Modular, configurable bioreactor system for manufacturing line

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 in particular to configurable manufacturing systems. In a configurable manufacturing system, the concept is to allow plug-and-play components to be added and removed from the system with little or no retooling of the system, and to allow robust communication to upstream and downstream equipment and technologies. Plug-and-play is of special concern for industrial systems that accommodate the operable coupling of interchangeable components whose footprints, properties, and thermal requirements vary from component type to component type. Current systems can be improved by allowing upstream and downstream communication with other components of the manufacturing line. Examples of components of a manufacturing line for tissue processing can include, but are not limited to, cell lysis systems, bioreactor systems, and tissue maintenance systems. Standard processes and communications between components along the manufacturing line allow for the addition and removal of components depending on the desired output. Thus, the manufacturing line of the present teachings contemplates various components, including those listed herein and others, operating simultaneously, sequentially, or in a recipe-determined, dynamically determined, and / or user-selected order. [Background technology]

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

[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 (hiPCS) (obtained by reprogramming somatic cells of human pluripotent stem cells (hPCS)) are cells that can be effectively used in the cell proliferation process. These cell types can self-renew and differentiate into specific cell types depending on their potency. Bioreactors (culture vessels) can be used to enable cell proliferation, especially since they can provide a 3D agitated, scalable, homogenized environment. Types of bioreactors that can be found in the manufacturing line can include stirred tanks, fixed beds, hollow fibers, rotating cells, rotating beds, and rocking motion bioreactors with disposable or durable components or combinations. Sensors can be associated with the bioreactors and controllers to establish operating ranges for temperature, acid / base levels, aeration, agitation speed, and culture medium flow rate. What is needed to enable reproducibility in cell growth on a production line is consistent internal control of the bioreactor system and communication of the status and other characteristics of the bioreactor system with other production line components.

[0004] Decellularization results in the removal of living tissue from an extracellular matrix (ECM) scaffold while retaining cellular integrity and homeostatic cues in the structure of the ECM. The scaffold can be physically or chemically decellularized. Chemicals used in decellularization include detergents such as sodium dodecyl sulfate (SDS), which lyse cells by disrupting phospholipid cell membranes, acids such as peracetic acid and bases such as sodium hydroxide, which solubilize cell membranes. 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 if decellularization is successful, the aspects of the ECM that remain intact after decellularization can be inspected 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 must not reach a preselected threshold of double-stranded DNA of a preselected fragment length and must not have any visible nuclear material. Mechanical properties, including elastic modulus and tensile strength, may be required to meet certain preselected criteria. The success of the decellularization method can be determined by the reduction of tissue immunogenicity, specifically genetic material and antigens. Insufficient reduction of immunogenicity may lead to in vivo rejection of the tissue. What is needed is a bioreactor system that includes internal controls to reach the desired decellularization outcome and includes a robust communication system that provides the status of decellularization and other properties to other components of the manufacturing line.

[0005] In recellularization, scaffolds are seeded with cells to form organs. Complete organ regeneration requires that parenchymal, vascular, and supportive components must be re-established prior to seeding with cells. Many types of cells have been considered for organ regeneration. For example, mesenchymal stem cells from bone marrow or adipose tissue, expanded to the correct numbers, can differentiate into various cell types, and scaffolds have been found to foster mesenchymal stem cell differentiation. Induced pluripotent stem cells can facilitate the use of patient-derived cells to provide a cell source for recellularization. Support cells, such as fibroblasts, can enhance certain types of cell function and therefore enhance recellularization. Successful seeding routes can be organ-dependent. For example, kidney scaffolds can be reseeded through the ureter or renal artery, with the renal artery route shown in some studies to result in higher cell distribution and retention than the ureter route. Recellularization requires a bioreactor control system that manages properties such as temperature, gas, pH, and pressure. Successful recellularization results in the required number of specific types of cells to form an entire tissue or organ. What is needed is a bioreactor system that includes internal controls to reach the desired recellularization outcome and includes a robust communication system that provides recellularization status and other characteristics to other components of the manufacturing line.

[0006] In systems designed to repeatably control the growth of mammalian cells by supplying nutrients, removing waste products, and controlling temperature and headspace gas mixing within the culture vessel, a modular design can be beneficial to take advantage of technological advances in the asynchronous nature of the system required to enable cell growth. Additionally, such systems can provide additional efficiencies by operating multiple cell culture vessel stations simultaneously. A major technology gap concerns issues related to the relative size of vessels required for autologous and allogeneic processes involving small tissues. There is a need for vessels capable of producing various amounts of cells without sacrificing the ability to monitor and control the growth process. Efficient use of certain commercial bioreactor systems requires large minimum volumes to encompass current sensors. Harvesting various volumes of cells is also made 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 realize the goal of efficiently automating the production of autologous and allogeneic tissue engineered constructs. Similarly, scaling up to larger vessels, for example as large as 100 liters or larger, is required. What is further needed is a system that may include both disposable and durable components. Disposable components such as tubing for supplying media, recirculating media, disposing of waste, seeding cells, and moving fluids and cells from one manufacturing line component to the next, as well as media reservoirs, and single-use sensors may be required. Durable components may include, for example, a system chassis, a user interface, a media chiller, a waste containment system, a gas management system, a pneumatic control block, and other components or subcomponents on the manufacturing line. Components that may be durable or disposable include, but are not limited to, growth and maturation bioreactors.

[0007] Another technology gap concerns issues surrounding the use of pumps and valves. In current systems, pumps and valves are arranged in fixed numbers, limiting the flexibility of the system and requiring the purchase of a different system if more or fewer pumps and valves are required for a particular application. Yet another technology gap involves the control of flexibly configured systems. For example, in systems 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 results to other components on the manufacturing line. For example, a system with multiple bioreactors can simultaneously decellularize a scaffold and prepare multiple types of cells to recellularize the scaffold. Each bioreactor and its associated pumps and valves can receive custom control flows and custom gas flows guided by monitoring conditions with the contents of the bioreactor. For each bioreactor, a variety of different pumps, valves, and sensors can be activated to achieve, in this example, tissue growth and scaffold preparation simultaneously.

[0008] Current systems such as Zoo Ning's Industrial Scale Optical Bioreactor No. CN201045139Y, a utility model (Ning) assigned to Yantai Haishangchuanqi Biotechnology Co., LTD., granted on April 9, 2009, expiring on September 19, 2014, describes bioreactors constructed from simple glass to accommodate 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, a form of networked control system in which a set of devices and controlling software are arranged in a hierarchical tree, with the links in the tree implemented by a computer network. Wang describes the improvement of a flat-organized control system for bioreactors based on parallel distributed smart sensors and actuators as a simple solution for process control in bioreactors. Laboratory configurations such as the Sartorius AMBR® 15 Cell Culture Generation 2 can be changed by the operator both at the start and during the process. The system includes single-use vessels and an automated workstation, all disposed in a biological safety cabinet. Multiple bioreactor cultures are monitored in parallel. The system includes an automated liquid handler that can provide media, feed, and reagents to the vessels and facilitates in-line or offline sampling. Other devices such as Eppendorf's DASGIP® parallel bioreactor system allow advanced bioprocess control and automation.The system offers precise control of parameters, user-defined profiles, automation features, and configurable solutions to accommodate 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, control agitation, pH, and DO, and TMFC gas mixing of air, N2, O2, and CO2.

[0009] What is needed, however, is not currently provided is an industrial control system with at least one integrated programmable logic controller (PLC) in the closed bioreactor. Such a system would allow communication between the bioreactor PLC and other controllers along the production line. What is needed is a system with integrated sensors, a system that can accommodate changes in vessel size and shape, allowing for a smaller number of MFCs than the number of bioreactors. What is needed is a system that can accommodate the ability to grow one type of cell on one side of the bioreactor scaffold and another type on the other side, combining the two types of cells. Summary of the Invention [Means for solving the problem]

[0010] The system of the present teachings overcomes the technology gaps outlined herein. The system of the present teachings efficiently automates the production of autologous and allogeneic tissues by accommodating processes involving various sizes of cell culture vessels and small tissues. The vessels of the present teachings can produce various amounts of cells without sacrificing the ability to monitor and control the growth process. The vessels include a head plate that can accommodate the tubing needs of various organ types. The vessels of the present teachings are configured to accommodate sensor placement such that the volume of cells encompasses the sensor. The vessels are disposable to allow for various volumes of cells to be harvested, thus reducing dead volume cells at the bottom of the vessel. The vessels can also be durable. The vessels can be scaled down to accommodate small volumes of cells, for example, but not limited to, 0.1-3 liters, or scaled up to larger vessels, for example, up to 1,000 liters. The system can also accommodate a variable number of pumps and valves, which can be apportioned based on the particular application. The system includes at least one controller configured to control a variable number of valves and pumps, a variable number and size of vessels, and fluids and gases associated with a desired process. Because the vessels can be configured with different types of cells, tissues, and / or scaffolds, the controller is configured to control various processes performed in the vessels in parallel. The system is configured to separately control each culture vessel station while they operate simultaneously. For example, a system with multiple culture vessel stations can simultaneously decellularize a scaffold and prepare multiple types of cells to recellularize the scaffold. Each culture vessel and its associated pumps, valves, and gas systems can undergo custom control flows guided by monitoring of 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 the present teachings includes pressure-controlled circulatory flow along with flow feedback and monitoring. The flow paths can be altered as the system operates.Another feature of the system of the present teachings is a gas control system configured to allow for a smaller number of mass flow controllers (MFCs) than the number of culture vessels, since each culture vessel is given a time window to receive gas therein. This feature allows for reduced gas consumption, less gas vented, and less wasted gas than a system with a one-to-one correspondence between MFCs and culture vessels. Yet another feature is that the system can accommodate growing one type of cell on one side of a culture vessel scaffold and another type on the other side, and can combine two types of cells. Uses for the system of the present teachings can include, but are not limited to, cell culture, media conditioning (i.e., adjusting the temperature of the media to a desired value and setting the pH and DO levels of the media), decellularizing and recellularizing scaffolds, generating blood, blood components, viruses for gene therapy, recombinant proteins, pharmaceuticals, vaccines, allergens, genes, antibodies, fermentation, drug combinations, cosmetics, and food, and converting raw materials into useful by-products. Other applications are also envisioned by the present teachings. The present invention provides, for example, the following: (Item 1) 1. A system for repeatably performing at least one type of tissue-related process as part of a manufacturing line, the system comprising: at least one culture vessel station, the at least one culture vessel station including at least one culture vessel of variable size, a fluid handling system, and a culture vessel control system, the at least one culture vessel station configured to accommodate performing the at least one type of tissue-related process; a gas management system configured to provide at least one type of gas to the at least one incubation vessel station; at least one controller configured to control the gas management system and the at least one culture vessel station to perform the at least one type of tissue-related process, the at least one controller configured to communicate with components on the manufacturing line using a standard industry communication protocol; A system comprising: (Item 2) 2. The system of claim 1, wherein the at least one variable-sized culture vessel comprises a disposable component. (Item 3) 2. The system of claim 1, wherein the at least one variable-sized culture vessel comprises a durable component. (Item 4) The at least one variable size culture vessel comprises: a vessel sleeve surrounding at least a section of the at least one variable-sized culture vessel, the vessel sleeve configured to transfer thermal energy to the at least one variable-sized culture vessel; a thermal sleeve operably coupled to the container sleeve, the thermal sleeve controlling an amount of thermal energy entering the container sleeve; a vessel clamp for stabilizing the at least one variable-sized culture vessel within the thermal sleeve; Item 2. The system of item 1, comprising: (Item 5) 2. The system of claim 1, wherein the fluid handling system is configured to move fluid through the at least one variable-sized culture vessel. (Item 6) 5. The system of claim 4, further comprising a sensor control system that controls the amount of thermal energy entering the container sleeve. (Item 7) 2. The system of claim 1, wherein the gas management system is configured to control the type and amount of gas entering the at least one variable-sized culture vessel. (Item 8) The container clamp comprises: a stretching device adapted to accommodate the height of the at least one variable sized culture vessel; 5. The system according to item 4, comprising: (Item 9) 5. The system of claim 4, further comprising an insulating layer between the container sleeve and an environment surrounding the container sleeve. (Item 10) an electrical cutoff device that senses when the thermal sleeve reaches at least one preselected threshold temperature, the electrical cutoff device configured to disable the addition of further thermal energy to the vessel sleeve; 5. The system of claim 4, further comprising: (Item 11) 5. The system of claim 4, further comprising at least one band clamp that secures the thermal sleeve to the container sleeve. (Item 12) The thermal sleeve comprises: 5. The system of item 4, comprising at least one expansion / contraction gap. (Item 13) Item 5. The system of item 4, further comprising a stabilizing pin configured to positionally secure the thermal sleeve to the container sleeve. (Item 14) 2. The system of claim 1, further comprising at least one temperature control element. (Item 15) 5. The system of claim 4, further comprising a thermally conductive material filling a space between an inner diameter of the vessel sleeve and an outer diameter of the at least one variable-sized culture vessel. (Item 16) 5. The system of claim 4, further comprising a plurality of vessel sleeves accommodating a plurality of sizes of the at least one variable-sized culture vessel. (Item 17) 2. The system of claim 1, further comprising a sensor system configured to monitor a variable-sized volume of cells within the at least one variable-sized culture vessel. (Item 18) The fluid handling system comprises: 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, the at least one controller controlling the at least one valve and the at least one pump; 6. The system according to item 5, comprising: (Item 19) The at least one controller: 2. The system of claim 1, comprising instructions configured to control a plurality of at least one culture vessel station to simultaneously perform independent tasks. (Item 20) 20. The system of claim 19, wherein a first of the at least one culture container station performs a first option of the at least one type of tissue-related process in parallel with a second of the at least one culture container station performing a second option of the at least one type of tissue-related process. (Item 21) 21. The system of claim 20, wherein a first type of at least one option of the at least one type of tissue-related process comprises an identical selection as a second option of the at least one type of tissue-related process. (Item 22) 21. The system of claim 20, wherein the first selection of the at least one type of tissue-related process comprises a different selection than the second selection of the at least one type of tissue-related process. (Item 23) 22. The system of claim 21, wherein a first type of the at least one type of tissue-related process comprises decellularization. (Item 24) 22. The system of claim 21, wherein a first type of the at least one type of tissue-related process comprises recellularization. (Item 25) 22. The system of claim 21, wherein a first type of the at least one type of tissue-related process comprises cellular maturation of the recellularized tissue. (Item 26) 22. The system of claim 21, wherein a first type of the at least one type of tissue-related process comprises perfusion of endothelial cells. (Item 27) The at least one controller: 2. The system of claim 1, further comprising determining a fluid flow path based at least on a recipe. (Item 28) The at least one controller: 2. The system of claim 1, further comprising dynamically determining a fluid flow path. (Item 29) The at least one controller: 2. The system of claim 1, further comprising determining a fluid flow path based on at least a user input. (Item 30) The at least one controller: 2. The system of claim 1, further comprising determining the fluid flow path based on a combination of at least a recipe, dynamically determined parameters, and user-provided parameters. (Item 31) The at least one type of tissue-related process comprises: 2. The system of claim 1, comprising a batch process. (Item 32) 32. The system of claim 31, wherein the batches are produced according to at least one industry standard process. (Item 33) said at least one industry standard process; Item 33. The system of item 32, comprising ANSI / ISA-88.01-1995. (Item 34) The standard industrial communication protocol is Item 33. The system of item 32, comprising an Ethernet / industrial protocol. (Item 35) The gas management system includes: at least one mass flow controller configured to receive a source of gas, the amount of the gas being controlled by the at least one controller; a mixing manifold configured to blend a plurality of types of gas from at least one mass flow controller, the amounts and types of the plurality of types of gas being controlled by the at least one controller; a distribution manifold that receives the blended gases and distributes the blended gases to the at least one culture vessel station according to commands from the at least one controller; Item 2. The system of item 1, comprising: (Item 36) 36. The system of claim 35, wherein the number of controllers of the at least one mass flow controller is independent of the number of vessels of the at least one culture vessel station. (Item 37) Item 37. The system of item 36, wherein the plurality of at least one mass flow controllers are configured to provide amounts and types of the plurality of types of gas to the plurality of at least one culture vessel station according to a cyclic delivery function. (Item 38) Item 38. The system of item 37, wherein the periodic delivery function is based at least on values ​​collected by a sensor associated with the at least one culture vessel station. (Item 39) a cone feature attached to a shaft of an agitation device in said at least one culture vessel, said cone feature substantially preventing cells from settling on said agitation device. Item 1. The system of item 1, further comprising: (Item 40) 1. A method for seeding multiple types of cells onto multiple areas of a scaffold, the method comprising: operatively coupling the scaffold to a rotation means within a bioreactor, the bioreactor configured to receive the multiple types of cells through multiple ports within the bioreactor; when a cell density of a first cell type of the plurality of types of cells in a selected culture vessel reaches a preselected threshold or after a preselected waiting period; (a) removing excess medium from the selected culture vessel; (b) washing first cells of the first cell type in the selected culture vessel; and (c) digesting the microcarrier / extracellular attached proteins in said selected culture vessel using a digestion solution; (d) suppressing the digestive solution; and (e) adjusting the concentration of the medium; pumping the first cells from the selected culture vessel into a first one of a plurality of areas of the scaffold within the bioreactor; When a first preselected time has elapsed, Treating a second cell type of said plurality of types of cells according to steps (a)-(e); pumping the second cell type from a second of the selected culture vessels to a second of the plurality of areas of the scaffold within the bioreactor, wherein the second cell type is generated in the second of the culture vessels; A method comprising: (Item 41) rotating the scaffold after the first area is seeded, said rotating positioning the scaffold to receive a second cell type of the plurality of types of cells on the second area. 41. The method of claim 40, further comprising: [Brief description of the drawings]

[0011] The foregoing features of the present disclosure will be more readily understood by reference to the following description, taken in conjunction with the accompanying drawings, in which:

[0012] [Figure 1A] FIG. 1A is a schematic block diagram of a configurable industrial manufacturing system that includes a system of the present teachings.

[0013] [Figure 1B]FIG. 1B is a schematic block diagram of a system of the present teachings.

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

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

[0016] [Figure 2A] FIG. 2A is a perspective view of an apparatus implementation of the system of the present teachings.

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

[0018] [Figure 3A] 3A-3E are schematic diagrams of implementations of the present teachings that include a downstream recirculation system. [Figure 3B] 3A-3E are schematic diagrams of implementations of the present teachings that include a downstream recirculation system. [Figure 3C] 3A-3E are schematic diagrams of implementations of the present teachings that include a downstream recirculation system. [Figure 3D] 3A-3E are schematic diagrams of implementations of the present teachings that include a downstream recirculation system. [Figure 3E] 3A-3E are schematic diagrams of implementations of the present teachings that include a downstream recirculation system.

[0019] [Figure 3F] 3F and 3G are perspective views of an impeller cone of the present teachings. [Figure 3G] 3F and 3G are perspective views of an impeller cone of the present teachings.

[0020] [Figure 4] FIG. 4 is a perspective view of a first side of an assembly of the present teachings that includes a commercial culture vessel.

[0021] [Diagram 5] FIG. 5 is a perspective view of a second side of an assembly of the present teachings that includes a commercial culture vessel.

[0022] [Figure 6] FIG. 6 includes bottom and top perspective views of an assembly of the present teachings that includes a commercial culture vessel.

[0023] [Figure 7] FIG. 7 is an exploded perspective view of a first side of an assembly of the present teachings that includes a commercial culture vessel.

[0024] [Figure 8] FIG. 8 is an exploded perspective view of a second side of an assembly of the present teachings that includes a commercial culture vessel.

[0025] [Figure 9] FIG. 9 is an exploded perspective view of a container sleeve and thermal sleeve configuration of the present teachings.

[0026] [Figure 10A] FIG. 10A is a perspective view of a first configuration of a container sleeve of the present teachings.

[0027] [Figure 10B] FIG. 10B is a perspective view of a second configuration of a container sleeve of the present teachings.

[0028] [Figure 11]FIG. 11 is a perspective view of a thermal sleeve configuration of the present teachings.

[0029] [Figure 12] FIG. 12 is a perspective exploded view of a vessel clamp of the present teachings and a commercial culture vessel.

[0030] [Figure 13] FIG. 13 is a perspective view of a container clamp of the present teachings.

[0031] [Figure 14] FIG. 14 is a schematic block diagram of an exemplary use of the gas management system of the present teachings.

[0032] [Figure 15A] 15A-15F are perspective views of components of an implementation of a gas management system of the present teachings. [Figure 15B] 15A-15F are perspective views of components of an implementation of a gas management system of the present teachings. [Figure 15C] 15A-15F are perspective views of components of an implementation of a gas management system of the present teachings. [Figure 15D] 15A-15F are perspective views of components of an implementation of a gas management system of the present teachings. [Figure 15E] 15A-15F are perspective views of components of an implementation of a gas management system of the present teachings. [Figure 15F] 15A-15F are perspective views of components of an implementation of a gas management system of the present teachings.

[0033] [Figure 16] FIG. 16 is a flow chart of the example controller process of the present teachings.

[0034] [Figure 17A] FIG. 17A is a schematic block diagram of an exemplary use of the system of the present teachings for decellularization of the heart.

[0035] [Figure 17B] FIG. 17B is a schematic diagram of an exemplary valve configuration for decellularizing the heart using the system of the present teachings.

[0036] [Figure 18A] 18A-18C are schematic diagrams illustrating how the system of the present teachings can be used to decellularize the heart. [Figure 18B] 18A-18C are schematic diagrams illustrating how the system of the present teachings can be used to decellularize the heart. [Figure 18C] 18A-18C are schematic diagrams illustrating how the system of the present teachings can be used to decellularize the heart.

[0037] [Figure 19A] 19A-19C are schematic block diagrams of an implementation of the present teachings in which multiple areas of a scaffold are seeded within the same bioreactor. [Figure 19B] 19A-19C are schematic block diagrams of an implementation of the present teachings in which multiple areas of a scaffold are seeded within the same bioreactor. [Figure 19C] 19A-19C are schematic block diagrams of an implementation of the present teachings in which multiple areas of a scaffold are seeded within the same bioreactor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description The system of the present teachings can follow a process specific to the contents of at least one culture vessel to produce a desired result in a controlled environment. The system can accommodate 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 that can produce consistent, reproducible results.

[0039] Referring now to FIG. 1A, the system of the present teachings may be a component of an industrial control system, an example of which is shown in FIG. 1A, where information from each component is accessible by other components of the system. The components of the industrial system benefit from conformance to 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 allows Ethernet to be used as a control protocol. The object libraries 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 (procedural), hardware (physical), and material changes (process) that are accomplished when the software runs on the hardware. Each model is organized into protocol levels. Across models, the protocol levels work cooperatively to produce a batch. For example, the procedural model includes four protocol levels: procedures, unit procedures, operations, and phases. The physical model includes two protocol levels: process cells and units, and optionally, equipment and control levels. The procedural model levels combine with the physical model levels to produce the process model levels: processes, process stages, process operations, and process actions.Compliance with industry-developed standards such as ISA-88 can increase ease of integration with other enterprise standards such as ANSI / ISA / 95. Standards such as ANSI / ISA-88 are used by the system of the present teachings to provide a consistent set of processes and terminology for producing batches of materials by subjecting a quantity 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 to use the system of the present teachings as a standalone system or integrated with a larger system. At least one integrated programmable logic controller (PLC) in the system can communicate with other controllers along the manufacturing line. For example, an industrial system for producing a finite amount (batch) of cells can include, for example, but not limited to, a cell thawing system 251, a culture vessel system that receives the thawed cells, and an incubation system 255 that receives results from any process performed in the culture vessel system. Other components of the industrial control system are also contemplated by this disclosure. A standard communication system 257 allows for data and control sharing between components of the illustrative industrial control system, as described herein with respect to Ethernet / IP. For example, the thaw controller 259 can communicate the status of the thaw operation to the biocontroller 106 through 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 thaw controller 259 so that the thawed cells can be replaced when the culture vessel system is ready for them. The controller 106 can provide its status and other information about the processes it is performing to the incubator controller 263, and can provide its own information to the controller 106.The components can be coordinately controlled by a system control means that can monitor and command the components by tracking standard communications between the components.

[0040] Referring now to FIG. 1B, the system of the present teachings is an automated culture vessel system intended for batch production. At a physical level, the system of the present teachings can include components such as, but not limited to, sensors, valves, motors / encoders, pumps, culture vessels, gas management, and controllers. These components are grouped into subsystems such as, but not limited to, fluid handling systems, culture vessel systems, gas management systems, and control systems. The control system, interchangeably referred to herein as PLC, can be organized in protocol levels of a procedural model according to the ISA-88 standard. The protocol levels, from the lowest level to the highest level in the protocol, include 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 the PLC 106. The equipment modules are logic that accommodates faults, operational thresholds, start / stop control, any basic function that the device will perform. The control modules can interface with one or more devices that need to cooperate to perform a function. A phase is defined as a state that is related to a control module but is also part of a recipe. A phase performs a specific function. An exemplary phase involves adding a volume of liquid to a culture vessel. The phase commands open a valve, start a pump, sum the flow until a preselected fluid volume is reached, stop the pump, and close the valve. Exemplary phases of the present teachings include, but are not limited to, pumping medium into the culture vessel, removing medium, heating the contents of the culture vessel, agitating the contents of the culture vessel, harvesting cells, and recirculating medium. A recipe is a combination of multiple phases to form a complete process.

[0041] Continuing with reference to FIG. 1B, ISA-88 states that a recipe contains five categories of information: header, equipment requirements, recipe, procedure, and other information. The procedure category combines other categories to provide a multi-level hierarchy of recipe procedure elements and contains logic. The control recipe defines the manufacture of a single batch of a specific product and reflects the process control. In some configurations, a PLC can execute the recipe in conjunction with a batch server and a human machine interface (HMI). The HMI can, for example, display the results of the recipe execution to an operator and can receive flow and configuration information modifications from the operator. In some configurations, a commercially available application can provide the implementation of the HMI and batch server. For example, Rockwell's FACTORYTALK® batch server software can run on a WINDOWS® server and drive the HMI and initiate the execution of the recipe on the PLC. The PLC can periodically access the sensor data to understand the current conditions in the culture vessel. In some configurations, the access period is, for example, about 100 ms, although other periods are also contemplated by the present disclosure. The value of the sensor data can activate manual or automatic reactions 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 the gas flow rate. Control is based on maintaining a set point in the culture vessel. During the decellularization process, the pump rate varies based on the current pressure reading with the goal of maintaining a constant pressure. The PLC takes action based on the recipe, which calls for a specific phase. A phase is a sequential operation where the next step cannot proceed until the current step permission, i.e., a condition that must be met before proceeding, is met. In some configurations, the fluid handling operation is a sequential or recipe-driven operation involving valves, pumps, flow sensors, or level sensors.Incoming sensor data is verified and fault checked, for example, but not limited to, by noise removal, data outlier detection, missing data imputation, and data aggregation. The incoming sensor data is used in state flows or sequences to output commands to motors, pumps, and valves to activate those devices in a certain order, for example, according to a recipe. Recipes can be dynamically changed, and the values ​​on which triggers in a recipe depend can also be changed dynamically, i.e., manually or automatically.

[0042] Continuing with reference to FIG. 1B, the separate culture vessels do not share any of the same resources or equipment, except for the MFC for gas supply. The same logic, though separate, exists for each culture vessel, which is why they can run the same or different processes simultaneously. Since constant gas supply is not required to maintain the pH / DO set points in the culture vessels, each culture vessel gets a time window for receiving gas in it. The process (cell maturation, proliferation, decellularization, recellularization) determines the pH and DO set points. The PLC monitors the time, reads the sensor data of the first culture vessel, and provides gas to the first culture vessel at the desired rate by opening the valve. At the end of the time window of the first culture vessel, the gas valve of the first culture vessel closes and the gas valve of the second culture vessel opens, and the PLC reads the sensor data from the second culture vessel and provides the desired gas mixture to the second culture vessel. Each culture vessel has its own valves, flow sensors, and pumps for fluid delivery. The PLC can run different recipes on each culture vessel simultaneously.

[0043] Referring now to FIG. 1C, an exemplary implementation of the system of the present teachings includes a culture vessel system and a gas management system 107 controlled by a controller 106. In configurations where there are multiple culture vessel systems, each can be equipped similarly to the 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 contemplated by the present teachings. 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 receive tubing appropriate to the task being performed by the culture vessel station. For example, if the heart is being recellularized, the lid can accommodate tubing connecting the descending aorta, pulmonary artery, and pulmonary vein with receptacles and / or nutrient sources outside the culture vessel system. Similarly, if the kidney is being recellularized, the lid can accommodate tubing that connects 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, valves, and sensors that are used to move fluids through the contents of the culture vessel 503. The culture vessel control system 111 maintains set points for various characteristics 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 set points 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 typed in or dynamically determined. The gas management system 107 is shared by all the culture vessel systems, each of which receives the gas mixtures it requires to maintain homeostasis of the contents of the culture vessel. The system of the present teachings can control and monitor the receipt and circulation of fluids and the provision of gases to fluids to enable desired outcomes regarding the contents of the culture vessel.In certain aspects, the controller 106 can simultaneously control processes performed at each of the multiple culture vessel stations 503. In certain aspects, the controller 106 can include multiple processors that can control the culture vessel stations 503. In certain aspects, a single gas management system 107 can control the flow of a single gas. In certain aspects, a single gas management system 107 can control the flow of multiple gases. In certain aspects, a single gas management system 107 can provide gas to a single culture vessel station 503. In certain aspects, a single gas management system 107 can provide gas to multiple culture vessel stations 503. In certain aspects, multiple gas management systems 107 can provide gas to a single culture vessel station 503. In certain aspects, multiple gas management systems 107 can provide gas to multiple culture vessel stations 503. The culture vessels 503 can include, but are not limited to, a configurable vessel assembly of the present teachings.

[0044] With continued reference to FIG. 1C, in some aspects, the system of the present teachings can decellularize a scaffold. In some aspects, the system of the present teachings can recellularize a scaffold. In some aspects, the system of the present teachings can provide an environment in which cells can mature and tissues can grow. In some aspects, the system of the present teachings can accommodate containers in sizes such as, but not limited to, 0.1-1,000 liters. In some aspects, the system of the present teachings can be used to generate biopharmaceutical products such as, but not limited to, vaccines, blood, blood components, allergens, genes, viruses for gene therapy, cosmetics, and proteins.

[0045] Referring again to FIG. 1B, the fluid handling system 108 moves media and other fluids through the contents of the culture vessel 503. The type of fluid, the pressure of the fluid, and the flow rate of the fluid are determined by a combination of factors, such as, but not limited to, a preselected process associated with the desired outcome, preselected set points for various properties required to produce the desired outcome, dynamic characterization, and user input. In some configurations, the system accesses a recipe that dictates the operations that will produce the desired outcome. Each operation is characterized by a set of phases or commands that are executed to accomplish a step in the process. In the fluid handling system 108, one operation can include accessing the type of fluid required to accomplish the step. Another can include configuring the PLC 106 with set points 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 can include configuring at least one pump to move the fluid through the culture vessel 503 and past a sensor. Fluid control can be governed by flow rate or pressure. Components of the fluid handling system 108 may include, but are not limited to, in-line flow and pressure sensors, liquid level sensors, pumps, and pinch valves for directing flow.

[0046] 1D, an implementation of the fluid handling system 108 (FIG. 1B) of the present teachings can 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, to assist with standard motor movements 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 caliber of the tubing, to waste 615. A pump pressure sensor 617 can enable control of the flow rate of the fluid 611 to 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 preselected range surrounding a preselected set point, e.g., 5 mmHg higher or lower, the pump speed is changed by a preselected percentage, either higher or lower, depending on the value of the pressure. For example, the pump speed can be changed by 10%, either higher to increase the pressure, or lower to decrease the pressure. The new pressure can be evaluated for a preselected amount of time, e.g., 5 seconds, and the process can be repeated. If the pressure reading is within the preselected range, no pump speed change is made. The pressure set point of 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 adjust the speed of the pump. In some configurations, a high cutout pressure switch can prevent the pump 619 from outputting extreme pressures. The pump pressure sensor 617 can be mounted on the inlet of the pump to monitor efficiency and improve the efficiency and reliability of the pump 619.

[0047] Referring now to FIG. 1E, a culture vessel or closed culture vessel system of the present teachings includes a configurable vessel assembly system that provides heating, location, 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 action required by the user. The system of the present teachings can culture and produce, for example, but not limited to, various cell types and fungal and bacterial cultures. The system can be used to produce viruses and proteins with cultured cells. The system can be configured for various shapes, sizes, and types of culture vessels, including, but not limited to, glass and plastic vessels that are durable and disposable. Exemplary types of culture vessels include, but are not limited to, stirred vessels, packed beds, roller bottles, oxygen permeable culture plates, microfluidic slides, or hydrogel-based culture vessels. The culture vessel system of the present teachings includes integrated sensors that communicate with a PLC as described elsewhere herein.

[0048] With continued reference to FIG. 1E, the configurable vessel assembly system of the present teachings can be part of a flexible configuration to accommodate various processes. A given cell growth process may require a specific vessel volume to expand to a desired number of cells, while an alternative process may require a different vessel volume. The vessel size can be established to fit the desired process without modifying anything other than the vessel sleeve in the flexible configuration, including the vessel heating element, electrical connections / wiring, control logic, calibration, and positioning adjustments, potentially. If a user wishes to change the vessel size, for example, from 2L to 0.5L, only a simple swap of components is required, nothing more. In some configurations, the user can simply remove the 2L vessel sleeve and replace it with a 0.5L vessel sleeve, each vessel sleeve having the same outer diameter. After tightening the clamp on the outside of the thermal sleeve surrounding the vessel sleeve and ensuring that the 0.5L vessel sleeve is secured, the assembly reconfiguration is complete and ready for operation with the 0.5L vessel. This can be done for a variety of sizes, including, but not limited to, 0.5L, 1L, 2L, 3L, and up to 100L. A commercial culture vessel can be mounted to the adapter ring of the assembly. The adapter ring is mounted into a temperature control ring or thermal sleeve, which has a clamping feature to ensure good thermal contact between the two components. The thermal sleeve can control the amount of thermal energy entering the vessel sleeve. The assembly can include a vessel clamp, which stabilizes the culture vessel within the thermal sleeve. If a different size vessel is desired, the different size vessel can be mounted into an appropriately sized vessel sleeve and disposed in the same thermal sleeve as the other size vessel. Thus, the basic system remains the same regardless of vessel size. A clamping heating ring can stabilize the vessel. The vessel clamp can optionally include a telescopic device. The telescopic device can accommodate the various heights of the various different culture vessels and vary the height of the specific culture vessel.The assembly can optionally include an insulating layer between the vessel sleeve and the environment surrounding the vessel sleeve, an electrical cutoff device that senses when the thermal sleeve reaches at least one preselected threshold temperature, the electrical cutoff device disabling the transfer of thermal energy to the vessel sleeve, and at least one band clamp that secures the thermal sleeve to the vessel sleeve. The thermal sleeve can optionally include an expansion / contraction gap. The assembly can optionally include a stabilizing pin that positionally secures the thermal sleeve to the vessel sleeve, at least one temperature control element, and a thermally conductive material that fills the space between the inner diameter of the vessel sleeve and the outer diameter of the culture vessel.

[0049] With continued reference to FIG. 1E, a configurable vessel assembly of the present teachings for treating cells in a controlled environment can include, but is not limited to, a vessel sleeve surrounding at least a portion of a culture vessel. The vessel sleeve can be configured to transfer thermal energy to the culture vessel. The assembly can include a thermal sleeve operably coupled to the vessel sleeve. The thermal sleeve can control the amount of thermal energy entering the vessel sleeve. The assembly can include a vessel clamp that stabilizes the culture vessel within the thermal sleeve. Optionally, the assembly can include a fluid handling system that moves 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 allows control of the amount of thermal energy entering the vessel sleeve. The vessel clamp can optionally include a telescopic device. The telescopic device can accommodate different heights of a variety of different culture vessels and vary the height of a particular culture vessel. The assembly can optionally include an insulating layer between the vessel sleeve and the environment surrounding the vessel sleeve, an electrical cutoff device that senses when the thermal sleeve reaches at least one preselected threshold temperature, the electrical cutoff device disabling the transfer of thermal energy to the vessel sleeve, and at least one band clamp that secures the thermal sleeve to the vessel sleeve. The thermal sleeve can optionally include an expansion / contraction gap. The assembly can optionally include a stabilizing pin that positionally secures the thermal sleeve to the vessel sleeve, at least one temperature control element, and a thermally conductive material that fills the space between the inner diameter of the vessel sleeve and the outer diameter of the culture vessel.

[0050] 1E, the culture vessel 601 can include thermal controls, agitation controls, 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. can also affect cell viability and cell metabolism. Maintaining the desired temperature is made possible by a temperature sensor 659, which reads the value of the temperature of the contents of the culture vessel 601 and then sends a signal to the culture vessel controller system 111 to adjust the temperature as needed through controlling heating / cooling devices, if present.

[0051] Continuing with reference to FIG. 1E, a level sensor 613 can monitor the liquid level in the incubation vessel 601. The liquid level can be used to control the amount of fluid added during the fluid transfer process and 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 incubation vessel 601 is much less than the density of the fluid 611, types of level sensors include, but are not limited to, glass level gauges, floats, displacers, bubblers, differential pressure transmitters, load cells, magnetic level gauges, capacitance 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 with still further reference to FIG. 1E, as an alternative to or in addition to the level sensor 613, a fluid flow meter 605 can measure the flow into and out of the culture vessel 601, for example, in mL / min. A summation function can be used during media transfer to calculate the total volume of fluid transferred by integrating the flow over the pump cycle. Fluid flow meter categories 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 rotameters. Velocity flow meters include Peet 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 use reciprocating piston meters, swinging disk meters, and rotary vane meters. Mass flow meters include heat flow meters and Coriolis flow meters. Either the level sensor 613 or the flow meter 605 can assist the dose control 603 by calculating the amount of fluid removed from or added to the culture vessel 601. The dose control 603 can track the volume added and stop adding further fluid when a desired set point is reached. Dosing potentially includes the ability to start / stop the pump under preselected circumstances, report shortfalls or excesses from capacity, and alert and report the status of delivery. Dosing devices fall into two main categories: gravimetric and volumetric. The choice of dosing device is based on the flow rate of the fluid and the desired flow rate of the fluid. Dosing can be automatic, programmable, or continuous, and can be controlled by a dosing valve, for example. In some configurations, dose control 603 can be accomplished by adding instructions to the level sensor 613 or flow meter 605 process.

[0053] With continued reference to FIG. 1E, in some configurations, the system can include thermal control. In one arrangement, the system can include active heating and passive cooling. At least one temperature control mechanism can be present within the vessel system itself, at least one temperature sensor can reside on the exterior of the vessel to sense the temperature of the temperature control mechanism, and at least one temperature sensor can reside inside the vessel to sense the temperature of the vessel contents. 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. of the contents of the culture vessel. In some configurations, temperature control can be performed using cascaded PID loops, with an inner loop controlling the temperature control mechanism and an outer loop controlling the temperature of the vessel contents. In some configurations, the temperature control mechanism can be powered by 110V. In such configurations, solid state relays can be used so that a 24V output card can pulse wave modulate the temperature control mechanism. Thermal control to a set point in the present teachings is achieved actively and passively. In some configurations, warming of the culture vessel contents is achieved actively, while cooling of the contents is achieved passively. Other configurations, such as active cooling and passive warming, and active cooling and warming, are also contemplated by the present teachings. In some configurations, the set point is a preselected value, such as, but not limited to, 37° C. In some configurations, warming can be limited to a preselected temperature, such as 40° C. Sensors report the temperature of the culture vessel 631 contents to the culture vessel control system 111, and the sensor data triggers the 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 the heater 655, which heats the fluid in the culture vessel 601. Data from the sensor RTD 659 and heater RTD 657 is received by the PLC 631 through the culture vessel control system 111 via an RTD I / O card 629 designed to receive such data.In some configurations, control of temperature is performed by a cascaded PID loop, with an inner loop controlling the temperature (PWM) of the heater 655 and an outer loop controlling the temperature of the contents of the culture vessel 601. The output of the PID drives the power input to the heater 655. Other methods of thermal control are also contemplated by the present disclosure. For example, a digital temperature sensor or a silicon-based linear thermistor can be used to measure the temperature of the sensor and the contents of the culture vessel 601.

[0054] With continued reference to FIG. 1E, the culture vessel 601 may contain tissues that require movement for proper growth. For example, the contents of the culture vessel 601 may require agitation to improve gas perfusion and possibly thermal control, depending on the specific process and desired outcome. Other reasons for agitation of the contents of the culture vessel 601 may include producing an even distribution of gas bubbles, producing small gas bubbles, maximizing residence time of gas in the fluid by driving gas bubbles to the bottom of the culture vessel, reincorporating nutrients from the top of the culture vessel back into the contents of the culture vessel 601, and providing a uniform nutrient and temperature profile throughout the contents. Agitation devices include mechanical agitation devices, for example, driven by brush or brushless DC motors 663. Mechanical agitation devices are categorized by the flow direction in which the fluid is mixed, for example, axial, radial, combined, and dispersive flow, and may include, for example, propellers and impellers. Propeller size and pitch are features selected based on the application. Exemplary impellers include pitched blade (for axial flow), Rushton (for radial flow), angled pitched blade (compound flow), and helical (dispersed flow). In some configurations, the agitator 661 can include a marine propeller attached to a shaft coupled to a stepper motor 663, the combination being used to support applications such as cellular processes. In some configurations, the PLC 631 controls and monitors the status of the motor 663 by using commands transmitted and data received over a communication medium and protocol such as, but not limited to, Ethernet IP 625. The speed and direction of the propeller is selected based on the application.

[0055] Continuing with yet further reference to FIG. 1E, the success of an application using the culture vessel 601 can rely on monitoring various characteristics of the system and the contents of the culture vessel 601. As described herein, sensors can be advantageously positioned to measure aspects of an ongoing process. For example, turbidity, pH, DO, glucose, and lactate levels can be measured by in-line 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, through a serial connection 665 (FIG. 1C), a USB connection (not shown), an Ethernet IP connection 625 (FIG. 1C), or wirelessly. In some configurations, glucose and lactate levels can be collected as fluid circulates through the tubing and culture vessel 601 of the system.

[0056] Continuing with reference to FIG. 1E, the configurable vessel assembly system of the present teachings can be part of a flexible configuration to accommodate a specific cell culture process. A given growth process may require a specific vessel volume to expand to a desired number of cells, while an alternative process may require a different vessel volume. The size of the culture vessel 601 can be established to fit the desired process, potentially without modifying anything other than the vessel sleeve in the flexible configuration. The heating elements, electrical connections / wiring, control logic, calibration, and positioning adjustments of the culture vessel 601 can remain the same across a change in the physical size of the culture vessel 601. If a user wishes to change the vessel size, for example, from 2L to 0.5L, only a simple swap of components is required, nothing more. In some configurations, the user can simply remove the 2L vessel sleeve and replace it with a 0.5L vessel sleeve and the culture vessel 601, each vessel sleeve having the same outer diameter. After tightening the clamp on the outside of the thermal sleeve surrounding the vessel sleeve to ensure that the 0.5L vessel sleeve is secured, the assembly reconfiguration is complete and ready for operation with the 0.5L vessel. This can be done for a variety of sizes, including but not limited to, 0.5L, 1L, 2L, 3L, and up to 100L. The commercial culture vessel can be mounted to the adapter ring of the assembly. The adapter ring is mounted into a temperature control ring, which has a tightening feature to ensure good thermal contact between the two components. If a different size vessel is desired, the different size vessel can be mounted into an appropriately sized vessel sleeve and disposed in the same thermal sleeve as the other size vessel. Thus, the basic system remains the same regardless of vessel size. The clamping heating ring can stabilize the vessel.

[0057] Now referring to FIG. 1F, the gas management system 107 (FIG. 1B) can include a gas source 609 per mass flow controller (MFC) 608 or functionally equivalent device that combines mass flow sensors and control valves to control the flow rate of gas to a desired amount without being affected by changes in the conditions of use or gas pressure. The MFC 608 can be analog or digital based on the number of types of gas being controlled. The gas source 609 can include one or several types of gas, and the type selected can be determined by the desired outcome from the process enabled by the system of the present teachings. The gas management system 107 (FIG. 1B) can include a gas manifold 607 that receives gas from all selected gas sources and delivers a single gas stream to the culture vessel whose turn it is to receive the selected gas. The PLC 631 (FIG. 1C) selects the type of gas to be provided to the culture vessel 101 (FIG. 1B) (also referred to herein as a bioreactor) to achieve a gas balance that will enable the desired outcome. The system may include multiple MFCs 608 and associated gas sources, but not necessarily as many MFCs 608 and gas sources as there are culture vessel stations 601 (FIG. 1C). The gas management system 107 (FIG. 1B) manages gas flow from available MFCs 608 / gas sources 609 to meet the needs of the components of the culture vessel 601 (FIG. 1C), regardless of the number of each component, as described elsewhere herein. In one aspect, the MFCs 608 are used to measure the total mass flow rate of gas flowing through a closed conduit. In one aspect, the gas management system 107 (FIG. 1B) may include pressure regulators and filters associated with various types of gases to prepare the gas flow for the MFCs 608. In one aspect, the compressed air may be exposed to a combination filter, possibly filtering particles and various types of gases from the compressed air stream. The present teachings contemplate combinations of filters for all types of gases being supplied to the system, as well as other types of filters.In one aspect, oxygen, nitrogen, and carbon dioxide are provided to the culture vessel in various amounts dictated by the PLC 631 (FIG. 1C). In one aspect, the gas management system 107 (FIG. 1B) can include a gas manifold with automatic switching to ensure 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 can include a gas mixer block to provide a mixture of available gases to the culture vessel 601 (FIG. 1C). The PLC 631 (FIG. 1C) accesses and processes instructions to achieve a desired outcome from sources such as, but not limited to, recipes, user input, and sensor input.

[0058] Referring now to FIG. 1G, sensors located throughout the system of the present teachings can provide analog and digital inputs / outputs that can arrive at the PLC 631 through serial, parallel, or communication ports. Input / output (I / O) from the sensors can be enabled by commercially available or custom I / O cards that provide an interface to the PLC 631. The I / O cards can be connected to the PLC 631 through a backplane and can be configurable by the PLC 631. The I / O can also be enabled by communication cards, such as an Ethernet IP card 625, that enable the use of communication protocols. Such device communication is routed through an Ethernet switch that is connected to a port of the PLC 631. In some configurations, the PLC 631 is wired directly to a block of I / O cards and connectors. In some configurations, the electrical signals can include, but are not limited to, 4-10 mA current loop (analog), RS-485 (serial), or 0-24V (digital). In some configurations, the Ethernet connection uses an RJ45 connector and the serial connection uses a DB-9 connector. Depending on the sensor, the PLC 631 can allow for, for example, scaling and configuration of data transmission / reception protocols. The PLC 631 includes code to build a control loop around the sensor based on the desired use of the system. The sensor can be passive or can control a pump based on, for example, pressure. The sensor can detect, for example, but not limited to, tissue temperature, fluid pressure, fluid temperature, and fluid properties. Sensors can be mounted throughout the system, for example, temperature sensors associated with the bioreactor 601 (FIG. 1C) and fluid 611 (FIG. 1C), and fluid pressure sensors can be mounted throughout the system. The sensors provide data to the PLC 631 (FIG. 1C) and potentially receive commands from the PLC 631 (FIG. 1C), either wired or wirelessly, both of which can be secured from outside or man-in-the-middle interference.Some sensors may be disposable, configured to contact the fluid 611 (FIG. 1C), while others may be durable, configured to read data without contact. A spot sensor may be mounted, for example, on the outside of the bioreactor 601 (FIG. 1C). In some configurations, a non-contact fluid pressure sensor 617 (FIG. 1C) may be mounted in-line anywhere on the tubing used to route the fluid 611 (FIG. 1C), for example, but not limited to, by a luer lock end. In some configurations, the sensor in communication with the PLC 631 (FIG. 1C) may sense 0-60 psi along the fluid path. A signal from the sensor, such as, for example, but not limited to, a 4-20 mA current loop, analog voltage, or digital voltage, may be communicated from the sensor to the PLC 631 (FIG. 1C) through, for example, an analog input module 627.

[0059] 2A-2C, a hardware implementation of a system of the present teachings is shown. Four culture vessel implementations 707 are shown. Also shown is an implementation of elements of fluid handling system 108 (FIG. 1B), including a fluid delivery enclosure 715 associated with the culture vessel implementations 707 as shown in FIGS. 1B and 1E. Specifically, fluid delivery enclosure (FDE) 715 includes valves 701, pump 703, and tube guides 713. Fluid flow lines are shown elsewhere herein. Also shown is a cable / sleeve snorkel 705, as more fully described at least in U.S. Patent Application No. 29 / 758,774, filed November 18, 2020, to White et al., entitled "Cable / Tube Sleeve and Snorkel," and U.S. Patent Application No. 17 / 522,003, filed November 9, 2021, to White et al., entitled "Cable / Tube Sleeve and Snorkel," both of which are 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 incubation station is movable for positioning along the assembly line. A clean room is not required, as there is substantially no exposure of the contents of the incubation vessel 707 or its associated fluids and inner tubing to the environment. Each bioreactor channel, i.e., culture vessel assembly 707, valves 701, and pumps 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 preselected number of FDEs, each of which can have a unique configuration. Each FDE can include several pumps 703, valves 701, and tube routing guides 713. In some configurations, the system of the present teachings can accommodate 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] 3A-3E, schematic diagrams of an exemplary implementation of the fluid and gas lines and components of the system of the present teachings used for cell maturation are shown. FIGS. 3A-3B show components of a gas management system described elsewhere herein. In this implementation, compressed air 801 (FIG. 3A) is routed through an air filter, such as a biological filter, and regulator 806 (FIG. 3A) to a pneumatic block 809 (FIG. 3B). Nitrogen 803 (FIG. 3A), oxygen 805 (FIG. 3A), and carbon dioxide 807 (FIG. 3A) are routed through a pressure valve 802 (FIG. 3A) and a pressure regulator 804 (FIG. 3A), through a filter 810 (FIG. 3B), and to a thermal MFC 811 (FIG. 3B). The MFC 811 (FIG. 3B) is commanded (by a controller) to provide a gas volume that will produce the correct gas mixture 812 (FIG. 3C) for the specific activity occurring in the bioreactor 821 (FIG. 3D).

[0061] 3C-3D, shown are components of a fluid management system described elsewhere herein. FIG. 3C shows fluid source providing and pumping components. In some configurations, growth medium 813 (FIG. 3C), buffer solution 815 (FIG. 3C), and trypsin 817 (FIG. 3C) are possible fluids that may be selectively pumped into bioreactor 821 (FIG. 3D) by pump 819 (FIG. 3C). In this example, container 814 (FIG. 3C) is suspended from load sensor 820 (FIG. 3C), which may measure the volume of fluid delivered as fluid, such as growth medium 813 (FIG. 3C), is delivered. The amount of fluid desired to be delivered can be controlled by use of data provided to a controller by load sensor 820 (FIG. 3C). Illustratively, the quality of growth medium 813 (FIG. 3C) is maintained by chiller 816A (FIG. 3C). Note that the delivery volumes of both the buffer solution 815 (FIG. 3C) and the trypsin 817 (FIG. 3C) may be measured, for example, by a load sensor 820 (FIG. 3C) as described herein. Fluid provided from a source is pumped by a pump 819 (FIG. 3C) controlled by a controller. The pump 819 (FIG. 3C) may include, for example, a non-contact pump that has a high suction lift while providing a gentle pumping action to avoid hemolysis. Such a pump may pump slurries, be reversible, and provide accurate dosing. Measurements of fluid pressure and air in the fluid are made, for example, but not limited to, by an in-line pressure transducer 816 (FIG. 3C) and an in-line air bubble sensor 818 (FIG. 3C) and provided to the controller for monitoring and future control of the pump 819 (FIG. 3C).

[0062] Referring now to FIG. 3D, gases and fluids are provided to and pumped from the bioreactor 821 to provide the products of the cell maturation process to meet the needs further down the production line. For example, shown in FIG. 3D-3E is a configuration that can provide recirculation to allow incubation. The gas blend provided as shown in FIG. 3B is filtered by a gas filter 822 (FIG. 3D) to remove viruses and microorganisms from the gas blend as it proceeds to the bioreactor 821 (FIG. 3D). Filtration can be accomplished, for example, but not limited to, filters sized for unwanted particulates, inertial separators, or electrostatic separation techniques, or a combination. Fluids are also provided to the bioreactor 821. As they are added, the bioreactor 821 is vented, 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 are condensed. The configuration of the system is such that gravity pulls condensate back into the bioreactor 821 (FIG. 3D) and therefore only air is vented to the atmosphere. In this way, the filter 824 remains dry and allows for a continuous flow of air, plus the culture vessel remains hydrated. An exemplary cooler 828 (FIG. 3D) can include, but is not limited to, a Peltier device. The filter 824 (FIG. 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 (FIG. 3D) can be configured to remove contaminants expected to be in the environment of the bioreactor 821 (FIG. 3D) and also protect the environment from contamination. For example, in some configurations, contaminants as small as 0.2 microns can be removed. An exemplary filter can be constructed, for example, from polypropylene and PTFE and can allow the pressure within the system to equalize with the outside atmospheric pressure. Such filters can be, for example, bidirectional, hydrophobic, and autoclavable.The levels of fluids in the bioreactor 821 (FIG. 3D) can be measured and provided to a controller to monitor the operation of the system, as described elsewhere herein. For example, characteristics of the contents of the bioreactor 821 (FIG. 3D) can be measured, such as, but not limited to, pH and DO, sensors for which are shown in FIG. 3D.

[0063] Continuing with reference to FIG. 3D, the temperature of the contents of the bioreactor 821 (FIG. 3D) can be controlled by a combination of a heat maintenance device 830, a bioreactor heat sensor 834 (FIG. 3D), a bioreactor contents heat sensor 836 (FIG. 3D), and a controller to which these data are routed. Depending on the desired process, the reactions in the contents of the bioreactor 821 (FIG. 3D) can be endothermic, exothermic, or static. Thus, the heat maintenance device 830 (FIG. 3D) must be capable of raising or lowering the temperature of the contents or maintaining a static thermal regime. The heat maintenance device 830 (FIG. 3D) can take the form of, for example, a heat exchanger in a coil, a heating blanket, or a jacket recirculation system. The bioreactor heat sensor 834 (FIG. 3D) in conjunction with the bioreactor contents heat sensor 836 (FIG. 3D) can provide the data required by the controller to provide thermal control to the contents.

[0064] 3D and 3F, the bioreactor 821 (FIG. 3D) can be durable and constructed from, for example, stainless steel or glass, or can be single-use, possibly pre-sterilized, and manufactured from polymeric materials. The material specifications of the bioreactor 821 (FIG. 3D) can affect thermal control and can be considered by the controller when determining the desired temperature of the contents. The contents of the bioreactor 821 (FIG. 3D) can require movement, which can be provided, for example, by stirring (pushing), suction (pushing), agitation, use of baffles, or by providing gas flow and removing waste products such as carbon dioxide, depending on the process being performed within the bioreactor 821 (FIG. 3D). Movement can be driven from a top or bottom drive device, for example, mechanically or magnetically driven. The agitator 832 (FIG. 3D) is one way to provide such movement. The impeller can include characteristics such as axial and radial fluid flow, and the propeller can include various shapes such as pitched blades or marine. An appropriate type of propeller / impeller can be selected based on the application. An impeller cone feature 852 (FIG. 3F) is disposed through a shaft cavity 856 (FIG. 3F) on the shaft of the impeller, conforming to the top surface of the impeller. The cone feature 852 (FIG. 3F) prevents cells or equivalent from settling on the flat surface of the impeller when agitation is completed, and allows the cells or equivalent to flow off the impeller and settle to the bottom of the culture vessel, where they can be collected by a dip tube. The cone feature 852 (FIG. 3F) includes a hole 854 (FIG. 3F) for receiving a set screw configured to hold the cone feature 852 (FIG. 3F) in place on the shaft of the impeller. The cone feature 852 (FIG. 3F) includes an interference fit around the shaft of the impeller and does not include any flat area. Impeller fins 880 (Figure 3G) are attached around the cone feature 852 (Figure 3G) at an angle to encourage the cells to slide to the bottom of the culture vessel to direct them to be captured by the tubing during harvesting.

[0065] Continuing with reference to FIG. 3D, in this configuration, samples can be taken from fluids within the system in the fluid lines entering and exiting the bioreactor 821 (FIG. 3D). Additional sampling ports at different locations are also envisioned by the system of the present teachings. A pump 838 can enable recirculation in a system such as that shown in FIGS. 3D-3E, for example, where the contents of the bioreactor 821 (FIG. 3D) are pumped downstream to allow for use of cells produced by the system, or pumped to waste 823 (FIG. 3D). In the configuration shown, harvested cells and media can be pumped downstream, and media from downstream processes can be recirculated back to the bioreactor 821 (FIG. 3D). This configuration includes tubes that extend into the bioreactor 821 (FIG. 3D) to perform various functions, and the amount they extend can be adjusted depending on 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 cells without removing medium. Another tube can extend further into the mixture and can be used to remove cells for use in downstream processes, such as seeding other bioreactors. Another use for the longer tube could be to completely remove medium from the bioreactor 821 prior to adding fresh medium or a different type of medium.

[0066] Now referring to FIG. 3E, the downstream process is shown. In an exemplary configuration, an incubator 851 (FIG. 3E) contains a bioreactor 859 (FIG. 3E). Carbon dioxide 857 (FIG. 3E) is provided to the incubator 851 (FIG. 3E) to establish the environment of the bioreactor 859 (FIG. 3E). In the incubator 851 (FIG. 3E), the bioreactor 859 (FIG. 3E) is rotated by a motor 861 as an example of what a consumer of a batch produced by the system of the present teachings may do. Cells and medium 853 (FIG. 3E) produced by the system of the present teachings are also provided to the bioreactor 859 (FIG. 3E). Medium and possibly other outputs 855 (FIG. 3E) are recirculated to the bioreactor 821 (FIG. 3D). The characteristics of the inflow and outflow fluids (to the bioreactor 859 (FIG. 3E)) can be monitored. For example, pressure and air bubbles in the inflow fluid can be measured and adjusted to ensure that cells will arrive at their destination intact, and pH, DO, and air bubbles in the outflow medium can be measured and adjusted before re-entering the bioreactor 821 (FIG. 3D). The system of the present teachings contemplates other types of properties that may be measured.

[0067] 4-6, the culture vessel system 101 (FIG. 1B) can include features that allow culture vessels of various sizes to be used and various environments to be established around the culture vessel. The culture vessel system 101 (FIG. 1B) can include features that hold the culture vessel in place, for example, to maintain an interface with a thermal management system.

[0068] Continuing with reference to FIGS. 4-6, various sizes of culture vessels can be accommodated in the culture vessel system 101 (FIG. 1B). The exemplary embodiment shown in FIGS. 6 and 7 illustrates a culture vessel 102 captured in a vessel clamp 120 (shown and described in more detail herein with respect to FIG. 13). The culture vessel 102 can include a commercially available bioreactor or a custom-designed bioreactor. The system of the present teachings can accommodate various sizes of culture vessels, and thus the culture vessel 102 is one example. The telescopic sleeve 123 and the support post 125 of the vessel clamp 120 (FIG. 13) can allow the height of the vessel clamp 120 (FIG. 13) to be extended / retracted to accommodate various culture vessel heights. The height of the exemplary culture vessel 102 dictates the slight extension of the telescopic support post 125, which is locked in place by a fastener set in the cavity 132.

[0069] 4-6, vessel clamp 120 (FIG. 13) can stabilize vessel sleeve 115 (FIG. 10) and draw culture vessel 102 into vessel sleeve 115 (FIG. 10A) and thus thermal sleeve 117 (FIG. 11) to ensure thermal conductivity between culture vessel 102 and thermal sleeve 117 (FIG. 11). The culture vessel clamp can ensure that the position of the culture vessel relative to the vessel sleeve and temperature control system is maintained to enable uniform temperature control of the contents of the culture vessel.

[0070] With continued reference to FIGS. 4-6, the temperature management system can be configured to maintain a desired temperature of the contents of the culture vessel. In some configurations, the temperature management can be partially or completely integrated with the culture vessel 102. In one arrangement, a vessel sleeve 115 (FIG. 10A) can enable thermal conductivity between the culture vessel 102 and a thermal sleeve 117 (FIG. 11). The culture vessel 102 can be secured to a vessel clamp 120 (FIG. 13) by a fitting that surrounds a cap 204 of the culture vessel 102. The fitting can include multiple interconnected parts that can be configured to accommodate the size of the cap 204. For example, the fitting can include a vessel clamp ring stand 129 that can be mounted on a telescoping strut 125 and provide a mounting surface for a bracket base 128. The cap 204 can be rested on the vessel clamp ring stand 129 and clamped vertically by at least one head plate bracket 127 that can be operably coupled to the bracket base 128. In some configurations, the bracket base 128 and the head plate bracket 127 can be formed as a single component. In some configurations, the bracket base 128 and the vessel clamp ring stand 129 can be formed as a single component or can be separate pieces. In some configurations, the vessel clamp ring stand 129, bracket base 128, and head plate bracket 127 can be formed as a single component or can be separate pieces. In some arrangements, there are three head plate brackets 127. More or fewer head plate brackets 127 can be mounted on a suitably configured vessel clamp ring stand 129 depending at least on the desired positional security of the culture vessel 102. The vessel clamp 120 can be operably coupled to the vessel sleeve 115 (FIG. 10A) by a cap ring 118.

[0071] Continuing with reference to FIGS. 4-6, the insulating layer 121 can provide thermal insulation between the container sleeve 115 (FIG. 10A) and the environment. The container sleeve 115 (FIG. 10A) can be surrounded by a thermal sleeve 117, which can be securely coupled to the container sleeve 115 (FIG. 10A) by, for example, but not limited to, a strap 143. The thermal sleeve 117 can include a fastening 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 (FIG. 10A) can be maintained by the use of, for example, easily inserted fasteners 129A (FIG. 4), such as, for example, but not limited to, a dowel pin, screw, bolt, or locking pin. The fasteners 129 are selected to allow float as the container sleeve 115 (FIG. 10A) expands / contracts in the vertical axis.

[0072] Continuing with reference to FIGS. 4-6, the thermal sleeve 117 can include a thermal control means 131 and a thermal sensor 155 (FIG. 5). The thermal control means 131 can 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 can be secured 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 can be a single component or separate components operably coupled by, for example, a fastener. The thermal sensor 155 (FIG. 5) can be used to disable the thermal control means 131. The thermal sensor 155 (FIG. 5) can include, for example, but not limited to, a thermal switch with manual or automatic reset, a thermal fuse, or a positive temperature coefficient thermistor.

[0073] 7-9, components of an exemplary system implementing features of the present teachings are shown in partially disassembled form. The culture vessel 102 is shown operatively coupled with a vessel clamp 120 (FIG. 13) as a first step in assembling the culture vessel 102 into an operational configuration. The vessel clamp 120 (FIG. 13) and the culture vessel 102 can be lowered into the vessel sleeve 117 and clamped in place before temperature management can take place. The cap ring 118 can be lowered into the inner geometry 116 over the insulating layer 121 and fastened to the vessel sleeve 115 (FIG. 10A) in a fastening cavity 128A located on the vessel sleeve surface 114. The vessel sleeve 115 (FIG. 10A) and thermal sleeve 117 are configured to receive the culture vessel 102, i.e., can be attached together and mounted on a mounting surface. Once the culture vessel 102 is moved into the inner geometry 116, the strap 143 can be tightened and the fastener 129A can be inserted.

[0074] Referring now to FIG. 9, a configuration is shown in exploded form that allows for various sizes of culture vessels to be used in a system implementing features of the present teachings. To minimize customization requirements, the thermal sleeve 117 can 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, the single component can include a cavity that can allow for the use of all sizes of culture vessels by allowing the addition of the amount of filler material needed to maintain the position of the culture vessel within the thermal / vessel component. The filler material can include any thermally conductive material that can be pressed against the culture vessel through, for example, tightening of the straps 143. The thermal sleeve 117 can include a gap 133 that can allow for space needed to accommodate the thermal expansion of the vessel sleeve 115 and tightening of the thermal sleeve 117 around the vessel sleeve 115. A thermal cutout can be mounted, for example, in the fastener cavity 137.

[0075] 10A, the vessel sleeve 115 can include an inner diameter surface 116 that can be constructed in some configurations to match the geometry of the culture vessel 102 (FIG. 12). For example, if the culture vessel 102 (FIG. 4) includes a tapered geometry 104 (FIG. 12), the inner diameter surface 116 can be tapered to match the culture vessel 102 (FIG. 12). The vessel sleeve 115 can vary in thickness depending on the size of the culture vessel. Temperature maintenance of the culture vessel 102 (FIG. 12) can be accomplished by positioning a thermal sleeve 117 (FIG. 11) around the vessel sleeve 115, positioning the vessel sleeve 115 around the culture vessel 102, and securing the entire configuration in place with a vessel clamp 120 (FIG. 13). The thermal sleeve 117 (FIG. 11) can accommodate any size culture vessel 102 because its diameter does not vary with the diameter of the culture vessel 102. Instead, in some configurations, the interior geometry 116 of the container sleeve 115 varies. The container sleeve 115 can include a thermal ring cavity 112 that can receive a thermal compressible ring (not shown). The thermal ring can be traced from the thermal ring cavity 112 to the interior surface of the cavity 116 and fastened in any suitable manner. The container sleeve 115 is designed to be easily removed, with the thermal sleeve 117 left in place.

[0076] 10B, in some configurations, the vessel sleeve 115A can include a general shape that can be used with a variety of culture vessel sizes and shapes. The generally shaped vessel sleeve 115A can be combined with a conformal and thermally conductive material (not shown) that can ensure a uniformly flush interface between the culture vessel 102 (FIG. 12) and the vessel sleeve 115A.

[0077] 11, the thermal sleeve 117 can be operatively coupled with the thermal modification means 131 and electrical leads (not shown) to power the thermal modification means 131, which can be, for example, a commercially available heater. The thermal modification means 131 conducts heat into the thermal sleeve 117, which then conducts heat into the container sleeve 115 (FIG. 10A). In some configurations, the thermal sleeve 117 can include a gap 133 that can accommodate expansion and contraction of the thermal sleeve 117. In some configurations, the gap 133 can be surrounded by a tapered edge 135, which results in forming a flat mounting surface. In some configurations, for example, an angle bracket (not shown) can be attached on one leg of the bracket at a connection point 138 to the thermal sleeve 117. The other leg of the angle bracket can be used to secure the thermal sleeve 117 to a mounting base (not shown). In some configurations, a thermal cutout sensor 155 (FIG. 9) can be attached to the thermal sleeve 117 at attachment point 137. In some configurations, the thermal cutout 155 can be set 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 the present teachings is to heat the contents of the culture vessel as fast as possible without raising the temperature of the contents above a preselected threshold, such as 37° C. If the temperature of the cutout sensor 155 (FIG. 9) exceeds a preselected threshold, it opens the power circuit and cuts off power to the thermal correcting means 131. In some configurations, the thermal sleeve 117 includes a temperature sensor that allows the sensor control system 105 (FIG. 1B) to use incoming sensor data that senses the temperature of the temperature correcting means 131 and adjust the power to the temperature correcting means 131. The goal of high degree of control over the temperature of the thermal sleeve 117, and therefore the contents of the culture vessel 101 (FIG. 1B), is to bring the contents to a preselected temperature as quickly as possible without damaging the contents. The thermal sleeve 117 can include stress relief notches 139 that can allow the thermal sleeve 117 to expand, contract, and flex to conform when needed.The thermal sleeve may include a cavity 141 that may receive a stabilization device, such as, for example, but not limited to, a dowel pin, a screw, a locking pin, or a bolt. The strap 143 may be tightened using, for example, but not limited to, a strap fastener 157.

[0078] 12, the vessel clamp 120 and the culture vessel are shown before they are coupled, with the vessel clamp 120 shown in exploded form. A bracket base 128 can be operatively coupled to the vessel clamp ring stand 129 at the projection 228. The bracket base 128 and projection 228 are sized to surround the cap 204, and the vessel clamp ring stand 129 is sized to accommodate the diameter of the cap 204. Other configurations are also possible. For example, the projection 228 can include a sliding cavity that can allow the bracket base 128 to be positioned for different size caps. Still further, the vessel clamp ring stand 129 can include a horizontally flexible, but vertically rigid material that can expand / contract with the diameter of the culture vessel, but provide a rigid mounting platform for the bracket base 128 and the telescoping column 125. The telescoping sleeve 123 can be mounted to the cap ring 118 at the mounting cavity 229. Alternatively, the telescoping sleeve 123 and the cap ring 118 can form a single component. Other variations are possible, for example, some telescoping sleeves 123 can be fastened to the cap ring 118 while others are manufactured as part of the cap ring 118.

[0079] 13, the vessel clamp 120 can include an insulating layer 121, a telescopic sleeve 123, a telescopic post 125 connected to a vessel clamp ring stand 129, and possibly a head plate bracket 127 spring-mounted to the vessel clamp ring stand 129. The telescopic post 125 and the telescopic sleeve 125 operate in a coordinated manner to allow the vessel clamp 120 to extend / contract, thereby accommodating culture vessels of various heights. In particular, the culture vessels can be drawn towards the temperature control means provided by the thermal sleeve 117 (FIG. 11), allowing efficient temperature control of the contents of the culture vessel. The desired vessel height can be secured by inserting a fastener into the thermal sleeve 117 (FIG. 11) through the sliding cavity 132. Setting the desired height can be used to accommodate vessels of various heights and ensure that the vessel is high enough for contact with the thermal sleeve, but still keeps the vessel high enough for visual inspection of the fluid contents when the system is powered on. The fastener can set the height of the vessel clamp based on the height of the vessel. The vessel clamp can be adjusted to have proper contact with the vessel ring. It may be desirable to expose a portion of the vessel to view the contents of the vessel over time. The vessel clamp can be adjusted to hold the vessel above the bottom of the vessel sleeve to accomplish exposing a portion of the vessel for visual inspection. Between the bottom of the vessel sleeve and the vessel, heat emanating from the vessel, vessel sleeve, and thermal sleeve can be trapped to maintain thermal uniformity around the vessel. In some configurations, the thermal sleeve can maintain a desired temperature of the contents of the vessel when the thermal sleeve is in contact with 1-2 inches or more of the side of the vessel. In some configurations, an insulating layer (not shown) can be located at the bottom of the vessel sleeve. The vessel clamp 120 can be operably coupled to the insulating layer 121. The insulating layer 121 is constructed from a material that does not encourage thermal conduction between the base surface on which the vessel clamp 120 is mounted and, ultimately, the contents of the culture vessel.The insulating layer 121 is operably coupled to the vessel sleeve 115 (FIG. 10A) through a cap ring 118. The cap ring 118 covers the insulating layer 121 and presents a clean interface to the culture vessel assembly. To securely position the culture vessel 102 (FIG. 12) for operational use, the vessel sleeve 115 (FIG. 10A) is placed into a thermal sleeve 117 (FIG. 11), which may be previously secured to a base chassis structure and coupled to a power supply and data I / O devices. In some configurations, to use a different culture vessel, e.g., to use a smaller sized vessel, the vessel clamp 120 and vessel sleeve 115 (FIG. 10A) can be removed and the vessel sleeve 115 (FIG. 10A) can be replaced with an appropriately sized sleeve, while the thermal sleeve 117 (FIG. 11) along with its data and power connections remains in place. No modification of the thermal management system is required to modify the culture vessel.

[0080] Referring again to FIG. 1B, pH and DO are important performance factors in the growth and maturation of cells in culture. Cell growth occurs in tissue culture vessels where cells are surrounded by growth medium, a substance that contains growth factors, serum, and other additives. Cell growth is the process of producing cells from a single cell. The pH level and amount of DO and other cell characteristics are maintained by the controlled addition of gases such as oxygen, nitrogen, and carbon dioxide by a gas management system 107.

[0081] Referring now to FIG. 14, in some configurations, the gas management system of the present teachings can include a configurable number of active MFCs, possibly differing from the configurable number of active bioreactors, i.e., a complement of MFCs for all gases is not required per bioreactor. When a constant gas overlay is not required, each active bioreactor is given a time slot during which it can receive the required gas mixture. Gases can settle into the bioreactor contents during times when no gas is being provided to the bioreactor. If applicable, the time gap between subsequent gas delivery to a particular bioreactor is configurable, as are the number of MFCs, the number of active bioreactors, and the amount and type of gas provided to a particular bioreactor during a time slot given to the bioreactor. These variables are set, for example, in a recipe and enabled by a PLC. Many possible combinations of gas delivery are also envisioned by the present teachings.

[0082] With continued reference to FIG. 14, the gas management system controls gas resources across multiple culture vessels. In some configurations, sensors and mass flow controllers (MFCs) that control the amount of oxygen, nitrogen, and carbon dioxide exposed to the contents of the culture vessels, for example but not limited to, are the primary features of the gas management system. In some configurations, the MFCs receive a mixture of gases, at least in part, depending on data from sensors associated with a particular culture vessel when the controller is controlling and monitoring multiple culture vessels. The sensors can be mounted on the culture vessel or elsewhere, depending on the configuration of the system. In some configurations, sparging can be used to introduce gases into the contents of the culture vessel. In some configurations, gas management is enabled by electronic solenoids driven by digital output cards on the PLC chassis. In some configurations, the MFCs communicate with the PLC 301 via Ethernet IP and the sensors communicate with the PLC 301 via RS-485, a serial protocol. In some aspects, a gateway is used to convert the serial protocol to Ethernet IP. Other communication methods and protocols are also contemplated by the present disclosure.

[0083] Continuing with reference to FIG. 14, gas management can be used to regulate the pH of the culture vessel contents. For example, some cell lines grow within a pH range of 7.0 to 7.4. Additions to the culture vessel contents can initially set the pH of the contents to the desired range. For example, the culture medium can include a bicarbonate buffer. As the culture vessel contents convert glucose to lactic acid, 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 unless lactic acid accumulates 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 overlaying carbon dioxide to lower the pH and overlaying nitrogen to allow the pH to rise. A PID loop drives the desired (variable) gas flow rate to the MFC. In one aspect, one gas, such as, but not limited to, carbon dioxide or nitrogen, is activated at a time depending on whether the pH is above or below a desired set point.

[0084] With continued reference to FIG. 14, oxygen gas (DO) dissolved in blood is consumed by cells in the culture vessel and requires replenishment by a gas management system. Some types of cell culture are carried out with a DO in the range of, for example, 20-50% oxygen saturation. In some configurations, air or nitrogen and oxygen can be added automatically under the control of a controller based on a comparison of a sensor reading of DO in at least the contents of the culture vessel to a preselected set point. In one aspect, a set point for DO in the culture vessel is achieved by layering in oxygen to increase the percent dissolved oxygen in the vessel, or layering in nitrogen to lower it. In one aspect, bidirectional control is achieved using a PID loop with each MFC. In one 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 set point. When DO exceeds the set point, 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 aspect, the contents of the culture vessel can simply consume oxygen until a set point is reached.

[0085] Continuing with reference to FIG. 14, the gas management system 107 can achieve a compact physical footprint and optimal equipment cost by using a different number of MFCs than the bioreactor 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 a specific type of liquid or gas at a specific range of flow rates. The description of the present teachings is not limited to the limitations of currently available MFCs. For example, MFCs that are not limited to handling a specific type of gas or a range of flow rates can be envisioned. Self-calibrating MFCs are envisioned by the description. In one arrangement, the MFCs can include devices that can programmably handle multiple gas and pressure or flow rate inputs that provide precise and repeatable outputs, such as, for example, but not limited to, the Brooks SLA 5800 series, connected by piping and filters to achieve a 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 one aspect, the MFCs can include one MFC for O2, one MFC for N2, one MFC for CO2, and one MFC for compressed air, although any number of MFCs with any assortment of source gases are possible. The number of culture vessels in the configuration is a function of the number of cell cultures being grown or tissues being maintained. In one aspect, the MFCs can deliver a potentially mixed gas to multiple bioreactors at scheduled time intervals. Using a single set of MFCs for all bioreactors to blend the gas mixture, and then sequentially, timed, and / or intermittently layering or sparging each bioreactor minimizes the number of MFCs required and minimizes gas consumption, exhaust, and waste.

[0086] Continuing with reference to FIG. 14, in one arrangement, the controller 106 can maintain the set point 303 by commanding the gas management system 107 to provide gas to the culture vessel 305 at various flow rates proportional to the control loop error. The percentage depends on the flow rate and varies among the gases. For example, for nitrogen and oxygen, the percentage is higher than for carbon dioxide. The control loop error is calculated from a PID loop dedicated to each bioreactor. The PID loop 303 can be used to drive the desired (variable) flow rate to the MFC. The controller 301 sends a set point based on the PID loop error to the gas management system 107, which includes the MFC. For example, Gas Flow Rate = C x Control Loop Error where C=f (flow rate, type of gas) Control loop error = set point - measured data. A proportional controller is set to work with a specific cell type. PID tuning parameters can be empirically determined by starting with the cell type and working backwards. In some arrangements, proportional control can be used without integral or derivative aspects. In some arrangements, steady state control (low deviation from set point) can be achieved by using either proportional, integral, or derivative control separately or in combination.

[0087] Continuing with reference to FIG. 14, the pH set point within the culture vessel 305 is maintained by overlaying CO2, which allows for a reduction in pH (more acidic), and N2, which allows for an increase in pH. Gassing can maintain a steady pH until glucose and lactate levels become the overwhelming pH driving factors. As explained 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) (incorporated herein by reference in its entirety), after a period of time, a medium change will be required to maintain the pH set point due to glucose consumption and lactate production. When the pH measurement is <7.2, a medium change may be necessary. In some arrangements, if continuous gassing cannot change the characteristics of the cell culture, an immediate change of medium will be initiated. In some arrangements, glucose and lactate can also be monitored.

[0088] Continuing with reference to FIG. 14, the set point of dissolved oxygen (DO) in the culture vessel 305 can be achieved by overlaying O2 to increase DO% or overlaying N2 to decrease DO%. The set point value is process specific depending on the type of cells being propagated 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) (incorporated herein by reference in its entirety), some cells or tissues grow in oxygen-rich environments while others grow in oxygen-deficient environments. In addition, gas transport laws dictate the amount of oxygen that dissolves into solution and ultimately reaches the cells. In one arrangement, recipes can be used to adjust the pH and DO set points according to the current values ​​of the various measured parameters, and the recipes are made available to the controller 106, allowing the system to accommodate various pH and DO set points derived from cell types. As with the control of pH, bidirectional control would be achieved using two PID loops 303 for each set point. Bidirectional control provides a response to positive and negative errors relative to the set point, for example adding oxygen when below the set point and adding nitrogen when above the set point.

[0089] Continuing with reference to FIG. 14, in one aspect, the system can include multiple culture vessels 305 for growing cells and tissues simultaneously. Since there can be multiple vessels used at one time, a control strategy is developed to deliver a specific gas mixture to each vessel 305. When the rate of change of pH and DO levels is slow enough that constant gas overlay is not required, gas can be overlayed into the culture vessel 305 for a period of time, and then gas injection can be suspended while gas diffusion into the contents occurs. A pause period of preselected time can be built into the process to account for processes / contents that require more than a preselected gas delivery time. When gas is being delivered, a PID loop dictates the set point for the monitored characteristic, such as, but not limited to, pH and DO. In one aspect, each culture vessel includes a separate and / or dedicated PID loop. Providing a gas overlay periodically is a successful strategy, at least in part, because the affected characteristic has a relatively slow rate of change. Isolation of gas delivery to one culture vessel is achieved by actuating a solenoid valve downstream of the MFC to control the direction of the gas mixture.

[0090] Continuing with reference to FIG. 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 change the mixture, if necessary, to accommodate the contents of the second culture vessel 305, and then instruct the gas management system 107 to deliver gas to the second culture vessel 305. This process can be repeated for each culture vessel 305 in the system. The culture vessel 305 can be revisited with additional gas injections, possibly a different mixture, depending on the specific requirements of the culture vessel contents and / or depending on the parameter measurement results. Isolation of gas delivery to one culture vessel 305 can be achieved by activating a specific solenoid valve based on a timed schedule. Coordination of MFC set points and valve states can be driven by a sequencing routine in the controller. The sequence gives each culture vessel a period to read pH / DO through the pH / DO sensors (Figure 1), deliver gas, and move on to the next culture vessel. This time sharing allows for the sharing of hardware components, minimizing gas consumption, exhaust, and waste.

[0091] 14, for example, if there are six culture vessels 305, each requiring gas delivery over 5 minutes, a cycle of cycling through the culture vessels 305 and supplying them with gas would require 30 minutes. For those 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 an exemplary process, the set points to each MFC can be changed every 5 minutes depending on the error output of the pH and DO PID loops 303 for each culture vessel 305. The controller 106 calculates the error from the PID loops / probes in each culture vessel 305 and makes settings to correct the gas for the next gas window.

[0092] Continuing with reference to FIG. 14, condensation of liquid in the gas lines exiting the culture vessel can potentially foul the gas management equipment. In some aspects, an air dryer can be used to strip the incoming gas from liquid impurities. In some aspects, a heating system, such as, for example, a Peltier heating system, can prevent condensation of liquid in the gas lines 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 materials including two dissimilar conductors or semiconductors. An increase in temperature occurs at the junction, where, for example, copper is transformed into bismuth, and a decrease in temperature occurs at the junction, where, for example, bismuth is transformed into copper. In some aspects, the Peltier heating system includes a thermoelectric heater and a fan. In some aspects, a controller provides commands to the fan and thermoelectric heater through a digital output associated with the controller.

[0093] 15A-15F, components of an exemplary gas management system are shown. Specifically, an enclosure housing the MFC and gas manifold is shown in FIG. 15A. A shroud 905 and a base 901 form an enclosure that protects the gas management system of the present teachings. Extending from the shroud 905 is a pressure relief valve 907 that is attached to a mixing manifold 917 (FIG. 15D). The shroud 905 (FIG. 15B) is partially removed to show the distribution manifold 915 (FIG. 15B / C). Gas entering the MFC inlet can be filtered by a particulate filter 918 (FIG. 15C). The distribution manifold 915 (FIG. 15B / C) receives gas according to the type and amount required for a particular process as defined by the PLC 106 (FIG. 1B). Each gas train incorporates a connection to an MFC 925 (FIG. 15E), a particulate filter 918 (FIG. 15E), and a check valve. There are as many gas trains as there are source gases in the system. Gases from each gas train (MFC) are combined in a mixing manifold 917 (FIG. 15D) and distributed to the desired bioreactors through a distribution manifold 915 (FIG. 15D). The distribution manifold 915 (FIG. 15D) is the outlet from the gas mixer to the bioreactors. The distribution manifold 915 (FIG. 15F) allows for distribution of source gases by PLC 106 (FIG. 1B) command. The distribution manifold 915 (FIG. 15F) includes features such as a pneumatic manifold fixture 933 / 935 (FIG. 15F), an exemplary five-station unit 937 (FIG. 15F), and an exemplary two-way normally closed solenoid valve 931 (FIG. 15F). The system of the present teachings contemplates larger or smaller units to accommodate more or fewer gas sources.

[0094] Referring now to FIG. 16, a controller (PLC) 106 (FIG. 1) receives data from sensors associated with the culture vessel, fluid management system, and gas management system, and uses the data to control system functions to achieve a desired outcome, such as, but not limited to, tissue generation, scaffold decellularization, and scaffold recellularization. The PLC 106 (FIG. 1B) is wired (or wirelessly) integrated to control other parts of the system without requiring command translation. The PLC 106 (FIG. 1B) can detect faults in the incoming sensor data and, after validation is complete, use the data to enable state changes, for example, to select a sequence of output commands to motors, pumps, and valves. In some configurations, the order of state changes and / or the sequence of commands can be driven by a recipe. The recipe itself can be changed by user input or by the PLC 106 (FIG. 1B) based on conditions in the system. Other ways in which a recipe can be changed or control can proceed are also envisioned by the present teachings.

[0095] Continuing with reference to FIG. 16, the PLC 106 (FIG. 1B) of the present teachings follows a general flow for all processes per culture vessel station (including the culture vessel 503 (FIG. 1B), the fluid handling system 108 (FIG. 1B), and the culture vessel control 111 (FIG. 1B)) that may be performed by the system of the present teachings. The PLC 106 (FIG. 1B) may simultaneously manage different processes performed in the culture vessel of the present teachings. Thus, a version of the method 950 may be performed in the PLC 106 (FIG. 1B) for each culture vessel. The method 950 may include, but is not limited to, step 951 of receiving a selection of a process to be initiated. Exemplary processes include, but are not limited to, cell maturation, organ decellularization, and organ recellularization. The selection may be made by, for example, a user, for example, but not limited to, through a secure remote communication line, a local wired line, or a secure wireless wireless connection. The HMI 630 (FIG. 1C) is one way that a user may select a desired process. A display associated with the HMI 630 (FIG. 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, active or not. Alternatively, the HMI 630 (FIG. 1C) can include multiple monitors, each supporting an active culture vessel. Still further, the processes can be interrelated, so that process selection occurs automatically or semi-automatically, and the user can confirm the selection before the process execution proceeds. For example, a decellularization process can automatically or semi-automatically start a recellularization process. The method 950 can include a step 953 of accessing a recipe associated with the selected process. When there are multiple possible recipes for the selected process, user input can be required to choose the desired recipe.Alternatively, the PLC 106 (FIG. 14) can select and access the most appropriate recipe based on previous or parallel processes executed by one or more of the bioreactors in the system. User interaction can be provided to validate the selection, depending on the process and characteristics associated with the recipe.

[0096] Continuing with reference to FIG. 16, the method 950 can include a step 955 of accessing, calculating, receiving, or prompting for parameters associated with the selected process and / or the selected recipe. For example, set points for temperature, pH, and DO can be provided. The user can type in the required values ​​or select to use default values, or the user can choose to exit the loop entirely, as the system can select default values ​​or calculate values ​​based on prior or parallel activity in the system. The method 950 can include a step 957 of starting the selected recipe using the parameters. The recipe start sets up a processing loop that moves through the recipe until all phases have been performed, then checks for additional recipes, and finally returns to the beginning to receive a process selection. In the processing loop, the method 950 can include a step 959 of executing a phase from the recipe, for example, executing instructions to open a valve, pump medium, or activate an agitator. The elements of the ISA-88 phase module are provided in the system of the present teachings by, for example, but not by way of limitation, a program that opens valves, starts pumps, totals flows using sensors until a preselected amount is reached, stops pumps, and closes valves. The phase control of the present teachings arbitrates ownership of equipment based on preselected criteria. For example, in some configurations, when running an automation phase, all devices are set to be controlled by the PLC unless there are manual steps in the sequence. Essentially, all equipment is owned by the PLC during the automation phase and recipe. In some configurations, an operator takes ownership of a device of interest under specific circumstances. When the operator is done using the device, the phase is resumed and the PLC takes ownership of the device. In some configurations, possible modes include operator, external, and maintenance. Other possible modes are also contemplated by the present teachings. Modes are associated with users who may lock ownership of equipment.The owner user must release the lock before the controller can grant ownership to another user. In the system of the present teachings, multiple phases can be run simultaneously. For example, a heating phase can be run simultaneously with an agitation phase and a recirculation phase. In some configurations, each culture vessel has dedicated equipment and software, allowing simultaneous unrelated operations on different culture vessel contents.

[0097] Continuing with reference to FIG. 16, data from sensors in the system can indicate, for example, when a phase is completed, or the PLC 106 (FIG. 1B) can move to the next phase after a preselected amount of time has elapsed. The recipe can dictate how each phase should be handled, or possibly leave this to calculations made by the PLC 106 (FIG. 1B) based on collected sensor data as the process progresses. The method 950 can include step 961 of receiving data from sensors associated with the culture vessel station where the process is being performed, and step 963 of adjusting properties and parameters, if necessary, based on the sensor data. Many such examples 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, the sensor data can indicate that a phase is completed. At 965, if there are more phases in the recipe, the method 950 can include returning to step 959 and executing from that point. If there are no further phases to execute in the recipe at 965, and if there are other recipes associated with the process at 967, the method 950 may include returning to step 953 and executing from that point. If there are no other recipes associated with the process at 967, the method 950 may include returning to step 951 and executing from that point.

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

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

[0100] 17A-17B, 18A-18C, and 19A-19B, various applications of the present system for decellularizing a heart are shown along with various component layouts. Referring to FIG. 17A, a simplified fluid flow diagram of the system of the present teachings is shown with respect to a heart during decellularization. In particular, a solution 351, such as nutrients in the form of a medium, is selected by valve 353 and pumped by pump 355 past sensor 357, selectively to either waste 359 or descending aorta 361. As fluids diffuse through the heart 371, they either pass from the pulmonary artery 363 to waste 359 or are recirculated back through the heart 371. Fluids exiting the heart 371 pass through the pulmonary vein 365 to a pressure vessel 367 to either waste 359 or recirculation. A pressure vessel is a leak-proof container that stores liquids or gases at higher or lower than atmospheric pressure. The pressure vessel 367 provides back pressure to the fluid exiting the bioreactor 369. In some configurations, the back pressure can come from indirect pressure through head height at an elevation above the bioreactor 369. The present teachings contemplate other methods of use of the pressure vessel 367 and other configurations for producing back pressure. In some configurations, the pressure vessel 367 is vented and effluent flows to waste 359. Other configurations are also contemplated by the present teachings. In some configurations, the pressure vessel 367 can maintain a fixed pressure in the pulmonary veins / left ventricle and encourage a higher percentage of flow through the coronary arteries and out the pulmonary artery. This pressure can keep the heart in an expanded state. Other configurations, such as a pressure control loop, are also contemplated by the present teachings. Various shapes of pressure vessels can be used depending on the type of gas used to provide pressure and the amount of pressure required. For example, pressure vessel types include, but are not limited to, cylindrical, conical, spherical, horizontal, or vertical, which may be crowned by heads of various shapes. For example, non-spherical pressure vessels require heads. Head types may include, for example, hemispherical shapes and shallow shaped (dish) heads (semi-elliptical or dished).

[0101] 17B, ​​for a specific decellularization configuration, the solution 351 can include, but is not limited to, buffered saline (PBS), detergent 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 can include, but are not limited to, detergent sodium deoxycholate (SD / SDC), detergent Triton X-100, zwitterionic solutions such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), trypsin / ethylenediaminetetraacetic acid (EDTA), and deoxyribonuclease (DNAse). SDS is a detergent known to denature proteins. Peracetic acid is used for sterilization of the final scaffold. Denaturing proteins involves breaking many of the weak links or bonds, e.g., hydrogen bonds, within the protein molecule. Bonding is responsible for the ordered structure of proteins, therefore denatured proteins have a looser, more random structure and are likely to be insoluble. PBS is a phosphate buffered solution, which is 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 sulfate. Valves 353 in this configuration include a mixture of normally closed solenoid valves and manual valves. In some configurations, the manual valves can be associated with certain source fluids, such as, for example, but not limited to, hypotonic NACl, heparinized PBC, and PBC / peracetic acid. In some configurations, the manual valves can be used to help prevent leakage during the set-up and disassembly process and are not used in the automated process. Manual valves can be used on containers that hold solutions that will be used in the process, and are a way for these vessels to be filled, transported, and deployed offline without the risk of leaking or contaminating the solutions in the source.The selected solution is pumped by pump 355 through pressure sensor 357 and a flow / bubble meter to either waste 359 or bioreactor 369, specifically the descending aorta 361. Pump 355 can include a peristaltic type pump that meets the desired pump characteristics described elsewhere herein.

[0102] 18A-18C, an exemplary decellularization recipe and associated valve operations are shown. FIG. 18A and Tables III and IV illustrate how a system with two fluid pumps and 12 fluid valves can be implemented to decellularize an organ such as a heart and pump fluids to a collection container. With reference to FIG. 18A, in this exemplary use, fluids are pumped from a first pump from a source of heparinized PBS, hypertonic NACl, PBS / peracetic acid, SDS, Triton, PBS, and deionized (DI) water, past a pressure gauge, and into the descending aorta. Fluids within the heart are routed to waste or collection containers. With reference to FIG. 18B, the phases executed to accomplish decellularization of the heart according to an exemplary recipe are listed. The first four phases involve clearing blood from the heart using heparinized PBS pumped alternately by volume and by time / pressure by the first pump while the second pump controls the output to a preselected pressure. It should be noted that the system of the present teachings allows for variation in pump volume, pressure, and time values, and therefore the values ​​listed in FIG. 18B are exemplary only. In the fifth phase, the remainder of the blood is washed out of the heart with DI water. The next phase involves lysing the cells in the heart with NaCl and then washing out the disrupted cells with DI water. Cell debris is then removed from the heart by repeated applications of SDS and DI water. In this recipe, there are three iterations of the debris removal sequence, involving two applications of SDS, followed by one application of DI water. The final phase of material uses Triton to remove residual SDS. The final two phases involve washing the remaining scaffold with DI water and PBS. FIG. 18C lists the valves shown in FIG. 18A that are used for each of the phases listed on FIG. 18B.

[0103] 19A-19C, another feature of the system of the present teachings includes growing one type of cells on one side of a culture vessel scaffold and another type on the other side, combining two types of cells. Such a configuration can include a source fluid, at least one culture vessel station including a culture vessel 503A / B, a fluid handling system 108, a pump 256, a culture vessel control system 111, a gas management 107, a biocontroller 106, a waste 274, a bioreactor 272, a rotation 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 rotation feature 270 can move the seeded area to a position where the cells will be encouraged to rest and multiply. The vent 280 or other exit path can be configured to prevent pressure buildup. A system such as that shown in FIG. 19A can be used to grow cells, such as suspension cells, aggregates, or cells on microcarriers. When cell density reaches a target threshold, the cells are allowed to settle, excess medium is removed, and a wash solution 252, such as, but not limited to, PBS, is added / removed. Trypsin 254 is added to digest microcarrier / extracellular attached proteins, high protein medium 262 or inhibitors are added to inhibit trypsin 254, medium 262 is added / removed to adjust its concentration, and the cells are then pumped to one side of the scaffold 266. Additional medium 262 can be used to clear out any cells in the tubing dead volume. The other side of the scaffold 266 is seeded as soon as the first side of the scaffold 266 has been completed attached by cells simultaneously growing in the second culture vessel 503B, for example, or at a later time if the first layer developed in the first culture vessel 503A needs time to mature. Medium exchange in the bioreactor 272 is performed, for example, continuously through a flow loop or periodically using an air vent.The second bioreactor (or third bioreactor) can be configured to perform medium exchange in the bioreactor 272. The choice of option is based at least on process timing, medium composition, and tissue size / metabolism. Sensors (shown elsewhere) are installed in the culture vessel 503A / B, the medium vessel 262, the tissue bioreactor 272, and / or in any of the fluid paths (e.g., tissue bioreactor inlet / outlet 276 / 278). In some configurations, thermal control over the medium 262 is maintained in the thermal device 264. The rotating means 270 can rotate the scaffold 266 about the rotation axis 268. In some configurations, waste products can be routed away from the bioreactor 272 into a waste collection 274. In some configurations, double-sided seeding can 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 times. The cells produced in the bioreactor 272 and a portion of the waste products can return to the culture vessel 503 and waste collection 274, respectively. Shown in Figure 19C is a rotation means 270 as described herein. In some configurations, cells grown outside the culture vessel system depicted in Figures 19A-19C can be provided to the bioreactor 272 through a specially configured port. In such a configuration, multiple types of cells, both cells grown in the culture vessel 503 (Figure 19B) and cells grown elsewhere, can be introduced into the bioreactor 272 to seed 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 completed to form more complex tissues.

[0104] A system for repeatably performing at least one type of tissue-related process as part of a manufacturing line, the system comprising: at least one culture vessel station including a variable-sized culture vessel, a fluid handling system, and a culture vessel control system, the at least one culture vessel station configured to accommodate performing at least one type of tissue-related process; a gas management system configured to provide 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 to perform the at least one type of tissue-related process, the controller configured to communicate with components on the manufacturing line using a standard industrial communication protocol. In the system as described herein, the at least one variable-sized culture vessel comprises a disposable component. In the system as described herein, the at least one variable-sized culture vessel comprises a durable component. In the system as described herein, the at least one variable-sized culture vessel comprises a vessel sleeve surrounding at least a portion of the at least one variable-sized culture vessel, the vessel sleeve configured to transfer thermal energy to the at least one variable-sized culture vessel, a thermal sleeve operably coupled to the vessel sleeve, the thermal sleeve controlling an amount of thermal energy entering the vessel sleeve, and a vessel clamp stabilizing the at least one variable-sized culture vessel within the thermal sleeve. In the system as described herein, the fluid handling system is configured to move fluid through the at least one variable-sized culture vessel. The system as described herein further comprises a sensor control system that controls an amount of thermal energy entering the vessel sleeve. In the system as described herein, the gas management system is configured to control the type and amount of gas entering the at least one variable-sized culture vessel.In the system as described herein, the vessel clamp comprises a telescoping device, the telescoping device adapted to the height of at least one variable-sized culture vessel. The system as described herein further comprises a thermal insulation layer between the vessel sleeve and an environment surrounding the vessel sleeve. The system as described herein further comprises an electrical cutoff device that senses when the thermal sleeve reaches at least one preselected threshold temperature, the electrical cutoff device configured to disable the addition of further thermal energy to the vessel sleeve. The system as described herein further comprises at least one band clamp that secures the thermal sleeve to the vessel sleeve. In the 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 vessel 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 that fills a space between an inner diameter of the vessel sleeve and an outer diameter of the at least one variable-sized culture vessel. The system as described herein further comprises a plurality of vessel sleeves adapted to accommodate a plurality of sizes of the at least one variable-sized culture vessel. The system as described herein further comprises a sensor system configured to monitor a variable-sized volume of cells in the at least one variable-sized culture vessel. In the system as described herein, the fluid handling system comprises a variable number of at least one valve and at least one pump configured to move fluids in and out of the at least one variable-sized culture vessel station, the at least one controller controlling the at least one valve and the at least one pump. In the system as described herein, the at least one controller comprises instructions configured to control a plurality of at least one culture vessel stations performing independent tasks simultaneously.In a system as described herein, a first of the at least one culture vessel station performs a first type of at least one type of tissue-associated process in parallel with a second of the at least one culture vessel station performing a second type of at least one type of tissue-associated process. In a system as described herein, the first type of at least one type of tissue-associated process comprises an identical type to the second type of at least one type of tissue-associated process. In a system as described herein, the first type of at least one type of tissue-associated process comprises a different type from the second type of at least one type of tissue-associated process. In a system as described herein, the first type of at least one type of tissue-associated process comprises decellularization. In a system as described herein, the first type of at least one type of tissue-associated process comprises recellularization. In a system as described herein, the first type of at least one type of tissue-associated process comprises cellular maturation of the recellularized tissue. In a system as described herein, the first type of at least one type of tissue-associated process comprises perfusion of endothelial cells. In the system as described herein, the at least one controller includes determining the fluid flow path based at least on a recipe. In the system as described herein, the at least one controller includes dynamically determining the fluid flow path. In the system as described herein, the at least one controller includes determining the fluid flow path based at least on user input. In the system as described herein, the at least one controller includes determining the fluid flow path based at least on a combination of a recipe, dynamically determined parameters, and user provided parameters. In the system as described herein, the at least one type of tissue related process includes generating a batch. In the system as described herein, the batch is produced according to at least one industry standard process.In the system as described herein, the at least one industry standard process comprises ANSI / ISA-88.01-1995. In the system as described herein, the standard industry communication protocol comprises Ethernet / Industrial Protocol. In the system as described herein, the gas management system comprises at least one mass flow controller configured to receive a source of gas, the amount of gas being controlled by the at least one controller, a mixing manifold configured to blend a plurality of types of gas from the at least one mass flow controller, the amount and type of the plurality of types of gas being controlled by the at least one controller, and a distribution manifold that receives the blended plurality of gases and distributes the blended plurality of gases to at least one incubation station according to commands from the at least one controller. In the system as described herein, the number of the at least one mass flow controller is independent of the number of the at least one incubation vessel station. In the system as described herein, the plurality of at least one mass flow controllers are configured to provide amounts and types of the plurality of types of gas to the plurality of at least one culture vessel station according to a periodic delivery function. In the system as described herein, the periodic delivery function is based at least on values ​​collected by a sensor associated with the at least one culture vessel station. The system as described herein further comprises a cone feature attached to a shaft of an agitation device in the culture vessel, the cone feature substantially preventing cells from settling on the agitation device.A method for seeding multiple types of cells onto multiple areas of a scaffold, the method comprising: operatively coupling the scaffold to a rotating means in a bioreactor, the bioreactor configured to receive the multiple types of cells through multiple ports in the bioreactor; when a cell density of a first cell type of the multiple types of cells in a first culture vessel reaches a preselected threshold or after a preselected waiting period, (a) removing excess medium from the culture vessel; (b) washing the first cells of the first cell type in the culture vessel using a digestion solution; and (c) culturing the first cells of the first cell type using a digestion solution. (d) digesting the microcarrier / extracellular attached proteins in the culture vessel, (d) inhibiting the digestion solution, (e) adjusting the concentration of the medium, (e) pumping the first cells from the culture vessel to a first area of ​​the plurality of areas of the scaffold in the bioreactor, and (f) treating a second cell type of the plurality of cell types according to steps (a)-(e) when a first preselected time has elapsed, and (g) pumping the second cells from the culture vessel to a second area of ​​the plurality of areas of the scaffold in the bioreactor, where the second cell type is generated in the second culture vessel. The method as described herein may further include rotating the scaffold after the first area is seeded, the rotating positioning the scaffold to receive the second type of the 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 present disclosure. Therefore, the present disclosure is intended to embrace all such alternatives, modifications, and variations. In addition, while some exemplary configurations of the present disclosure are shown in the drawings and / or discussed herein, the present disclosure is not intended to be limited thereto, as the present disclosure is as broad as the art will permit, and the present specification is intended to be read as well. Thus, the above description should not be interpreted as limiting, but merely as an illustration of a particular configuration. In addition, those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto. Other elements, steps, methods, and techniques that are insubstantial different from those described above and / or in the appended claims are also intended to be within the scope of the present disclosure.

[0106] The drawings are presented only to demonstrate certain embodiments of the present disclosure. In addition, the drawings described are illustrative only and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to a particular scale for illustrative purposes. In addition, elements shown in the drawings having the same numbers may be the same elements or similar elements, depending on the context.

[0107] When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. When referring to a singular noun, when an indefinite or definite article is used, such as "a", "an", or "the", this includes the plural of that noun, unless something else is specifically stated. Thus, the term "comprising" should not be interpreted as being limited to the items listed thereafter, as it does not exclude other elements or steps, and thus the scope of the expression "a device comprising items A and B" should not be limited to a device consisting of only components A and B.

[0108] Moreover, the terms "first," "second," "third," and the like, whether used in the description or claims, are provided to distinguish between similar elements and not necessarily to describe a sequential or chronological order. It is to be understood that terms so used are synonymous under appropriate circumstances (unless expressly disclosed otherwise) and that the exemplary configurations of the disclosure described herein are capable of operation in sequences and / or arrangements other than those described or illustrated herein.

Claims

[Claim 1] The invention described in this specification.

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