the assembly of this instruction, including a commercial culture vessel, on the first side.

[0024] [Figure 8] Figure 8 is an exploded perspective view of the assembly of this instruction, including a commercial culture vessel, from the second side.

[0025] [Figure 9] Figure 9 is an exploded perspective view of the container sleeve and thermal sleeve configurations of this instruction.

[0026] [Figure 10A] Figure 10A is a perspective view of the first configuration of the container sleeve of this instruction.

[0027] [Figure 10B] Figure 10B is a perspective view of the second configuration of the container sleeve in this instruction.

[0028] [Figure 11]Figure 11 is a perspective view of the configuration of the thermal sleeve in this instruction.

[0029] [Figure 12] Figure 12 is an exploded perspective view of the container clamp and commercial culture vessel used in this instruction.

[0030] [Figure 13] Figure 13 is a perspective view of the container clamp described in this instruction.

[0031] [Figure 14] Figure 14 is a schematic block diagram illustrating the exemplary use of the gas management system described in this instruction.

[0032] [Figure 15A] Figures 15A-15F are perspective views of the components of the gas management system implementation described in this instruction. [Figure 15B] Figures 15A-15F are perspective views of the components of the gas management system implementation described in this instruction. [Figure 15C] Figures 15A-15F are perspective views of the components of the gas management system implementation described in this instruction. [Figure 15D] Figures 15A-15F are perspective views of the components of the gas management system implementation described in this instruction. [Figure 15E] Figures 15A-15F are perspective views of the components of the gas management system implementation described in this instruction. [Figure 15F] Figures 15A-15F are perspective views of the components of the gas management system implementation described in this instruction.

[0033] [Figure 16] Figure 16 is a flowchart of the exemplary controller process in this teaching.

[0034] [Figure 17A] Figure 17A is a schematic block diagram illustrating an exemplary use of the system described in this instruction for cardiac decellularization.

[0035] [Figure 17B] Figure 17B is a schematic diagram of an exemplary valve configuration for decellularizing the heart using the system described in this instruction.

[0036] [Figure 18A] Figures 18A-18C are schematic diagrams illustrating how the system of this instruction may be used to decellularize the heart. [Figure 18B] Figures 18A-18C are schematic diagrams illustrating how the system of this instruction may be used to decellularize the heart. [Figure 18C] Figures 18A-18C are schematic diagrams illustrating how the system of this instruction may be used to decellularize the heart.

[0037] [Figure 19A] Figures 19A-19C are schematic block diagrams of the implementation of this instruction, in which multiple areas of the scaffold are seeded within the same bioreactor. [Figure 19B] Figures 19A-19C are schematic block diagrams of the implementation of this instruction, in which multiple areas of the scaffold are seeded within the same bioreactor. [Figure 19C] Figures 19A-19C are schematic block diagrams of the implementation of this instruction, in which multiple areas of the scaffold are seeded within the same bioreactor. [Modes for carrying out the invention]

[0038] Detailed explanation The system described herein can follow a process specific to the contents of at least one culture vessel to produce desired results in a controlled environment. The system can adapt to culture vessels of various sizes and shapes, various configurations and numbers of valves, pumps, and sensors, and various types and numbers of fluids and gases, thus enabling a plug-and-play system capable of producing consistent and reproducible results.

[0039] Referring here to Figure 1A, the system in this instruction may be a component of an industrial control system, an embodiment of which is shown in Figure 1A, where information from each component is accessible by other components of the system. Components of industrial systems benefit from compliance with industry-agreed standards such as the Ethernet® / IP protocol and ANSI / ISA-88.01-1995 (ISA-88). The Ethernet® / IP protocol, or Ethernet® Industrial Protocol, is an industrial network protocol that enables Ethernet® to be used as a control protocol. The object library and device profiles associated with Ethernet® / IP enable plug-and-play interoperability between complex devices and support real-time I / O messaging, configuration, and diagnostics over the same network. Ethernet® / IP provides information and control messaging services (see Brooks, EtherNet / IP: Industrial Protocol White Paper, IEEE, EFTA 2001 (October 2001)). ISA-88 consists of models and terminology for structuring production processes and developing equipment controls. ISA-88 is organized into three models: software (procedures), hardware (physical), and material changes (processes) that are performed when the software is executed on the hardware. Each model is organized at the protocol level. Across the models, the protocol levels work together to produce a batch. For example, the procedure model includes four protocol levels: procedure, unit procedure, operation, and phase. The physical model includes two protocol levels: process cell and unit, and optionally, equipment and control levels. The procedure model levels, in combination with the physical model levels, produce the process model levels: process, process stage, process operation, and process action.Compliance with industrial development standards such as ISA-88 can increase the ease of integration with other enterprise standards such as ANSI / ISA / 95. Standards such as ANSI / ISA-88 are used by the system in this teaching to provide a consistent set of processes and terminology for producing batches of material by subjecting a given amount of input material to an ordered set of processing activities over a finite period of time using one or more sets of equipment (see https: / / www.plcacademy.com / isa-88-s88-batch-control-explained / ). The standards provide flexibility for using the system in this teaching, either as a standalone system or integrated into a larger system. At least one integrated programmable logic controller (PLC) in the system can communicate with other controllers along the production line. For example, an industrial system for producing a finite quantity (batch) of cells may include, for example, a cell thawing system 251, a culture vessel system for receiving the thawed cells, and an incubation system 255 for receiving the results from any processes performed in the culture vessel system. Other components of the industrial control system are also assumed by this disclosure. A standard communication system 257 enables data and control sharing between components of the illustrative industrial control system, as described herein with respect to Ethernet® / IP. For example, the thawing controller 259 can communicate the status of its thawing operation to the biocontroller 106 via the standard communication system 257, so that the controller 106 can schedule the processes it expects to perform on the thawed cells. Similarly, the controller 106 can exchange its status with the thawing controller 259 so that the thawed cells can be replaced when the culture vessel system is ready for them. The controller 106 can provide the incubator controller 263 with its status and other information about the processes it is performing, and can provide its own information to the controller 106.Components can be coordinately controlled by system control means that monitor and command them by tracking standard communications between components.

[0040] Referring here to Figure 1B, the system of this instruction is an automated culture vessel system intended for batch production. At a physical level, the system of this instruction may include, but is not limited to, components such as sensors, valves, motors / encoders, pumps, culture vessels, gas management, and controllers. These components may be grouped into subsystems such as fluid handling systems, culture vessel systems, gas management systems, and control systems. The control system, synonymously referred to herein as PLC, can be organized at the protocol level of a procedure model conforming to the ISA-88 standard. The protocol level includes, from the lowest to the highest level in the protocol, device modules and control modules that use the device modules to generate control logic, phases, and recipes (or operations). The device modules establish communication between the device and PLC106. The device modules are logic adapted to faults, operating thresholds, start / stop control, and any basic functions that the device will perform. The control modules may interface with one or more devices that need to cooperate to perform a function. Phases are defined as states that are related to control modules but are also part of a recipe. A phase performs a specific function. An exemplary phase involves the step of adding a certain volume of liquid to a culture vessel. A phase command opens a valve, starts a pump, totalizes the flow until a pre-selected fluid volume is reached, stops the pump, and closes the valve. Exemplary phases in this instruction include, but are not limited to, the steps of pumping culture medium into a culture vessel, removing the medium, heating the contents of the culture vessel, agitating the contents of the culture vessel, harvesting cells, and recirculating the medium. A recipe is a combination of multiple phases that form a complete process.

[0041] Continuing to refer to Figure 1B, ISA-88 states that a recipe contains information in five categories: header, equipment requirements, formulation, procedure, and other information. The procedure category combines the other categories to provide a multi-level hierarchy of recipe procedure elements and contains logic. A control recipe defines the production of a single batch of a specific product and reflects process control. In some configurations, the PLC can execute recipes in conjunction with a batch server and a human-machine interface (HMI). The HMI can, for example, display the results of recipe execution to the operator and receive modifications of flow and configuration information from the operator. In some configurations, commercially available applications can provide implementations of the HMI and batch server. For example, Rockwell's FACTORYTALK® batch server software can run on a WINDOWS® server, drive the HMI, and initiate recipe execution on the PLC. The PLC can periodically access sensor data to understand the current conditions in the culture vessel. In some configurations, the access period is, for example, about 100 milliseconds, but other periods are also assumed by this disclosure. Sensor data values ​​can activate manual or automatic responses by the PLC. For example, in a configuration where pH / dissolved oxygen (DO) is maintained via a proportional-integral-derivative (PID) loop and gas control, the PLC opens / closes valves and sets gas flow rates. The control is based on maintaining a setpoint within the culture vessel. During the decellularization process, the pump rate changes based on the current pressure reading with the goal of maintaining a constant pressure. The PLC takes action based on a recipe, which calls a specific phase. A phase is a sequential operation where the next step cannot proceed until the current step permission, i.e., conditions that must be met before proceeding, are met. In some configurations, fluid handling operations are sequential or recipe-driven operations involving valves, pumps, flow sensors, or level sensors.Incoming sensor data is verified and fault-checked by, for example, but not limited to, noise reduction, outlier detection, missing data imputation, and data aggregation. The incoming sensor data is used in a state flow or sequence to output commands to motors, pumps, and valves, for example, to activate those devices in a certain order according to a recipe. Recipes can be modified dynamically, and the values ​​on which triggers in a recipe depend can also be modified dynamically, i.e., manually or automatically.

[0042] Continuing to refer to Figure 1B, the separate culture vessels do not share any of the same resources or equipment, except for the MFC for gas supply. Although each culture vessel is separate, the same logic exists, which is why they can perform the same or different processes simultaneously. Since constant gas supply is not required to maintain the pH / DO setpoint in the culture vessel, each culture vessel has a time window for receiving gas within it. The processes (cell maturation, proliferation, decellularization, recellularization) determine the pH and DO setpoints. The PLC monitors the time, reads sensor data from the first culture vessel, and supplies gas to the first culture vessel at the desired rate by opening a valve. At the end of the time window for the first culture vessel, the gas valve of the first culture vessel closes, the gas valve of the second culture vessel opens, and the PLC reads sensor data from the second culture vessel and supplies the desired gas mixture to the second culture vessel. Each culture vessel has its own valve, flow sensor, and pump for fluid delivery. The PLC can run different recipes simultaneously on each culture vessel.

[0043] Referring here to Figure 1C, an exemplary implementation of the system of this instruction includes a culture vessel system controlled by a controller 106 and a gas management system 107. In configurations with multiple culture vessel systems, each can be equipped in the same way as others and used to perform various tasks. Each culture vessel system includes a culture vessel 503, a fluid handling system 108, and a culture vessel control system 111. Other configurations are also assumed by this instruction. The culture vessel 503 includes a lidded container equipped with a sensor, at least one fluid inlet, at least one fluid outlet, and at least one gas inlet. The lid can accommodate tubing appropriate for the task being performed by the culture vessel station. For example, if a heart is being recellularized, the lid can accommodate tubing connecting the descending aorta, pulmonary artery, and pulmonary vein to receptacles and / or nutrient sources outside the culture vessel system. Similarly, if the kidney is being recellularized, the lid may accommodate tubing connecting the arteries, veins, and urethra to receptacles and / or nutrient sources outside the culture vessel system. The fluid handling system 108 includes at least one pump, a valve, and a sensor used to move fluid through the contents of the culture vessel 503. The culture vessel control system 111 maintains setpoints for various properties of the contents of the culture vessel 503 by monitoring sensor data associated with the contents of the culture vessel 503. The culture vessel control system 111 provides these setpoints and sensor data to the controller 106 and receives commands from the controller. The controller 106 has access to recipes or other forms of command structures whose execution implements the tasks that the culture vessel system is to perform. The controller 106 can adapt to changes in recipes, whether they are manually entered or dynamically determined. The gas management system 107 is shared by all culture vessel systems, each receiving the gas mixture required to maintain homeostasis of the contents of the culture vessel. The system described in this instruction can control and monitor the reception and circulation of fluids and the supply of gases to the fluid, enabling desired results regarding the contents of the culture vessel.In one aspect, the controller 106 can simultaneously control processes performed in each of the multiple culture vessel stations 503. In another aspect, the controller 106 may include multiple processors capable of controlling the culture vessel stations 503. In one aspect, a single gas management system 107 can control the flow of a single gas. In another aspect, a single gas management system 107 can control the flow of multiple gases. In another aspect, a single gas management system 107 can supply gas to a single culture vessel station 503. In another aspect, a single gas management system 107 can supply gas to multiple culture vessel stations 503. In another aspect, multiple gas management systems 107 can supply gas to a single culture vessel station 503. In another aspect, multiple gas management systems 107 can supply gas to multiple culture vessel stations 503. The culture vessel 503 may include, but is not limited to, the configurable vessel assemblies of this teaching.

[0044] Continuing to refer to Figure 1C, in some respects, the system of this teaching can decellularize scaffolds. In some respects, the system of this teaching can recellularize scaffolds. In some respects, the system of this teaching can provide an environment in which cells can mature and tissues can grow. In some respects, the system of this teaching can be adapted to containers of sizes such as, for example, 0.1 to 1,000 liters, but not limited to these. In some respects, the system of this teaching can be used to generate biopharmaceutical products such as, for example, vaccines, blood, blood components, allergens, genes, viruses for gene therapy, cosmetics, and proteins, but not limited to these.

[0045] Referring again to Figure 1B, the fluid handling system 108 moves culture medium and other fluids through the contents of the culture vessel 503. The type of fluid, the fluid pressure, and the fluid flow rate are determined by a combination of factors, e.g., pre-selected processes associated with desired outcomes, pre-selected setpoints for various properties required to produce the desired outcomes, dynamic characterization, and user input. In some configurations, the system accesses a recipe that determines the actions that will produce the desired outcomes. Each action is characterized by a set of phases or commands that are performed to accomplish a step in the process. In the fluid handling system 108, one action may include a step to access the type of fluid required to accomplish the step. Another may include a step to configure the PLC 106 with setpoints for various properties such as the pH of the circulating fluid, the dissolved oxygen (DO) content of the circulating fluid, and the temperature of the circulating fluid. Yet another may include a step to configure at least one pump to move the fluid through the culture vessel 503 past sensors. Fluid control can be governed by flow rate or pressure. The components of the fluid handling system 108 may include, but are not limited to, inline flow and pressure sensors, liquid level sensors, pumps, and pinch valves for directing the flow.

[0046] Referring here to Figure 1D, an implementation of the fluid handling system 108 (Figure 1B) of this teaching may include at least one fluid pump 619 per culture vessel 601, operably coupled with a stepper motor 621 and a motor drive 623. The motor drive 623 can communicate with a PLC 631, for example, by an Ethernet® IP connection 625, and can assist with standard motor operations such as motor start, stop, direction, rate, alarms, and status. The pump 619 can circulate the fluid 611 through the culture vessel 601, possibly through tubing that limits fluid contact to the inner diameter of the tubing, to the waste 615. A pump pressure sensor 617 can enable control of the fluid flow rate of the fluid 611 into the culture vessel 601 based on an in-line pressure reading upstream of the tissue in the culture vessel 601. In this configuration, if the pressure reading is outside a pre-selected range surrounding a pre-selected setpoint, for example, 5 mmHg higher or lower, the pump speed is changed by a pre-selected percentage, either higher or lower, depending on the pressure value. For example, the pump speed may be changed by 10%, either higher to increase the pressure or lower to decrease the pressure. The new pressure can be evaluated over a pre-selected time, for example, 5 seconds, and the process can be repeated. If the pressure reading is within the pre-selected range, no pump speed change is made. The pressure setpoint for the function is determined by the recipe. The pump pressure sensor 617 converts the outlet pressure from the pump 619 into an electrical signal that the variable speed pump uses to regulate the pump speed. In some configurations, a high cutout pressure switch can be used to prevent the pump 619 from outputting extreme pressures. The pump pressure sensor 617 may be mounted above the pump inlet to monitor efficiency and improve the efficiency and reliability of the pump 619.

[0047] Referring here to Figure 1E, the culture vessel or closed culture vessel system of this teaching includes a configurable vessel assembly system that provides heating, positioning, and structural support for the culture vessel 601 and also provides a method for including a vessel that is appropriately sized for a given process with little or no operation required by the user. The system of this teaching can, for example, culture and produce various cell types and fungal and bacterial cultures. The system can be used to produce viruses and proteins using cultured cells. The system can be configured for various shapes, sizes, and types of culture vessels, including, but not limited to, glass and plastic containers that are durable and disposable. Exemplary types of culture vessels may include, but not limited to, stirring vessels, packed beds, roller bottles, oxygen-permeable culture plates, microfluidic slides, or hydrogel culture vessels. The culture vessel system of this teaching includes an integrated sensor that communicates with a PLC, as described elsewhere in this specification.

[0048] Continuing to refer to Figure 1E, the configurable container assembly system of this instruction can be part of a flexible configuration to adapt to various processes. A given cell proliferation process may require a specific container volume to expand to a desired number of cells, while alternative processes may require different container volumes. The container size can be established to suit the desired process without modifying anything other than the container sleeve in the flexible configuration, possibly including the heating elements, electrical connections / wiring, control logic, calibration, and positioning adjustments of the container. If the user wishes to change the container size, for example, from 2L to 0.5L, only a simple swap of components is required, and nothing more. In some configurations, the user can simply remove the 2L container sleeve and replace it with a 0.5L container sleeve, each having the same outer diameter. After tightening the clamps on the outside of the heating sleeve surrounding the container sleeve to ensure that the 0.5L container sleeve is secured, the assembly reconfiguration is complete and ready for operation with the 0.5L container. This can be done with respect to various sizes, but is not limited to, for example, 0.5L, 1L, 2L, 3L, and up to 100L. Commercial culture vessels can be mounted on the adapter ring of the assembly. The adapter ring is mounted in a temperature control ring or thermal sleeve having a tightening feature to ensure good thermal contact between the two components. The thermal sleeve can control the amount of thermal energy entering the container sleeve. The assembly may include a container clamp to stabilize the culture vessel within the thermal sleeve. If different sized vessels are desired, different sized vessels can be mounted in appropriately sized container sleeves and placed in the same thermal sleeve as other sized vessels. Thus, the basic system remains the same regardless of the vessel size. A clamp-type heating ring can stabilize the vessel. The container clamp may optionally include an expandable device. The expandable device can adapt to various heights of various different culture vessels and can vary the height of the specific culture vessel.The assembly may optionally include an insulating layer between the container sleeve and the environment surrounding it, an electrical shutoff device for sensing when the thermal sleeve reaches at least one pre-selected threshold temperature, the electrical shutoff device for disabling the transfer of thermal energy to the container sleeve, and at least one band clamp for securing the thermal sleeve to the container sleeve. The thermal sleeve may optionally include an expansion / contraction gap. The assembly may optionally include a stabilizing pin for positionally securing the thermal sleeve to the container sleeve, at least one temperature control element, and a thermally conductive material for filling the space between the inner diameter of the container sleeve and the outer diameter of the culture vessel.

[0049] Continuing to refer to Figure 1E, the configurable container assemblies of this instruction for processing cells in a controlled environment may include, but are not limited to, a container sleeve enclosing at least a portion of the culture vessel. The container sleeve may be configured to transfer thermal energy to the culture vessel. The assembly may include a thermal sleeve operably coupled to the container sleeve. The thermal sleeve can control the amount of thermal energy entering the container sleeve. The assembly may include a container clamp for stabilizing the culture vessel within the thermal sleeve. Optionally, the assembly may include a fluid handling system for moving fluid through the culture vessel and a sensor control system operably coupled to the thermal sleeve. The culture vessel control system 111 can provide data that enables control of the amount of thermal energy entering the container sleeve. The container clamp may optionally include a stretchable device. The stretchable device can adapt to various heights of various different culture vessels and can vary the height of a specific culture vessel. The assembly may optionally include an insulating layer between the container sleeve and the environment surrounding it, an electrical shutoff device for sensing when the thermal sleeve reaches at least one pre-selected threshold temperature, the electrical shutoff device for disabling the transfer of thermal energy to the container sleeve, and at least one band clamp for securing the thermal sleeve to the container sleeve. The thermal sleeve may optionally include an expansion / contraction gap. The assembly may optionally include a stabilizing pin for positionally securing the thermal sleeve to the container sleeve, at least one temperature control element, and a thermally conductive material for filling the space between the inner diameter of the container sleeve and the outer diameter of the culture vessel.

[0050] Continuing to refer to Figure 1E, the culture vessel 601 may include thermal control, agitation control, and sensors to control the properties of the contents of the culture vessel 601, such as temperature, pH, pO2, agitation, and pressure. In some processes, a temperature of 37°C is optimal for cell maintenance. Temperatures slightly above or below 37°C may also affect cell viability and cellular metabolism. Maintaining a desired temperature is made possible by a temperature sensor 659, which reads the temperature value of the contents of the culture vessel 601 and then transmits a signal to the culture vessel controller system 111, thereby adjusting the temperature as needed by controlling a heating / cooling device, if present.

[0051] Continuing to refer to Figure 1E, the level sensor 613 can monitor the liquid level in the culture vessel 601. The liquid level can be used to control the amount of fluid added during the fluid transfer process and to generate an alert if the level is too high or too low. Assuming that the density of vapor in the space not occupied by the fluid 611 in the culture vessel 601 is much lower than the density of the fluid 611, the types of level sensors include, but are not limited to, glass level gauges, floats, displacers, bubblers, differential pressure transmitters, load cells, magnetic level gauges, capacitive transmitters, magnetostrictive level transmitters, optical, vibration, ultrasonic, laser level transmitters, and radar level transmitters. In some configurations, the level sensor can provide a transducer output signal in the form of a 4-20 mA current loop to the PLC 631, for example, through an analog input module 627.

[0052] Continuing to refer to Figure 1E, as an alternative to, or in addition to, the level sensor 613, a fluid flow meter 605 can measure the flow entering and exiting the culture vessel 601, for example, in mL / min units. A totalization function can be used during culture medium transfer to calculate the total volume of fluid transferred by integrating the flow over the pump cycle. Categories of fluid flow meters include, but are not limited to, differential pressure, velocity, volumetric, mass flow, and open channel. Differential pressure flow meters include orifice plates, venturi tubes, flow nozzles, and variable area rotometers. Velocity flow meters include Piet tubes, calorimetric flow meters, turbine flow meters, vortex flow meters, electromagnetic flow meters, ultrasonic Doppler flow meters, and time-of-flight flow meters. Volumetric flow meters can include reciprocating piston meters, oscillating disk meters, and rotating vane meters. Mass flow meters include thermal flow meters and Coriolis flow meters. Either the level sensor 613 or the flow meter 605 can assist dose control 603 by calculating the amount of fluid removed from or added to the culture vessel 601. Dose control 603 can track the added volume and, once a desired setpoint is reached, can stop the addition of further fluid. Dosage may, as a possibility, include the ability to start / stop the pump, report any shortages or overages from the allowable volume, and alert and report the status of delivery under pre-selected circumstances. Dosage devices fall into two main categories: gravimetric and volumetric. The selection of a dosage device is based on the fluid flow rate and the desired fluid rate. Dosage can be automatic, programmable, or continuous and can be controlled, for example, by a dosing valve. In some configurations, dose control 603 can be performed by adding commands to the level sensor 613 or flow meter 605 processing.

[0053] Continuing to refer to Figure 1E, in some configurations, the system can include thermal control. In one configuration, the system can include active heating and passive cooling. At least one temperature control mechanism can reside within the container system itself, at least one temperature sensor can reside outside the container to sense the temperature of the temperature control mechanism, and at least one temperature sensor can reside inside the container to sense the temperature of the contents of the container. In some configurations, the desired temperature for the contents of the culture vessel is in the range of 34–39°C. In some configurations, the temperature threshold can include a temperature of 65°C for the contents of the culture vessel. In some configurations, temperature control is performed using a cascaded PID loop, where the inner loop controls the temperature control mechanism and the outer loop controls the temperature of the contents of the container. In some configurations, the temperature control mechanism can be powered by 110V. In such configurations, a solid-state relay or a 24V output card can be used to pulse-wave modulate the temperature control mechanism. Thermal control to a set point in this teaching is achieved actively and passively. In some configurations, heating of the contents of the culture vessel is achieved actively, while cooling of the contents is achieved passively. Other configurations, such as active cooling and passive heating, and active cooling and heating, are also assumed by this instruction. In some configurations, the setpoint is a pre-selected value, such as 37°C, but is not limited to this. In some configurations, heating can be limited to a pre-selected temperature, such as 40°C. Sensors report the temperature of the contents of the culture vessel 631 to the culture vessel control system 111, and these sensor data trigger thermal control. In some configurations, a temperature probe 659 is mounted on the head plate of the culture vessel 601. The temperature probe 659 reads the temperature of the fluid in the culture vessel 601. A heater RTD 657 is mounted on heater 655, which heats the fluid in the culture vessel 601. Data from the sensor RTD659 and heater RTD657 is received by the PLC631 via the culture vessel control system 111 through the RTD I / O card 629, which is designed to receive such data.In some configurations, temperature control is performed by a cascaded PID loop, where the inner loop controls the temperature (PWM) of the heater 655, and the outer loop controls the temperature of the contents of the culture vessel 601. The PID output drives the power input to the heater 655. Other methods of thermal control are also envisioned in this disclosure. For example, a digital temperature sensor or a silicon-based linear thermistor may be used to measure the temperature of the sensor and the contents of the culture vessel 601.

[0054] Continuing to refer to Figure 1E, the culture vessel 601 may contain tissue that requires migration for proper growth. For example, the contents of the culture vessel 601 may require agitation, depending on the specific process and desired outcome, to improve gas perfusion and, potentially, thermal control. Other reasons for agitating the contents of the culture vessel 601 may include producing a uniform dispersion of gas bubbles, producing small gas bubbles, maximizing the residence time of gas in the fluid by driving gas bubbles to the bottom of the culture vessel, reincorporating nutrients to return from the top of the culture vessel into the contents of the culture vessel 601, and providing a uniform nutrient and temperature profile throughout the contents. The agitation device may include, for example, a mechanical agitation device driven by a brush or brushless DC motor 663. Mechanical agitation devices are categorized by the flow direction in which the fluid is mixed, e.g., axial, radial, compound, and dispersed flow, and may include, for example, propellers and impellers. Propeller dimensions and pitch are features selected based on the application. Exemplary impellers include pitch blades (for axial flow), rushton (for radial flow), angled pitch blades (for compound flow), and helical (for dispersed flow). In some configurations, the agitator 661 may include a marine propeller mounted on a shaft coupled to a stepper motor 663, and the combination is used to support applications such as cell processes. In some configurations, the PLC 631 controls the motor 663 and monitors its status by using commands transmitted and received data through communication media and protocols such as Ethernet® IP625, but not limited to these. The propeller speed and direction are selected based on the application.

[0055] Continuing to refer to Figure 1E, the success of applications using the culture vessel 601 can depend on the various characteristics of the system and the monitoring of the contents of the culture vessel 601. As described herein, sensors can be advantageously positioned to measure aspects of the process in progress. For example, turbidity, pH, DO, glucose, and lactate levels can be measured by inline sensors 669 that provide data to the culture vessel control system 111. Such sensors can be invasive, minimally invasive, or non-invasive. Data from the sensors can be provided from the culture vessel control system 111 to the PLC 631, for example, but not limited to, serial connection 665 (Figure 1C), USB connection (not shown), Ethernet® IP connection 625 (Figure 1C), or wirelessly. In some configurations, glucose and lactate levels can be collected as the fluid circulates through the tubing of the system and the culture vessel 601.

[0056] Continuing to refer to Figure 1E, the configurable container assembly system of this instruction can be part of a flexible configuration to adapt to a specific cell culture process. A given proliferation process may require the specific container volume to expand to a desired number of cells, while an alternative process may require a different container volume. The size of the culture container 601 can potentially be established to fit the desired process without modifying anything other than the container sleeve in the flexible configuration. The heating elements, electrical connections / wiring, control logic, calibration, and positioning adjustments of the culture container 601 can remain identical across changes in the physical size of the culture container 601. If the user wishes to change the container size, for example, from 2L to 0.5L, only a simple swap of components is required, and nothing more. In some configurations, the user can simply remove the 2L container sleeve and replace it with a 0.5L container sleeve and culture container 601, each container sleeve having the same outer diameter. After tightening the clamps on the outside of the heat sleeve surrounding the container sleeve to ensure the 0.5L container sleeve is secured, the assembly reconstruction is complete and ready for operation with the 0.5L container. This can be done for various sizes, but is not limited to, 0.5L, 1L, 2L, 3L, and up to 100L. Commercial culture vessels can be mounted on the adapter ring of the assembly. The adapter ring is mounted within a temperature control ring with a tightening feature to ensure good thermal contact between the two components. If different sized containers are desired, different sized containers can be mounted in appropriately sized container sleeves and placed within the same heat sleeve as other sized containers. Thus, the basic system remains the same regardless of container size. A clamp-type heating ring can be used to stabilize the container.

[0057] Referring here to Figure 1F, the gas management system 107 (Figure 1B) may include a gas source 609 for each mass fluid controller (MFC) 608 or functionally equivalent device, which combines a mass fluid sensor and a control valve to control the gas flow rate to a desired amount, unaffected by changes in operating conditions or gas pressure. The MFC 608 may be analog or digital, depending on the number of gas types being controlled. The gas source 609 may contain one or more types of gas, the type to be selected may be determined by the desired outcome from the process enabled by the system of this teaching. The gas management system 107 (Figure 1B) may include a gas manifold 607 that receives gas from all selected gas sources and supplies a single gas flow to the culture vessel, which is its turn to receive the selected gas. The PLC 631 (Figure 1C) selects the type of gas to be supplied to the culture vessel 101 (Figure 1B) (also referred to herein as the bioreactor) and achieves gas equilibrium which will enable the desired outcome. The system may include multiple MFC608s and associated gas sources, but not necessarily the same number as the number of culture vessel stations 601 (Figure 1C). The gas management system 107 (Figure 1B) manages the gas flow from the available MFC608s / gas sources 609 to meet the component requirements of the culture vessels 601 (Figure 1C), regardless of the number of each component, as described elsewhere in this specification. In one aspect, the MFC608s are used to measure the total mass flow rate of the gas flowing through closed conduits. In another aspect, the gas management system 107 (Figure 1B) may include pressure regulators and filters associated with various types of gas to prepare the gas flow for the MFC608s. In another aspect, compressed air may be exposed to a combination filter that filters out particles and various types of gas from the compressed air flow, as is possible. This teaching assumes combinations of filters and other types of filters for all types of gas supplied to the system.In one aspect, oxygen, nitrogen, and carbon dioxide are supplied to the culture vessel in various amounts determined by the PLC631 (Figure 1C). In another aspect, the gas management system 107 (Figure 1B) includes a gas manifold with automatic switching to ensure a continuous gas supply. Switching can be enabled mechanically or pneumatically, and the system can be alerted when switching occurs so that a new gas source can be connected. The system may include a gas mixer block to provide a mixture of available gases to the culture vessel 601 (Figure 1C). The PLC631 (Figure 1C) accesses and processes instructions to achieve desired results from sources such as, but not limited to, recipes, user inputs, and sensor inputs.

[0058] Referring here to Figure 1G, the sensors, positioned throughout the system in this teaching, can provide analog and digital inputs / outputs that can arrive at the PLC631 via serial, parallel, or communication ports. Inputs / outputs (I / O) from the sensors can be enabled by commercially available or custom I / O cards that provide an interface to the PLC631. I / O cards can be connected to the PLC631 via a backplane and may be configurable by the PLC631. I / O can also be enabled by communication cards, such as the Ethernet® IP card 625, which enables the use of communication protocols. Such device communication is routed through an Ethernet® switch connected to a port on the PLC631. In some configurations, the PLC631 is wired directly to a block of I / O cards and connectors. In some configurations, electrical signals can include, but are not limited to, 4-10mA current loops (analog), RS-485 (serial), or 0-24V (digital). In some configurations, Ethernet® connectivity uses RJ45 connectors, and serial connectivity uses DB-9 connectors. Depending on the sensor, the PLC631 can enable the configuration of, for example, scaling and data transmission / reception protocols. The PLC631 includes code for building control loops around the sensors based on the desired use of the system. Sensors can be passive or, for example, control pumps based on pressure. Sensors can detect, for example, tissue temperature, fluid pressure, fluid temperature, and fluid properties. Sensors are mounted throughout the system; for example, a temperature sensor may be associated with the bioreactor 601 (Figure 1C) and fluid 611 (Figure 1C), and a fluid pressure sensor may be mounted throughout the system. Sensors can provide data to the PLC631 (Figure 1C) and, potentially, receive commands from the PLC631 (Figure 1C) either wired or wirelessly, and both can be secured from external or man-in-the-middle interference.Some sensors may be disposable and configured to make contact with the fluid 611 (Figure 1C), while others may be durable and configured to read data non-contact. Spot sensors can be mounted, for example, on the outside of the bioreactor 601 (Figure 1C). In some configurations, non-contact fluid pressure sensors 617 (Figure 1C) can be mounted in series anywhere on the tubing used to route the fluid 611 (Figure 1C), for example, by Luer lock ends, but not limited to. In some configurations, sensors communicating with PLC 631 (Figure 1C) can sense 0 to 60 psi along the fluid path. Signals from the sensors, for example, 4 to 20 mA current loops, analog voltages, or digital voltages, can be communicated from the sensors to PLC 631 (Figure 1C) through, for example, an analog input module 627.

[0059] Referring here to Figures 2A-2C, the hardware implementation of the system of this teaching is shown. Four culture vessel implementations 707 are shown. Also shown is the implementation of elements of the fluid handling system 108 (Figure 1B), which includes a fluid delivery encapsulation 715 associated with the culture vessel implementations 707 as shown in Figures 1B and 1E. Specifically, the fluid delivery encapsulation (FDE) 715 includes a valve 701, a pump 703, and a pipe guide 713. The fluid flow line is shown elsewhere in this specification. Also shown is the cable / sleeve snorkel 705, which is fully described by at least White et al.'s U.S. Patent Application No. 29 / 758,774, “Cable / Tube Sleeve and Snorkel,” filed November 18, 2020, and White et al.'s U.S. Patent Application No. 17 / 522,003, “Cable / Tube Sleeve and Snorkel,” filed November 9, 2021 (both incorporated by reference). A connector bank 713A is shown. Electrical connections between the sensor and the PLC are routed through the connector bank 713A. A waste collector 708 and a human-machine interface display 711 are shown. In this implementation, the culture station is movable for positioning along the assembly line. A cleanroom is not required because there is virtually no exposure of the contents of the culture vessel 707 or its associated fluids and internal tubing to the environment. Each bioreactor channel, i.e., the culture vessel assembly 707, valve 701, and pump 703, can be configured differently based on the desired outcome. In some configurations, the FDEs are mechanically stackable and mate together using interlocking plates and latches. Each bioreactor channel can be associated with a pre-selected number of FDEs, each having a unique configuration. Each FDE can include several pumps 703, valves 701, and tubing routing guides 713. In some configurations, the system of this teaching can be adapted to multiple pump sizes and fluid valves.The size and number of valves and pumps can be mixed and matched based on the desired outcome.

[0060] Referring here to Figures 3A-3E, schematic diagrams of exemplary implementations of the fluid and gas lines and components of the system of this instruction used for cell maturation are shown. Figures 3A-3B show components of a gas management system described elsewhere in this specification. In this implementation, compressed air 801 (Figure 3A) is routed to a pneumatic block 809 (Figure 3B) through an air filter and regulator 806 (Figure 3A), such as a biological filter. Nitrogen 803 (Figure 3A), oxygen 805 (Figure 3A), and carbon dioxide 807 (Figure 3A) are routed to a thermal MFC 811 (Figure 3B) through a filter 810 (Figure 3B) via a pressure valve 802 (Figure 3A) and a pressure regulator 804 (Figure 3A). The MFC 811 (Figure 3B) is commanded (by the controller) to supply a gas volume that will produce the correct gas mixture 812 (Figure 3C) for the specific activities that will occur in the bioreactor 821 (Figure 3D).

[0061] Referring here to Figures 3C-3D, the components shown are those of a fluid management system described elsewhere in this specification. Figure 3C shows the fluid source providing and pumping components. In some configurations, possible fluids are growth medium 813 (Figure 3C), buffer solution 815 (Figure 3C), and trypsin 817 (Figure 3C), which can be selectively pumped into the bioreactor 821 (Figure 3D) by pump 819 (Figure 3C). In this embodiment, container 814 (Figure 3C) is suspended from a load sensor 820 (Figure 3C) that can measure the volume of fluid delivered as the fluid, such as growth medium 813 (Figure 3C), is delivered. The amount of fluid to be delivered can be controlled by using data provided to the controller by the load sensor 820 (Figure 3C). Exemplarily, the quality of the growth medium 813 (Figure 3C) is maintained by a refrigerator 816A (Figure 3C). It should be noted that the delivery volumes of both the buffer solution 815 (Figure 3C) and the trypsin 817 (Figure 3C) can be measured, for example, by a load sensor 820 (Figure 3C) as described herein. The fluid supplied from the source is pumped by a pump 819 (Figure 3C) controlled by a controller. Pump 819 (Figure 3C) may include, for example, a non-contact pump that has a high suction head while providing a gentle pumping action to avoid hemolysis. Such a pump can pump a slurry, is reversible, and can provide precise dosing. Measurement of fluid pressure and air in the fluid is performed, for example, by an inline pressure transducer 816 (Figure 3C) and an inline bubble sensor 818 (Figure 3C), and is provided to the controller for monitoring and future control of pump 819 (Figure 3C).

[0062] Referring here to Figure 3D, gases and fluids are supplied to and pumped from the bioreactor 821 to provide products of the cell maturation process to meet the needs further down the production line. For example, as shown in Figure 3D-3E, a configuration may provide recirculation to enable incubation. The supplied gas mixture, as shown in Figure 3B, is filtered by a gas filter 822 (Figure 3D) to remove viruses and microorganisms from the gas mixture as it proceeds to the bioreactor 821 (Figure 3D). Filtration can be performed by, for example, a filter sized for unwanted particulate matter, an inertial separator, or electrostatic separation techniques, or a combination thereof. Fluids are also supplied to the bioreactor 821. As they are added, the bioreactor 821 is ventilated, for example, to the atmosphere. As the escaping air leaves the bioreactor 821, it is cooled by an exhaust air conditioner 826 so that the fluids condense. The system is configured such that gravity pulls condensate back into the bioreactor 821 (Figure 3D), and thus only air is vented to the atmosphere. In this way, the filter 824 remains dry, allowing for continuous airflow, and in addition, the culture vessel remains hydrated. The exemplary cooler 828 (Figure 3D) may include, but is not limited to, a Peltier device. The filter 824 (Figure 3D), through which the vented air must pass, is used to prevent contaminants from entering the bioreactor 821 from the atmosphere. Thus, the filter 824 (Figure 3D) can be configured to remove contaminants that are expected to be present in the environment of the bioreactor 821 (Figure 3D), and also to protect the environment from contamination. For example, in some configurations, contaminants as small as 0.2 microns can be removed. The exemplary filter can be constructed from, for example, polypropylene and PTFE, and can allow the pressure within the system to be equal to the external atmospheric pressure. Such filters may be, for example, bidirectional, hydrophobic, and autoclavable.The fluid level within the bioreactor 821 (Figure 3D) can be measured and provided to a controller to monitor the operation of the system, as described elsewhere in this specification. For example, but not limited to, properties of the contents of the bioreactor 821 (Figure 3D), such as pH and DO, can be measured, and the sensors for these are shown in Figure 3D.

[0063] Continuing to refer to Figure 3D, the temperature of the contents of the bioreactor 821 (Figure 3D) can be controlled by a combination of a heat maintenance device 830, a bioreactor thermal sensor 834 (Figure 3D), a bioreactor contents thermal sensor 836 (Figure 3D), and a controller to which this data is routed. Depending on the desired process, the reaction within the contents of the bioreactor 821 (Figure 3D) may be endothermic, exothermic, or static. Therefore, the heat maintenance device 830 (Figure 3D) must be capable of raising or lowering the temperature of the contents or maintaining a static thermal state. The heat maintenance device 830 (Figure 3D) may take the form of, for example, a heat exchanger in a coil, a heating blanket, or a jacket recirculation system. The bioreactor thermal sensor 834 (Figure 3D), together with the bioreactor contents thermal sensor 836 (Figure 3D), can provide the data required by the controller to provide thermal control to the contents.

[0064] Referring here to Figures 3D and 3F, the bioreactor 821 (Figure 3D) can be durable and constructed from, for example, stainless steel or glass, or single-use and possibly pre-sterilized, from polymer materials. The material specifications of the bioreactor 821 (Figure 3D) may affect thermal control and may be taken into consideration by the controller when determining the desired temperature of the contents. The contents of the bioreactor 821 (Figure 3D) may require movement depending on the process performed within the bioreactor 821 (Figure 3D), for example, by stirring (propulsion), suction (push), agitation, use of baffles, or by providing gas flow and removing waste products such as carbon dioxide. Movement may be driven from top or bottom drive devices, for example, mechanically or magnetically driven. A stirrer 832 (Figure 3D) is one method for providing such movement. The impeller may include properties such as axial and radial fluid flow, and the propeller may include various shapes such as pitch blades or marine. An appropriate type of propeller / impeller can be selected based on the application. An impeller cone feature 852 (Figure 3F) is positioned on the shaft of the impeller via a shaft cavity 856 (Figure 3F), coinciding with the upper surface of the impeller. The cone feature 852 (Figure 3F) prevents cells or equivalents from settling on the flat surface of the impeller when agitation is complete, allowing the cells or equivalents to flow off the impeller and settle at the bottom of the culture vessel, where they can be collected by an immersion tube. The cone feature 852 (Figure 3F) includes a hole 854 (Figure 3F) for housing a set screw, which is configured to hold the cone feature 852 (Figure 3F) in place on the shaft of the impeller. The cone feature 852 (Figure 3F) includes a compression fit around the shaft of the impeller and does not include any flat area. The imperafin 880 (Figure 3G) is attached to the bottom of the culture vessel at an angle that encourages the cells to slide, instructing them to be captured by tubes during harvesting.

[0065] Continuing to refer to Figure 3D, in this configuration, a sample can be taken from the fluid within the system in a fluid line that enters and exits the bioreactor 821 (Figure 3D). Further sampling ports at different locations are also assumed by the system of this instruction. Pump 838 can enable recirculation in the system, such as in the system shown in Figures 3D-3E, for example, in which the contents of the bioreactor 821 (Figure 3D) are pumped downstream to enable the use of cells produced by the system, or pumped to waste 823 (Figure 3D). In the configuration shown, the collected cells and culture medium can be pumped downstream, and the culture medium from the downstream process can be recirculated into the bioreactor 821 (Figure 3D). This configuration includes tubes extending into the bioreactor 821 (Figure 3D) to perform various functions, and the amount they extend can be adjusted according to the desired function. For example, when the agitator 832 is not activated, the cells and microcarriers will settle to the bottom of the bioreactor 821. One of the tubes can extend into the mixture but not to the bottom of the bioreactor 821 and can be used to remove the cells without removing the culture medium. Another tube can extend further into the mixture and can be used to remove the cells for use in downstream processes, such as seeding in other bioreactors. Another use for the longer tube may be to completely remove the culture medium from the bioreactor 821 prior to adding fresh culture medium or a different type of culture medium.

[0066] Referring here to Figure 3E, the downstream process is shown. In the exemplary configuration, an incubator 851 (Figure 3E) houses a bioreactor 859 (Figure 3E). Carbon dioxide 857 (Figure 3E) is supplied to the incubator 851 (Figure 3E) to establish the environment for the bioreactor 859 (Figure 3E). In the incubator 851 (Figure 3E), the bioreactor 859 (Figure 3E) is rotated by a motor 861, as an example of what a consumer of the batch produced by the system of this teaching may do. Cells and culture medium 853 (Figure 3E) produced by the system of this teaching are also supplied to the bioreactor 859 (Figure 3E). Culture medium and possibly other outputs 855 (Figure 3E) are recirculated to the bioreactor 821 (Figure 3D). The characteristics of the inflow and outflow fluids (to the bioreactor 859 (Figure 3E)) can be monitored. For example, the pressure and bubbles in the inflow fluid can be measured and adjusted to ensure that the cells arrive at their destination intact, and the pH, DO, and bubbles in the efflux medium can be measured and adjusted before re-entry into bioreactor 821 (Figure 3D). The system in this teaching assumes other types of properties that can be measured.

[0067] Referring here to Figure 4-6, the culture vessel system 101 (Figure 1B) may include features that allow the use of culture vessels of various sizes and the establishment of various environments around the culture vessels. The culture vessel system 101 (Figure 1B) may also include features that hold the culture vessels in place, for example, to maintain an interface with a thermal management system.

[0068] Continuing with reference to Figures 4-6, various sizes of culture vessels can be adapted in the culture vessel system 101 (Figure 1B). Exemplary embodiments shown in Figures 6 and 7 illustrate a culture vessel 102 that is captured within a vessel clamp 120 (more specifically shown and described herein with respect to Figure 13). The culture vessel 102 may include commercially available bioreactors or custom-designed bioreactors. The system of this teaching can be adapted to various sizes of culture vessels, and therefore, the culture vessel 102 is one embodiment. The telescopic sleeve 123 and support column 125 of the vessel clamp 120 (Figure 13) can be extended / retracted to accommodate the height of various culture vessels. The height of the exemplary culture vessel 102 determines the slight extension of the telescopic support column 125, which is locked in place by fasteners set within the cavity 132.

[0069] Continuing to refer to Figure 4-6, the container clamp 120 (Figure 13) stabilizes the container sleeve 115 (Figure 10), drawing the culture vessel 102 into the container sleeve 115 (Figure 10A), and thus into the heat sleeve 117 (Figure 11), ensuring thermal conductivity between the culture vessel 102 and the heat sleeve 117 (Figure 11). The culture vessel clamp ensures that the position of the culture vessel relative to the container sleeve and the temperature control system is maintained, enabling uniform temperature control of the contents of the culture vessel.

[0070] Continuing with Figure 4-6, the temperature control system can be configured to maintain a desired temperature for the contents of the culture vessel. In some configurations, the temperature control can be partially or completely integrated with the culture vessel 102. In one arrangement, a vessel sleeve 115 (Figure 10A) can allow thermal conduction between the culture vessel 102 and a heat sleeve 117 (Figure 11). The culture vessel 102 can be secured to a vessel clamp 120 (Figure 13) by a fitting that surrounds the cap 204 of the culture vessel 102. The fitting may include multiple interconnected parts that can be configured to adapt to the size of the cap 204. For example, the fitting may include a vessel clamp ring stand 129 that is mounted on a telescopic support 125 and can provide a mounting surface for a bracket base 128. The cap 204 can be resting on the vessel clamp ring stand 129 and vertically clamped by at least one headplate bracket 127 that can be operably coupled to the bracket base 128. In some configurations, the bracket base 128 and the headplate bracket 127 can be formed as a single component. In some configurations, the bracket base 128 and the container clamp ring stand 129 can be formed as a single component or may be separate parts. In some configurations, the container clamp ring stand 129, the bracket base 128, and the headplate bracket 127 can be formed as a single component or may be separate parts. In some arrangements, three headplate brackets 127 are present. More or fewer headplate brackets 127 can be mounted on the container clamp ring stand 129, which can be suitably configured at least according to the desired positional safety of the culture vessel 102. The container clamp 120 can be operably coupled to the container sleeve 115 (Figure 10A) by the cap ring 118.

[0071] Continuing to refer to Figure 4-6, the thermal insulation layer 121 can provide thermal insulation between the container sleeve 115 (Figure 10A) and the environment. The container sleeve 115 (Figure 10A) can be surrounded by a thermal sleeve 117, which can be securely coupled to the container sleeve 115 (Figure 10A) by, for example, a strap 143, but is not limited to this. The thermal sleeve 117 may include a tightening notch that can allow the thermal sleeve 117 to conform when the strap 143 is tightened. The position of the thermal sleeve 117 relative to the container sleeve 115 (Figure 10A) can be maintained by, for example, the use of an easily inserted fastener 129A (Figure 4), such as a dowel pin, screw, bolt, or retaining pin, but is not limited to this. The fastener 129 is selected to allow floating as the container sleeve 115 (Figure 10A) expands / contracts along its vertical axis.

[0072] Continuing to refer to Figure 4-6, the thermal sleeve 117 may include a thermal control means 131 and a thermal sensor 155 (Figure 5). The thermal control means 131 may include a heating pad, a thermal strip, a thermal tape, and / or a thermal sheath. The thermal sleeve 117 and the thermal control means 131 may be fixed to a mounting platform (not shown) by, for example, a bracket foot 147, a bracket 152, a fastener 151, and a fastener 145. The bracket foot 147 and the bracket 152 may be a single component or separate components operably coupled by, for example, a fastener. The thermal sensor 155 (Figure 5) may be used to disable the thermal control means 131. The thermal sensor 155 (Figure 5) may include, for example, a thermal switch with manual or automatic reset, a thermal fuse, or a positive temperature coefficient thermistor, with manual or automatic reset.

[0073] Referring here to Figure 7-9, components of an exemplary system implementing the features of this teaching are shown in a partially disassembled form. The culture vessel 102 is shown operably coupled with the vessel clamp 120 (Figure 13) as the first step in assembling the culture vessel 102 into a working configuration. The vessel clamp 120 (Figure 13) and the culture vessel 102 can be lowered into the vessel sleeve 117 and tightened into place before temperature control can be performed. The cap ring 118 can be lowered into the inner geometry 116 on the insulation layer 121 and fastened to the vessel sleeve 115 (Figure 10A) in a fastening cavity 128A located on the vessel sleeve surface 114. The vessel sleeve 115 (Figure 10A) and the heat sleeve 117 are configured to receive the culture vessel 102, i.e., they can be coupled together and mounted on the mounting surface. When the culture vessel 102 is moved into the inner geometric shape 116, the strap 143 can be tightened and the fastener 129A can be inserted.

[0074] Referring here to Figure 9, a configuration is shown in disassembled form that allows various sizes of culture vessels to be used in a system implementing the features of this teaching. To minimize custom requirements, the thermal sleeve 117 may be a separate component from the vessel sleeve 115. However, the functions performed by the thermal sleeve 117 and the vessel sleeve 115 can be performed by a single component. For example, a single component may include a cavity that allows for the use of culture vessels of all sizes by allowing for the addition of the amount of packing material required to maintain the position of the culture vessel within the thermal / vessel component. The packing material may include, for example, any thermally conductive material that can be pressed against the culture vessel through the tightening of the strap 143. The thermal sleeve 117 may include a gap 133 that allows for the space required to accommodate the thermal expansion of the vessel sleeve 115 and the tightening of the thermal sleeve 117 around the vessel sleeve 115. A thermal cutout may be mounted, for example, in a fastener cavity 137.

[0075] Referring here to Figure 10A, the container sleeve 115 may include an inner diameter surface 116 that, in some configurations, can be constructed to conform to the geometry of the culture vessel 102 (Figure 12). For example, if the culture vessel 102 (Figure 4) includes a tapered geometry 104 (Figure 12), the inner diameter surface 116 may be tapered to conform to the culture vessel 102 (Figure 12). The container sleeve 115 can vary in thickness depending on the size of the culture vessel. Temperature maintenance of the culture vessel 102 (Figure 12) can be achieved by positioning a heat sleeve 117 (Figure 11) around the container sleeve 115, positioning the container sleeve 115 around the culture vessel 102, and securing the entire configuration in place with a container clamp 120 (Figure 13). The heat sleeve 117 (Figure 11) can be adapted to any size culture vessel 102 because its diameter does not vary with the diameter of the culture vessel 102. Alternatively, in some configurations, the internal geometric shape 116 of the container sleeve 115 varies. The container sleeve 115 may include a thermal ring cavity 112 that can receive a thermally compressible ring (not shown). The thermal ring can be fastened in any preferred manner by traversing from the thermal ring cavity 112 along the inner surface of the cavity 116. The container sleeve 115 is designed to be easily removed, and the thermal sleeve 117 is left in place.

[0076] Referring here to Figure 10B, in some configurations, the container sleeve 115A may include a general shape that can be used with various culture vessel sizes and shapes. A commonly molded container sleeve 115A can be combined with a conformable and thermally conductive material (not shown) which can ensure a uniformly coplanar interface between the culture vessel 102 (Figure 12) and the container sleeve 115A.

[0077] Referring here to Figure 11, the heat sleeve 117 can be operably coupled to a heat correcting means 131 and conductors (not shown) to power the heat correcting means 131, which may be a commercially available heater. The heat correcting means 131 conducts heat into the heat sleeve 117, which in turn conducts heat into the container sleeve 115 (Figure 10A). In some configurations, the heat sleeve 117 may include a gap 133 that can accommodate the expansion and contraction of the heat sleeve 117. In some configurations, the gap 133 may be surrounded by a tapered edge 135, which results in the formation of a flat mounting surface. In some configurations, for example, a corner bracket (not shown) may be attached to one leg of the bracket at a connection point 138 to the heat sleeve 117. The other leg of the corner bracket may be used to secure the heat sleeve 117 to a mounting base (not shown). In some configurations, a thermal cutout sensor 155 (Figure 9) can be mounted on the thermal sleeve 117 at a mounting point 137. In some configurations, the thermal cutout 155 can be configured to ensure that the thermal sleeve 117 does not exceed, for example, 60°C. In some configurations, the desired temperature of the contents in the culture vessel is 37°C. One goal of the system of this teaching is to heat the contents of the culture vessel as quickly as possible without raising the temperature of the contents above a pre-selected threshold, such as 37°C. If the temperature of the cutout sensor 155 (Figure 9) exceeds a pre-selected threshold, it opens a power circuit and cuts off power to the thermal correction means 131. In some configurations, the thermal sleeve 117 includes a temperature sensor, which allows a sensor control system 105 (Figure 1B) to use incoming sensor data sensing the temperature of the temperature correction means 131 to adjust the power to the temperature correction means 131. The goal of the thermal sleeve 117, and therefore the high degree of control over the temperature of the contents of the culture vessel 101 (Figure 1B), is to bring the contents to a pre-selected temperature as quickly as possible without damaging them. The thermal sleeve 117 may include stress-relieving notches 139 that may allow the thermal sleeve 117 to expand, contract, and bend to conform to the shape when needed.The heat sleeve may include a cavity 141 that can receive a stabilizing device such as, for example, a dowel pin, screw, retaining pin, or bolt. The strap 143 may be tightened using, for example, a strap fastener 157.

[0078] Referring here to Figure 12, the container clamp 120 and the culture vessel are shown before they are joined, and the container clamp 120 is shown in disassembled form. The bracket base 128 can be operably joined to the container clamp ring stand 129 at the projection 228. The bracket base 128 and projection 228 are sized to surround the cap 204, and the container clamp ring stand 129 is sized to accommodate the diameter of the cap 204. Other configurations are also possible. For example, the projection 228 may include a sliding cavity that may allow the bracket base 128 to be positioned for caps of different sizes. Furthermore, the container clamp ring stand 129 may expand / contract with the diameter of the culture vessel, but may include a material that is horizontally flexible but vertically rigid, providing a rigid mounting platform for the bracket base 128 and the telescopic support 125. The telescopic sleeve 123 can be mounted on the cap ring 118 in the mounting cavity 229. Alternatively, the expansion sleeve 123 and the cap ring 118 can form a single component. Other variations are also possible; for example, some expansion sleeves 123 can be fastened to the cap ring 118, while others are manufactured as part of the cap ring 118.

[0079] Referring here to Figure 13, the container clamp 120 may include an insulating layer 121, an expandable sleeve 123, an expandable support 125 connected to a container clamp ring stand 129, and possibly a head plate bracket 127 spring-mounted on the container clamp ring stand 129. The expandable support 125 and the expandable sleeve 125 operate in a coordinated manner to allow the container clamp 120 to extend / contract, thereby accommodating culture vessels of various heights. In particular, the culture vessel can be drawn towards a temperature control means provided by a heat sleeve 117 (Figure 11), enabling efficient temperature control of the contents of the culture vessel. The desired vessel height can be fixed by inserting fasteners into the heat sleeve 117 (Figure 11) through a sliding cavity 132. Setting the desired height can be used to accommodate vessels of various heights and to ensure that the vessel is high enough to contact the heat sleeve, but still to ensure that the vessel is kept high enough for visual inspection of the fluid contents when the system is running. The fasteners can set the height of the container clamp based on the height of the container. The container clamp can be adjusted to have proper contact with the container ring. It may be desirable to expose a portion of the container to observe the appearance of the container's contents over time. The container clamp can be adjusted to hold the container above the bottom of the container sleeve to perform the task of exposing a portion of the container for visual inspection. Between the bottom of the container sleeve and the container, the heat emanating from the container, container sleeve, and heat sleeve can be contained to maintain thermal uniformity around the container. In some configurations, the heat sleeve can maintain a desired temperature of the container's contents if the heat sleeve is in contact with 1-2 inches or more above the side of the container. In some configurations, an insulating layer (not shown) can be located at the bottom of the container sleeve. The container clamp 120 can be operably coupled with the insulating layer 121. The insulating layer 121 is constructed from a material that does not promote thermal conductivity between the base surface on which the container clamp 120 is mounted and, ultimately, the contents of the culture vessel.The insulation layer 121 is operably coupled to the container sleeve 115 (Figure 10A) through a cap ring 118. The cap ring 118 covers the insulation layer 121 and presents a clean interface to the culture vessel assembly. To ensure the culture vessel 102 (Figure 12) is positioned for operational use, the container sleeve 115 (Figure 10A) is placed inside a thermal sleeve 117 (Figure 11), which may be pre-fixed to the base chassis structure and coupled to the power supply and data I / O devices. In some configurations, to use different culture vessels, for example, to use smaller sized vessels, the container clamp 120 and container sleeve 115 (Figure 10A) can be removed and the container sleeve 115 (Figure 10A) can be replaced with a sleeve of the appropriate size, but the thermal sleeve 117 (Figure 11), along with its data and power connections, remains in place. No changes to the thermal management system are required to change the culture vessel.

[0080] Referring again to Figure 1B, pH and DO are important performance factors in cell proliferation and maturation in the culture. Cell proliferation occurs in a tissue culture vessel where cells are surrounded by a growth medium, which is a substance containing growth factors, serum, and other additives. Cell proliferation is the process of producing cells from a single cell. pH levels, DO levels, and other cellular properties are maintained by the controlled addition of gases such as oxygen, nitrogen, and carbon dioxide via the gas management system 107.

[0081] Referring here to Figure 14, in some configurations, the gas management system of this teaching can include a configurable number of active MFCs, which may differ from the configurable number of active bioreactors, i.e., MFC complements for all gases are not required per bioreactor. When a certain gas layering is not required, each active bioreactor is given a time slot in which it can accept the gas mixture in which it is required. Gas may settle in the bioreactor contents during the time when no gas is being supplied to the bioreactor. Where applicable, the time gap between subsequent gas supplies to a specific bioreactor is configurable, as is the number of MFCs, the number of active bioreactors, and the amount and type of gas supplied to a specific bioreactor during the time slot given to the bioreactor. These variables are set, for example, in the recipe and enabled by the PLC. Many possible combinations of gas supplies are also assumed by this teaching.

[0082] Continuing to refer to Figure 14, the gas management system controls gas resources across multiple culture vessels. In some configurations, sensors and mass fluid controllers (MFCs) that control the amount of oxygen, nitrogen, and carbon dioxide exposed to the contents of the culture vessels are key features of the gas management system, for example, but are not limited to. In some configurations, the MFC receives a mixture of gases in response to data from sensors associated with a particular culture vessel, at least in part, when the controller is controlling and monitoring multiple culture vessels. The sensors may be mounted on the culture vessels or elsewhere, depending on the configuration of the system. In some configurations, sparging may be used to introduce gas into the contents of the culture vessels. In some configurations, gas management is enabled by an electronic solenoid driven by a digital output card on the PLC chassis. In some configurations, the MFC communicates with the PLC301 via Ethernet® IP, and the sensors communicate with the PLC301 via the serial protocol RS-485. In some aspects, a gateway is used to convert the serial protocol to Ethernet® IP. Other communication methods and protocols are also assumed by this disclosure.

[0083] Continuing to refer to Figure 14, gas control can be used to adjust the pH of the contents of a culture vessel. For example, some cell lines grow within a pH range of 7.0–7.4. Additions to the contents of the culture vessel can first set the pH of the contents to a desired range. For example, the culture medium can contain bicarbonate buffer. As the contents of the culture vessel convert glucose to lactate, carbon dioxide is produced, changing the pH and making the culture medium more acidic. Adding gaseous carbon dioxide can increase dissolved carbon dioxide and decrease the pH. Adding air or nitrogen can decrease dissolved carbon dioxide and increase the pH, provided that lactate does not accumulate in the culture medium, in which case a base solution can be added to the contents to increase the pH. In one aspect, the pH set point in the culture vessel is achieved by doubling carbon dioxide to decrease the pH and nitrogen to allow the pH to increase. A PID loop drives the desired (variable) gas flow rate to the MFC. In one respect, one gas, for example, but not limited to, carbon dioxide or nitrogen, is activated at once depending on whether the pH is above or below a desired setpoint.

[0084] Continuing to refer to Figure 14, the oxygen gas (DO) dissolved in the blood is consumed by the cells in the culture vessel and requires replenishment by the gas management system. Several types of cell cultures are performed using DO in the range of 20–50% oxygen saturation, for example. In some configurations, air or nitrogen and oxygen can be automatically added under the control of a controller, based on a comparison of sensor readings of DO in the contents of the culture vessel with a pre-selected setpoint. In one aspect, the setpoint for DO in the culture vessel is achieved by adding oxygen to increase the dissolved oxygen percentage in the vessel, or by adding nitrogen to decrease it. In another aspect, bidirectional control is achieved using PID loops with each MFC. In another aspect, the addition of nitrogen, air, and / or oxygen is based on the difference between the amount of DO, as measured by a sensor submerged in the contents of the culture vessel, and the desired setpoint. When the DO exceeds the setpoint, nitrogen can be added to the culture vessel through a sparger to volatilize some of the oxygen out of the contents of the culture vessel. In one respect, the contents of the culture vessel can simply consume oxygen until they reach a set point.

[0085] Continuing to refer to Figure 14, the gas management system 107 can achieve a compact physical footprint and optimal equipment cost by using a bioreactor and a different number of MFCs in some configurations. MFCs can measure and control the flow of liquids and gases. Commonly used types of MFCs are designed and calibrated to control specific types of liquids or gases within a particular range of flow rates. The description in this teaching is not limited to the MFCs currently available. For example, MFCs not limited to handling specific types of gases or a certain range of flow rates can be imagined. Self-calibrated MFCs are assumed in this description. In some configurations, MFCs may include devices that can programmatically handle multiple gas and pressure or flow rate inputs, resulting in precise and reproducible output, such as, but not limited to, Brooks SLA 5800 series, connected by piping and filters to achieve the desired footprint and cost in a standalone enclosure. The number of MFCs required is a function of the number of gas sources required for the configuration. In some respects, an MFC can include one MFC for O2, one for N2, one for CO2, and one for compressed air, but any number of MFCs with any combination of source gases is possible. The number of culture vessels in the configuration is a function of the number of cell cultures being grown or the number of tissues being maintained. In some respects, an MFC can deliver mixed gases to multiple bioreactors at scheduled time intervals. Using a single set of MFCs for all bioreactors to formulate the gas mixture, and then performing sequential, timely, and / or intermittent overlaying or sparging to each bioreactor, minimizes the number of MFCs required and minimizes gas consumption, exhaust, and waste.

[0086] Continuing to refer to Figure 14, in a given sequence, the controller 106 can maintain a setpoint 303 by instructing the gas management system 107 to supply gas to the culture vessel 305 at various flow rates proportional to the control loop error. The rate depends on the flow rate and differs among the gases. For example, with respect to nitrogen and oxygen, the rate is higher than that of carbon dioxide. The control loop error is calculated from a dedicated PID loop for each bioreactor. The PID loop 303 can be used to drive the desired (variable) flow rate to the MFC. The controller 301 transmits a setpoint based on the PID loop error to the gas management system 107, which includes the MFC. For example, Gas flow rate = C × control loop error, In the equation, C = f (flux rate, gas type), The control loop error is equal to the setpoint minus the measured data. The proportional controller is configured to work in conjunction with the specific cell type. PID tuning parameters can be determined empirically by starting with the cell type and considering them retrospectively. In some sequences, proportional control can be used without integral or differential aspects. In some sequences, steady-state control (low deviation from the setpoint) can be achieved by using proportional, integral, or differential control separately or in combination.

[0087] Continuing to refer to Figure 14, the pH setpoint in culture vessel 305 is maintained by overlaying CO2, which allows for a decrease in pH (more acidity), and N2, which allows for an increase in pH. Gas replenishment can maintain a steady pH until glucose and lactate levels become the overwhelming pH driving factors. As described in Michl et al., “Evidence-based guidelines for controlling pH in mammalian live-cell culture systems” COMMUNICATIONS BIOLOGY | 2:144 | https: / / doi.org / 10.1038 / s42003-019-0393-7 / www.nature.com / commsbio (2019) (which is incorporated herein by reference in its entirety), after a certain period, a medium change will be necessary to maintain the pH setpoint due to glucose consumption and lactate production. A medium change may be necessary when the pH measurement < 7.2. In some sequences, if continuous gas replenishment is unable to alter the characteristics of the cell culture, an immediate medium change will be initiated. In some sequences, glucose and lactate can also be monitored.

[0088] Continuing to refer to Figure 14, the setpoint for dissolved oxygen (DO) in the culture vessel 305 can be achieved by overlaying O2 to increase DO% or N2 to decrease DO%. The setpoint value is process-specific depending on the type of cells being proliferated or tissue being grown. As discussed in Place et al., “Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research,” Free Radical Biology and Medicine, 113: 311-322 (2017) (which is incorporated herein by reference as a whole), some cells or tissues grow in oxygen-rich environments, while others grow in oxygen-deficient environments. In addition, gas transport laws determine the amount of oxygen that dissolves in a solution and ultimately reaches the cells. In one sequence, a recipe can be used to adjust the pH and DO setpoints according to the current values ​​of various measurement parameters, and the recipe is made available to the controller 106, allowing the system to adapt to various pH and DO setpoints derived from cell types. Similar to pH control, bidirectional control will be achieved using two PID loops 303 for each setpoint. Bidirectional control provides a response to positive and negative errors to the setpoint, for example, by adding oxygen when below the setpoint and adding nitrogen when above the setpoint.

[0089] Continuing to refer to Figure 14, in one aspect, the system may include multiple culture vessels 305 for simultaneously growing cells and tissues. Since multiple vessels may be used at once, control strategies are developed for delivering specific gas mixtures to each vessel 305. When the rate of change of pH and DO levels is slow enough that constant gas overlaying is not required, the gas can be overlaid within the culture vessel 305 over a period of time, and then the gas injection can be interrupted while the gas diffuses into the contents. To consider processes / contents that require a pre-selected pause period that exceeds a pre-selected gas delivery time, this can be incorporated into the process. When the gas is being delivered, the PID loop determines setpoints for monitored properties such as pH and DO, but is not limited to these. In one aspect, each culture vessel includes a separate and / or dedicated PID loop. Periodically providing gas overlaying is a successful strategy, at least in part, because the properties affected have a relatively slow rate of change. Isolation of gas delivery to a single culture vessel is achieved by activating a solenoid valve downstream of the MFC to control the direction of the mixed gas.

[0090] Continuing to refer to Figure 14, the controller 106 can instruct the gas management system 107 to supply gas, possibly a mixture of available gases, to a specific culture vessel 305 over a specific time frame. The controller 106 can then instruct the gas management system 107 to modify the mixture, if necessary, to adapt it to the contents of a second culture vessel 305, and then instruct the gas management system 107 to deliver the gas to the second culture vessel 305. This process can be repeated for each culture vessel 305 in the system. The culture vessel 305 may be revisited with further gas injection, possibly a different mixture, depending on the specific requirements of the contents of the culture vessel and / or the results of parameter measurements. Isolation of gas delivery to a single culture vessel 305 can be achieved by activating a specific solenoid valve based on a fixed schedule. Coordination of MFC setpoints and valve states can be driven by a sequenced routine in the controller. The sequence reads the pH / DO through a pH / DO sensor (Figure 1) in each culture vessel, delivers gas, and provides time for moving to the next culture vessel. This time sharing allows for the sharing of hardware components and minimizes gas consumption, exhaust, and waste.

[0091] Referring to Figure 14, for example, if there are six culture vessels 305, each requiring gas delivery over a 5-minute period, the cycle of circulating through the culture vessels 305 and supplying them with gas would require 30 minutes. For culture vessels 305 where the gas diffusion rate is the same as the cycle time, the controller 106 can instruct the gas management system 107 to supply more gas to that culture vessel 305. In the exemplary process, the setting point for each MFC can be changed every 5 minutes depending on the error output of the pH and DO PID loop 303 for each culture vessel 305. The controller 106 calculates the error from the PID loop / probe in each culture vessel 305 and sets the gas to correct for the next gas window.

[0092] Continuing to refer to Figure 14, condensation of liquid in the gas line exiting the culture vessel can potentially contaminate gas control equipment. In one aspect, an air dryer can be used to volatilize the inflowing gas from liquid impurities. In another aspect, a heating system, such as a Peltier heating system, can prevent condensation of liquid in the gas line exiting the bioreactor. In an exemplary Peltier heating system, one junction is cooled while the other is heated, and an electric current is maintained in a circuit of material containing two dissimilar conductors or semiconductors. An increase in temperature occurs at the junction, where, for example, copper is converted to bismuth, and a decrease in temperature occurs at the junction, where, for example, bismuth is converted to copper. In one aspect, the Peltier heating system includes a thermoelectric heater and a fan. In another aspect, a controller provides commands to the fan and thermoelectric heater through a digital output associated with the controller.

[0093] Referring here to Figures 15A-15F, components of an exemplary gas management system are shown. Specifically, the enclosure housing the MFC and gas manifold is shown in Figure 15A. The shroud 905 and base 901 form the enclosure protecting the gas management system of this teaching. Extending from the shroud 905 is a pressure relief valve 907, which is attached to the mixing manifold 917 (Figure 15D). With the shroud 905 (Figure 15B) partially removed, the distribution manifold 915 (Figure 15B / C) is shown. The gas entering the MFC inlet can be filtered by a particulate filter 918 (Figure 15C). The distribution manifold 915 (Figure 15B / C) receives gas according to the type and amount required for a particular process, such as defined by PLC 106 (Figure 1B). Each gastrain incorporates connections to an MFC 925 (Figure 15E), a particulate filter 918 (Figure 15E), and a check valve. The number of gastrains is equal to the number of source gases present in the system. Gases from each gastrain (MFC) are blended in a mixing manifold 917 (Figure 15D) and distributed to the desired bioreactor through a distribution manifold 915 (Figure 15D). The distribution manifold 915 (Figure 15D) is the outlet from the gas mixer to the bioreactor. The distribution manifold 915 (Figure 15F) enables the distribution of source gases via PLC 106 (Figure 1B) commands. The distribution manifold 915 (Figure 15F) includes features such as pneumatic manifold fixtures 933 / 935 (Figure 15F), an exemplary 5-station unit 937 (Figure 15F), and an exemplary two-way normally closed solenoid valve 931 (Figure 15F). The system described in this instruction assumes larger or smaller units that can be adapted to larger or smaller gas sources.

[0094] Referring here to Figure 16, the controller (PLC) 106 (Figure 1) receives data from sensors associated with the culture vessel, fluid management system, and gas management system, and uses that data to control system functions to achieve desired outcomes such as, but not limited to, tissue development, scaffold decellularization, and scaffold recellularization. PLC 106 (Figure 1B) is integrated wired (or wirelessly) to control other components of the system without requiring command translation. PLC 106 (Figure 1B) can detect faults in incoming sensor data, and after verification, can use the data to enable state changes to select a sequence of output commands to, for example, motors, pumps, and valves. In some configurations, the sequence of state changes and / or commands can be driven by a recipe. The recipe itself can be modified by user input or by PLC 106 (Figure 1B) based on conditions in the system. Other ways in which recipes may be modified or control may proceed are also assumed in this teaching.

[0095] Continuing to refer to Figure 16, the PLC 106 (Figure 1B) of this instruction follows a general flow for all processes per culture vessel station (including culture vessel 503 (Figure 1B), fluid handling system 108 (Figure 1B), and culture vessel control 111 (Figure 1B)) that can be performed by the system of this instruction. The PLC 106 (Figure 1B) can simultaneously manage different processes performed in the culture vessels of this instruction. Thus, a version of Method 950 can be performed in the PLC 106 (Figure 1B) for each culture vessel. Method 950 may include, but is not limited to, a step 951 that receives a selection of a process to be initiated. Exemplary processes include, but is not limited to, cell maturation, organ decellularization, and organ recellularization. The selection may be made by a user, for example, through, but is not limited to, a secure telecommunication line, a local wired line, or a secure wireless connection. HMI 630 (Figure 1C) is one method by which a user may select a desired process. A display associated with the HMI630 (Figure 1C) can provide status information for each culture vessel. The display can be divided into several sections, for example, according to the number of active culture vessels, or according to the total number of culture vessels, whether active or not. Alternatively, the HMI630 (Figure 1C) can include multiple monitors, each supporting an active culture vessel. Furthermore, processes can be related to each other, so process selection can occur automatically or semi-automatically, and the user can approve the selection before process execution proceeds. For example, a decellularization process can automatically or semi-automatically initiate a recellularization process. Method 950 can include a step 953 to access a recipe associated with the selected process. When there are multiple possible recipes for the selected process, user input can be prompted to select the desired recipe.Alternatively, PLC106 (Figure 14) can select and access the most appropriate recipe based on previous or parallel processes performed by one or more of the bioreactors in the system. User interaction can be provided to validate the selection depending on the characteristics associated with the process and recipe.

[0096] Continuing to refer to Figure 16, Method 950 may include a step 955 to access, calculate, receive, or prompt for parameters related to the selected process and / or selected recipe. For example, setpoints for temperature, pH, and DO may be provided. The user may enter the requested values ​​or opt to use default values, or the user may exit the loop entirely, as the system may select default values ​​or calculate values ​​based on prior or parallel activities in the system. Method 950 may include a step 957 to start the selected recipe using the parameters. Recipe start sets up a processing loop that moves through the recipe until all phases are performed, then checks for further recipes, and finally returns to the beginning to receive the process selection. In the processing loop, Method 950 may include a step 959 to execute phases from the recipe, for example, a step to execute commands to open valves, pump culture medium, or activate a stirrer. The elements of the ISA-88 phase module are provided in the system of this teaching by a program that, for example, but is not limited to, opens a valve, starts a pump, totals the flow using sensors until a pre-selected amount is reached, stops the pump, and closes a valve. The phase control of this teaching negotiates ownership of equipment based on pre-selected criteria. For example, in some configurations, when an automated phase is executed, all devices are configured to be controlled by the PLC unless there are manual steps in the sequence. Essentially, all equipment is owned by the PLC during the automated phase and recipe. In some configurations, the operator acquires ownership of the device of interest under specific circumstances. When the operator has finished using the device, the phase is restarted and the PLC acquires ownership of the device. In some configurations, possible modes include operator, external, and maintenance. Other possible modes are also assumed by this teaching. Modes are associated with users who can lock ownership of equipment.The owner user must unlock the controller before it can transfer ownership to another user. In the system described in this instruction, multiple phases can be performed simultaneously. For example, the heating phase can be performed simultaneously with the stirring and recirculation phases. In some configurations, each culture vessel has dedicated equipment and software, enabling simultaneous, unrelated operations on different culture vessel contents.

[0097] Continuing to refer to Figure 16, data from sensors in this system may, for example, indicate when a phase is complete, or the PLC 106 (Figure 1B) may move to the next phase after a pre-selected amount of time has elapsed. The recipe may specify how each phase should be handled, or, as possible, it may be left to calculations performed by the PLC 106 (Figure 1B) based on the collected sensor data as the process progresses. Method 950 may include a step 961 of receiving data from sensors associated with a culture vessel station on which the process is being performed, and a step 963 of adjusting characteristics and parameters based on the sensor data, if necessary. Many such embodiments are provided herein. Adjusting the thermal profile of the contents of the culture vessel relies on such data sensing in and around the culture vessel station. In particular, sensor data may indicate that a phase is complete. If further phases exist in the recipe in 965, Method 950 may include returning to step 959 and performing steps from that point onward. If, in 965, there are no further phases to be performed in the recipe, and in 967, there are other recipes associated with the process, method 950 may return to step 953 and include steps to be performed from that point onward. If, in 967, there are no other recipes, method 950 may return to step 951 and include steps to be performed from that point onward.

[0098] In some configurations, exemplary phases for culturing cells are described in Table I. [Table 1]

[0099] In some configurations, exemplary phases for decellularizing the scaffold are described in Table II. [Table 2]

[0100] Referring here to Figures 17A–17B, 18A–18C, and 19A–19B, various applications of this system for decellularizing the heart are shown along with various component layouts. Referring to Figure 17A, a simplified fluid flow diagram of the system of this instruction is shown with respect to the heart during decellularization. In particular, a solution 351 of nutrients, etc., in the form of a culture medium, is selected by a valve 353 and pumped by a pump 355, passing through a sensor 357, to either the waste 359 or the descending aorta 361. As the fluid diffuses through the heart 371, it either proceeds to the waste 359 from the pulmonary artery 363 or is recirculated to return through the heart 371. The fluid exiting the heart 371 proceeds through the pulmonary vein 365 to a pressure vessel 367, and proceeds to either the waste 359 or recirculation. A pressure vessel is a leak-proof container that stores liquids or gases at or above atmospheric pressure. The pressure vessel 367 imparts back pressure to the fluid flowing out of the bioreactor 369. In some configurations, the back pressure can be derived from indirect pressure through the head height at an altitude above the bioreactor 369. This instruction assumes other methods of using the pressure vessel 367 and other configurations for producing back pressure. In some configurations, the pressure vessel 367 is vented and waste fluid flows into the waste 359. Other configurations are also assumed by this instruction. In some configurations, the pressure vessel 367 can maintain a fixed pressure in the pulmonary vein / left ventricle and facilitate a higher percentage of flow to exit the pulmonary artery through the coronary arteries. This pressure can keep the heart in an expanded state. Other configurations, such as pressure control loops, are also assumed by this instruction. Pressure vessels of various shapes can be used depending on the type of gas used to impart pressure and the amount of pressure required. For example, the types of pressure vessels include, but are not limited to, cylindrical, conical, spherical, horizontal, or vertical, and may be crowned with heads of various shapes. For example, non-spherical pressure vessels require a head. The types of heads may include, for example, hemispherical and shallow (dish-shaped) heads (semi-elliptical or dish-shaped).

[0101] Referring here to Figure 17B, regarding the specific decellularization composition, solution 351 may include, but is not limited to, buffer solution (PBS), washing sodium dodecyl sulfate (SDS) in distilled deionized (DI) water, hypertonic NaCl, hypotonic sodium chloride (NaCl), heparinized PBS, and PBS with peracetic acid. Other decellularization solutions may include, but is not limited to, washing sodium deoxycholate (SD / SDC), washing Triton X-100, zwitterionic solutions such as 3-[(3-coramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), trypsin / ethylenediaminetetraacetic acid (EDTA), and deoxyribonuclease (DNAse). SDS is a washing agent known to denature proteins. Peracetic acid is used for final scaffold sterilization. Denature of proteins involves breaking many weak links or bonds within the protein molecule, such as hydrogen bonds. Binding is involved in the ordered structure of proteins; therefore, denatured proteins are likely to have a looser, more random structure and be insoluble. PBS is a phosphate buffer solution, an aqueous salt solution containing disodium hydrogen phosphate, sodium chloride, and possibly potassium chloride and potassium dihydrogen phosphate, to help maintain a constant pH. Deionized water can be used to produce predictable and reproducible results because it lacks ions from mineral salts such as iron, calcium, and sulfates, for example. Valve 353 in this configuration includes a mixture of normally closed solenoid valves and manual valves. In some configurations, the manual valve can be associated with a source fluid such as, for example, hypotonic NACl, heparinized PBC, and PBC / peracetic acid, but is not limited to these. In some configurations, the manual valve can be used to help prevent leakage during the setup and disassembly processes and is not used in automated processes. Manual valves can be used on containers that hold solutions to be used in a process, and are a method for filling, transporting, and deploying these containers offline without the risk of leaking or contaminating the solutions in the source.The selected solution is pumped by pump 355 through a pressure sensor 357 and a flow meter / bubble meter to either the waste 359 or the bioreactor 369, specifically the descending aorta 361. Pump 355 may include a peristaltic pump that satisfies the desired pump characteristics described elsewhere in this specification.

[0102] Referring here to Figures 18A–18C, an exemplary decellularization recipe and associated valve operations are shown. Figure 18A and Tables III and IV illustrate how a system with two fluid pumps and twelve fluid valves may be performed to decellularize an organ such as the heart and pump the fluid into a collection container. Referring to Figure 18A, in this exemplary use, the fluid is pumped from the first pump through a pressure gauge into the descending aorta from sources of heparinized PBS, hypertonic NACl, PBS / peracetic acid, SDS, Triton, PBS, and deionized (DI) water. The fluid within the heart is routed to a waste or collection container. Referring to Figure 18B, the phases performed to carry out cardiac decellularization according to the exemplary recipe are enumerated. The first four phases involve a step of clearing blood from the heart using heparinized PBS, which is pumped alternately by volume and by time / pressure by the first pump, while the second pump controls the output to a pre-selected pressure. Note that the system in this instruction allows for variations in pump volume, pressure, and time values, and therefore the values ​​listed in Figure 18B are illustrative only. In the fifth phase, the remaining blood is washed away from the heart with DI water. The next phase involves lysing the cells in the heart with NaCl, and then washing away the destroyed cells with DI water. The cell debris is then removed from the heart by repeated applications of SDS and DI water. In this recipe, there are three repetitions of the debris removal sequence, each involving two applications of SDS followed by one application of DI water. The final phase of the material involves removing residual SDS using Triton. The last two phases involve washing away the remaining scaffold with DI water and PBS. Figure 18C lists the valves shown in Figure 18A, which are used for each phase listed above in Figure 18B.

[0103] Referring here to Figures 19A-19C, another feature of the system of this instruction is the step of growing one type of cells on one side of a culture vessel scaffold and another type on the other side, thereby combining the two types of cells. Such a configuration may include a source fluid, at least one culture vessel station including culture vessels 503A / B, a fluid handling system 108, a pump 256, a culture vessel control system 111, a gas management system 107, a biocontroller 106, a waste 274, a bioreactor 272, a rotating means 270, and a vent 280, all of which are described herein. In some configurations, the scaffold 266 is fixed in the middle of the bioreactor 272, and the medium / cell addition ports 276 / 278 enter the bioreactor 272. The rotating feature 270 can move the seeded area to a position where cells will be encouraged to rest and reproduce. The vents 280 or other outlet pathways may be configured to prevent pressure buildup. A system as shown in Figure 19A can be used to grow cells such as suspension cells, aggregates, or cells on microcarriers. When the cell density reaches a target threshold, the cells are allowed to settle, excess medium is removed, and a washing solution 252, e.g., PBS, but not limited to PBS, is added / removed. Trypsin 254 is added to digest microcarrier / extracellularly attached proteins, and high-protein medium 262 or an inhibitor is added to suppress trypsin 254, and medium 262 is added / removed to adjust its concentration, after which the cells are pumped to one side of the scaffold 266. Additional medium 262 may be used to clear any cells in the tubular dead volume. The other side of the scaffold 266 is seeded as soon as the first side of the scaffold 266 has finished adhering, for example, to cells simultaneously growing in the second culture vessel 503B, or at a later time if the first layer developed in the first culture vessel 503A requires time to mature. Culture medium exchange in the bioreactor 272 is performed, for example, continuously through the flow loop or periodically using vents.A second bioreactor (or third bioreactor) may be configured to perform medium exchange in bioreactor 272. The selection of the option is based at least on process timing, medium composition, and tissue size / metabolism. Sensors (shown elsewhere) are installed in the culture vessels 503A / B, medium vessel 262, tissue bioreactor 272, and / or in one of the fluid pathways (e.g., tissue bioreactor inlet / outlet 276 / 278). In some configurations, thermal control for the medium 262 is maintained in a thermal device 264. A rotating means 270 can rotate the scaffold 266 around a rotation axis 268. In some configurations, waste products may be routed away from bioreactor 272 into a waste collection 274. In some configurations, bilateral seeding may be performed using a single culture vessel station and a single set of source fluids. In such a system, as illustrated in Figures 19B and 19C, the culture vessel 503 can supply cells to both sides of the scaffold 266 at different time points. Some of the cells produced within the bioreactor 272 and some of the waste products can be returned to the culture vessel 503 and the waste collection 274, respectively. Shown in Figure 19C is a rotating means 270, as described herein. In some configurations, cells grown outside the culture vessel system depicted in Figures 19A–19C can be supplied to the bioreactor 272 through specially configured ports. In such configurations, multiple types of cells, both cells grown within the culture vessel 503 (Figure 19B) and cells grown elsewhere, can be introduced into the bioreactor 272 for seeding across multiple areas of the scaffold 266. The scaffold 266 is depicted as a rectangle, but can take any shape and size according to the size of the bioreactor 272. Furthermore, multiple scaffolds 266 can be combined after the seeding process is complete to form more complex tissues.

[0104] A system for repeatedly performing at least one type of tissue-related process as part of a production line, the system comprising at least one culture vessel station including a variable-size culture vessel, a fluid handling system, and a culture vessel control system, the at least one culture vessel station configured to adapt to steps for performing at least one type of tissue-related process, a gas management system configured to supply at least one type of gas to the at least one culture vessel station, and a controller configured to control the gas management system and the at least one culture vessel station for performing at least one type of tissue-related process, the controller configured to communicate with components on the production line using a standard industrial communication protocol. In a system as described herein, at least one variable-size culture vessel comprises disposable components. In a system as described herein, at least one variable-size culture vessel comprises durable components. In a system as described herein, at least one variable-size culture vessel comprises a vessel sleeve surrounding at least a portion of the at least one variable-size culture vessel, the vessel sleeve configured to transfer thermal energy to the at least one variable-size culture vessel; a thermal sleeve operably coupled to the vessel sleeve, the thermal sleeve controlling the amount of thermal energy entering the vessel sleeve; and a vessel clamp stabilizing the at least one variable-size culture vessel within the thermal sleeve. In a system as described herein, a fluid handling system is configured to move a fluid through the at least one variable-size culture vessel. The system as described herein further comprises a sensor control system that controls the amount of thermal energy entering the vessel sleeve. In a system as described herein, a gas management system is configured to control the type and amount of gas entering the at least one variable-size culture vessel.In a system as described herein, the container clamp comprises an expandable device, the expandable device adapting to the height of at least one variable-size culture vessel. The system as described herein further comprises an insulating layer between the container sleeve and the environment surrounding the container sleeve. The system as described herein further comprises an electrical shutoff device that senses when the thermal sleeve reaches at least one pre-selected threshold temperature, the electrical shutoff device configured to disable the addition of further thermal energy to the container sleeve. The system as described herein further comprises at least one band clamp for securing the thermal sleeve to the container sleeve. In a system as described herein, the thermal sleeve comprises at least one expansion / contraction gap. The system as described herein further comprises a stabilizing pin configured to positionally secure the thermal sleeve to the container sleeve. The system as described herein further comprises at least one temperature control element. The system as described herein further comprises a thermally conductive material filling the space between the inner diameter of the container sleeve and the outer diameter of at least one variable-size culture vessel. A system as described herein further comprises a plurality of vessel sleeves that adapt to a plurality of sizes of at least one variable-size culture vessel. A system as described herein further comprises a sensor system configured to monitor a variable-size volume of cells in at least one variable-size culture vessel. In a system as described herein, a fluid handling system comprises a variable number of at least one valve and at least one pump configured to move fluid in and out of at least one variable-size culture vessel station, wherein at least one controller comprises a variable number of at least one valve and at least one pump controlling at least one valve and at least one pump. In a system as described herein, at least one controller comprises commands configured to control a plurality of at least one culture vessel stations performing independent tasks simultaneously.In a system as described herein, a first culture vessel station performs a first type of at least one type of tissue-related process in parallel with a second culture vessel station performing a second type of at least one type of tissue-related process. In a system as described herein, the first type of at least one type of tissue-related process comprises the same type as the second type of at least one type of tissue-related process. In a system as described herein, the first type of at least one type of tissue-related process comprises a different type from the second type of at least one type of tissue-related process. In a system as described herein, the first type of at least one type of tissue-related process comprises decellularization. In a system as described herein, the first type of at least one type of tissue-related process comprises recellularization. In a system as described herein, the first type of at least one type of tissue-related process comprises cell maturation of the recellularized tissue. In a system as described herein, the first type of at least one type of tissue-related process comprises endothelial cell perfusion. In a system as described herein, at least one controller includes a step of determining a fluid flow path based on at least a recipe. In a system as described herein, at least one controller includes a step of dynamically determining a fluid flow path. In a system as described herein, at least one controller includes a step of determining a fluid flow path based on at least user input. In a system as described herein, at least one controller includes a step of determining a fluid flow path based on at least a combination of a recipe, dynamically determined parameters, and user-provided parameters. In a system as described herein, at least one type of organization-related process includes a step of generating a batch. In a system as described herein, the batch is produced according to at least one industry standard process.In a system as described herein, at least one industrial standard process comprises ANSI / ISA-88.01-1995. In a system as described herein, the standard industrial communication protocol comprises Ethernet® / Industrial Protocol. In a system as described herein, the gas management system comprises at least one mass fluid controller configured to receive a gas source, wherein the amount of gas is controlled by at least one controller; a mixing manifold configured to blend multiple types of gases from a plurality of at least one mass fluid controllers, wherein the amount and type of the multiple types of gases are controlled by at least one controller; and a dispensing manifold that receives the blended multiple gases and distributes the blended multiple gases to at least one culture station according to commands from at least one controller. In a system as described herein, the number of at least one mass fluid controllers is independent of the number of at least one culture vessel station. In a system as described herein, a plurality of at least one mass flow controllers are configured to provide a plurality of amounts and types of gases to a plurality of at least one culture vessel station according to a periodic delivery function. In a system as described herein, the periodic delivery function is based on values ​​collected by at least one sensor associated with the culture vessel station. The system as described herein further comprises a cone feature attached to the shaft of a stirring device in the culture vessel, the cone feature substantially prevents cells from settling on the stirring device.A method for seeding multiple types of cells on multiple areas of a scaffold, the method comprising the steps of: operably connecting the scaffold to a rotating means in a bioreactor, the bioreactor being configured to receive multiple types of cells through multiple ports in the bioreactor; and when the cell density of a first cell type of multiple types of cells in a first culture vessel reaches a pre-selected threshold or after a pre-selected waiting cycle, (a) removing excess medium from the culture vessel; (b) washing the first cells of the first cell type in the culture vessel; and (c) using a digestion solution The method includes (a) digesting microcarriers / extracellularly attached proteins in a culture vessel, (d) inhibiting the digestion solution, (e) adjusting the concentration of the culture medium, (b) pumping the first cells from the culture vessel to a first area of ​​a plurality of areas of a scaffold in a bioreactor, (c) processing a second cell type of a plurality of cell types according to steps (a)-(e) when a first pre-selected time has elapsed, and (b) pumping the second cells from the culture vessel to a second area of ​​a plurality of areas of a scaffold in a bioreactor, wherein the second cell type is produced in a second culture vessel. The method as described herein further includes, after the first area has been seeded, rotating the scaffold, the rotating step of positioning the scaffold to receive a second type of a plurality of cells on the second area.

[0105] Various alternatives and modifications can be devised by those skilled in the art without departing from the Disclosure. Therefore, the Disclosure is intended to encompass all such alternatives, modifications, and variations. In addition, while some exemplary configurations of the Disclosure are shown in the drawings and / or discussed herein, the Disclosure is not intended to be limited thereto, as the Disclosure is as broad as the art will allow and this Specification is also intended to be read carefully. Accordingly, the above description should not be construed as a limitation, but merely as an example of a particular configuration. Furthermore, those skilled in the art will imagine other modifications within the scope and spirit of the claims appended herein. Other elements, steps, methods, and techniques that are not substantially different from those described above and / or appended to the claims are also intended to be within the scope of the Disclosure.

[0106] The drawings are provided solely to demonstrate certain embodiments of the present disclosure. Furthermore, the drawings described are illustrative and non-limiting. In the drawings, for illustrative purposes, the sizes of some elements may be exaggerated and not drawn to a particular scale. Additionally, elements shown in the drawings with the same number may, depending on the context, be the same element or similar elements.

[0107] When the term “equipped with” is used in this description and claims, it does not exclude other elements or steps. When referring to a singular noun, if an indefinite or definite article, e.g., “a,” “an,” or “the,” is used, it includes the plural form of that noun unless something else specifically states otherwise. Thus, the term “equipped with” should not be interpreted as being limited to the items listed thereafter, and it does not exclude other elements or steps, and therefore the scope of the expression “a device equipped with items A and B” should not be limited to a device consisting only of components A and B.

[0108] Furthermore, the terms “First,” “Second,” “Third,” and their equivalents are provided to distinguish similar elements, whether used in the description or claims, and not necessarily to describe a sequential or chronological order. It should be understood that, where used in this manner, the terms are synonymous under appropriate circumstances (unless expressly otherwise disclosed), and that exemplary configurations of the disclosures described herein are capable of operating in sequences and / or arrangements other than those described or illustrated herein.

Claims

1. A method for seeding multiple types of cells on multiple areas of a scaffold, wherein the method is The scaffold is operably coupled to a rotating means within a bioreactor, the bioreactor being configured to receive the plurality of types of cells through a plurality of ports within the bioreactor, When the cell density of the first cell type of the plurality of cell types in the selected first culture vessel reaches a pre-selected threshold, or after a pre-selected waiting cycle, (a) Removing excess culture medium from the selected first culture vessel, (b) Washing the first cells of the first cell type in the selected first culture vessel, (c) Digesting the microcarriers / extracellularly attached proteins in the selected first culture vessel using a digestion solution, (d) Suppressing the digestive solution, (e) Adjusting the concentration of the culture medium, The first cells are pumped from the selected first culture vessel to a first area of ​​the multiple areas of the scaffold in the bioreactor. When the cell density of the second cell type of the plurality of cell types in the selected second culture vessel reaches a pre-selected threshold, or after a pre-selected waiting cycle, (a') Removing excess culture medium from the selected second culture vessel, (b') Washing the second cells of the second cell type in the selected second culture vessel, (c') Digesting the microcarriers / extracellularly attached proteins in the selected second culture vessel using a digestion solution, (d') To suppress the digestive solution, (e') Adjusting the concentration of the culture medium, The second cell type is pumped from the selected second culture vessel to a second area of ​​the scaffold within the bioreactor, wherein the second cell type is generated in the selected second culture vessel and pumped to the second area. Methods that include...

2. After the first area has been seeded, the scaffold is rotated, the rotation of which positions the scaffold to receive a second cell type of the plurality of cell types on the second area. The method according to claim 1, further comprising:

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