System and method for preparation of cells and tissues

Abstract

To provide a manufacturing system and method for creating multiple tissue constructs from cells.SOLUTION: A system can include a thaw subsystem (if the cells are provided in a frozen state), an expansion subsystem, a concentration subsystem, and a tissue maturation subsystem. Each of these subsystems is modular and can be reconfigured, and a process can be repeated depending on the specific tissue process being implemented. Multiple tissue types can be combined in multiple bioreactors. The activities of multiple bioreactors can be coordinated and controlled in an automated manner by a supervisor controller. The supervisor controller can receive user input at the start of the process, and can manage the process thereafter, alerting the user if user actions are required.SELECTED DRAWING: None

Description

Technical Field

[0001] Government license right This invention was made with government support under Contract No. F0008, awarded by the Advanced Reproduction Manufacturing Institute, and is subject to the terms of the contract under W911NF-17-3-003. The government has certain rights in this invention. (Cross - reference to related applications)

[0002] This utility patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 862,379, filed on June 17, 2019, entitled "System and Method for Cell Preparation" (Attorney Docket No. Z69), which is hereby incorporated by reference in its entirety.

Background Art

[0003] The present disclosure generally relates to tissue processing lines, and more specifically to tissue processing lines that can automate multiple simultaneous instances of allogeneic, syngeneic, and autologous tissue manufacturing processes. Single - use components and non - invasive sensing can be used to support commercial scale - up in a closed - flow path within a non - environmentally controlled ISO "gray space".

[0004] Manual tissue processing in the laboratory has been attempted for many years. The methods used for processing are typically plagued by non-reproducibility or non-consistency from run to run, due to the skilled manual nature of the work and the relatively poorly understood biological input materials. Current methods are also typically highly labor-intensive. However, the basic elements of tissue processing can include processes that can be automated. In particular, automation can eliminate human error and improve consistency between production runs. Automation must include tying together the process start timing associated with the heterogeneous processes required to implement the entire processing line and the biological processes that may underlie tissue generation. Monitoring of specific characteristics indicating the state of the tissue must also be continued and fed back into the system activity control loop.

[0005] Tissue processing can occur in stages. If cells arrive in a frozen state, they can be thawed. Cells arriving in a non-frozen state, i.e., thawed cells, may require expansion, concentration, and maturation steps. Typically, expansion occurs in static bioreactors located within flasks and incubators, which require manual media exchange. Typically, monitoring is visual and subjective. Transfer of cells from one stage to the next is typically done manually.

[0006] If the process starts with frozen cells, proper thawing of the cells, and, if necessary, revival and storage can assist in the success of cell use. To thaw the cells, a common manual method is, in part, to submerge the cell vial in a 37°C water bath. This manual method, among other problems, cannot meet automated manufacturing requirements, is highly subjective, and generates a risk of contamination. High-speed thawing is essential to prevent crystallization during thawing. This is followed by a controlled "reaction stop" of the medium to dilute the cryoprotectant without swelling (and bursting) the cells too much. An automated system can better control high-speed thawing than a manual process. Cell cultures can be lost through accidents, contamination, degradation or environmentally induced changes, and inappropriate monitoring. When operating in manual mode, the cells will be visually monitored to assess levels of confluence, any change in the color of the medium, etc.

[0007] What is needed is a manufacturing line that can automatically and consistently generate tissue. What is needed is a system in which the process enabling staging is automated and the movement of cells from one stage to the next is also automated. What is needed is a system in which the user does not determine when the cells need to be moved, when one stage has ended and another should begin, and does not perform cell transfer manually. What is needed is a system that produces an overall consistent result and avoids the problem of the cells / tissue being "ready" in the middle of the night or on weekends when the user is not monitoring them. What is needed is a system in which any feedback loop associated with monitoring and the monitored results is fully automated. Such a line would be scalable so that the same automated process can be used to generate many tissue constructs. The manufacturing line should consist of modular components and be reconfigurable based on the specific tissue processes required. The line should also be fluidly closed so that it can operate in a non-clean space without concern for contaminating the cells and tissue contained within it.

Summary of the Invention

Means for Solving the Problems

[0008] The system of the present disclosure can include, but is not limited to, a manufacturing line for producing tissue engineering medical products (TEMP), or more broadly, human cells, tissues, and cell- and tissue-based products (HCT / P). The manufacturing line can produce consistent results across tissue engineering operations and can address safety and quality issues because no human intervention is required during the cell maturation and incubation processes. The manufacturing line can include, but is not limited to, a station where cells are thawed, a station where the thawed cells are expanded, a station where the expanded cells are concentrated, and a station where the concentrated cells are incubated into tissue.

[0009] The method of the present disclosure for enabling automated fabrication of tissues can include, but is not limited to, receiving cells in at least one vial, automatically pumping the cells from the at least one vial to at least one first bioreactor, automatically controlling fluid delivery from the at least one first bioreactor to the at least one vial when the cells are transferred to the at least one first bioreactor, automatically generating a first preselected environment based on sensor data of critical process parameters to promote expansion of the cells within the at least one first bioreactor, automatically harvesting the cells from the microcarrier surface and arresting the harvesting enzyme, automatically pumping the expanded cells from the at least one first bioreactor to a concentrator when the expanded cells reach a preselected density, automatically concentrating the expanded cells, automatically pumping the concentrated cells into the at least one first bioreactor when a preselected event occurs, automatically pumping the cells from the at least one first bioreactor to at least one second bioreactor when the concentrated cells are suspended, and automatically generating a second preselected environment based on sensor data of critical process parameters to promote maturation of the concentrated cells into tissue within the at least one second bioreactor.

[0010] The method can optionally include the step of automatically receiving and thawing cells, which includes accessing an insulated container. The insulated container can store at least one vial of frozen cells and can include an insulated container lid and a vial holder. The method can further optionally include the step of automatically controlling, by a thawing controller, the step of removing the insulated container lid of the insulated container using a first gripper. The first gripper can be controlled by the thawing controller. The method can optionally include the step of automatically determining, by the thawing controller, the location of at least one vial in the vial holder, the step of automatically controlling, by the thawing controller, the step of positioning a second gripper at the location, and the step of automatically controlling, by the thawing controller, the step of removing at least one vial from the insulated container using the second gripper. The second gripper can be controlled by the thawing controller. The method can include the step of automatically controlling, by the thawing controller, the step of returning the insulated container lid to the insulated container using the first gripper. The first gripper can be controlled by the thawing controller. The method can include the step of automatically controlling, by the thawing controller, the step of installing at least one removed vial into a cell thawing device using the second gripper. The second gripper can be controlled by the thawing controller. The method can include the step of automatically determining, by the thawing controller and controlled by a thawing management device, the time when the frozen cells are thawed, and the step of automatically controlling, by the thawing controller, the step of removing at least one vial of the thawed cells using the second gripper. The second gripper can be controlled by the thawing controller. The method can include the step of automatically controlling, by the thawing controller, the step of transferring at least one vial of the thawed cells to a third gripper using the second gripper.The second gripper can be controlled by a thawing controller. The method automatically controls, by the thawing controller, a step of moving at least one vial of thawed cells to a first pre-selected location using an actuator operably coupled to a third gripper, the actuator being controllable by the thawing controller. The method automatically controls, by the thawing controller, a step of decontaminating the exterior of at least one vial by a decontamination system, the decontamination system being controllable by the thawing controller. The method automatically controls, by the thawing controller, a step of moving at least one vial of thawed cells to a second pre-selected location for access using an actuator, the actuator being controllable by the thawing controller. The method automatically controls, by the thawing controller, a step of accessing at least one vial by an access system controllable by the thawing controller, the access system including an access device, the access device being continuously energized during a pumping operation. The method can include automatically controlling, by the thawing controller, a step of pumping a solution into at least one vial by a pump controllable by the thawing controller, automatically controlling, by the thawing controller, a step of extracting thawed cells from at least one vial using advection, and automatically controlling, by the thawing controller, a step of discarding at least one vial. The first gripper and the second gripper can optionally constitute a single device. The first gripper, the second gripper, and the third gripper can optionally constitute a single device. The method can optionally include automatically determining, by a first device controllable by the thawing controller, the identification of at least one vial removed.The decontamination system can optionally include the step of decontaminating the outer surface of at least one vial. The solution can optionally include a neutralizing agent. The solution can optionally include a medium. The step of concentrating the expanded cells can optionally include the step of centrifuging the expanded cells.

[0011] The system of the present teachings for enabling automated tissue manufacturing is an expansion subsystem that automatically pumps cells from at least one vial to at least one first bioreactor, the expansion subsystem automatically closing a valve from at least one vial to at least one first bioreactor when the cells are moved to the at least one first bioreactor, the expansion subsystem automatically generating a first preselected environment to facilitate expansion of the cells within the at least one first bioreactor, the expansion subsystem automatically pumping the expanded cells from the at least one first bioreactor to a concentrator when the expanded cells reach a preselected density, the expansion subsystem can include, but is not limited to including, an expansion subsystem configured to generate resuspended cells from the concentrated cells. The system can include a concentration subsystem that automatically concentrates the expanded cells and a maturation subsystem that automatically pumps the resuspended cells into at least one second bioreactor when a preselected event occurs, the maturation subsystem automatically generating a second preselected environment based on monitored sensor data of critical process parameters to facilitate maturation of the concentrated cells within the at least one second bioreactor.

[0012] The concentration subsystem can optionally include a centrifugation device. The preselected event can optionally include the detection of a desired concentration. The first preselected environment can optionally include a first growth medium, which is continuously and automatically adjusted based on sensor data monitoring critical process parameters to maintain a first preselected level of growth medium characteristics. The second preselected environment can optionally include a second growth medium, which is continuously and automatically adjusted based on the monitoring of sensor data of critical process parameters to maintain a second preselected level of growth medium characteristics. The system can optionally include a thawing subsystem that receives frozen cells in at least one vial and automatically thaws the frozen cells.

[0013] The thawing subsystem optionally includes a thawing controller that controls the preparation of cells, cooling means for cooling the environment surrounding the frozen cells in at least one vial, a thermally insulated container for maintaining the frozen cells within a preselected temperature range, a thermally insulated container station having a thermally insulated container lid for retaining the environment, a lid gripper and a vial gripper, where the lid gripper moves the thermally insulated container lid under the control of the thawing controller, and the vial gripper moves at least one vial under the control of the thawing controller, at least one device for locating the respective positions of at least one vial under the control of the thawing controller, a thawing station for receiving at least one identified vial from the vial gripper under the control of the thawing controller, where the thawing station includes a thawing device for storing at least one identified vial, the thawing device for thawing the cells in at least one vial, the thawing station includes a thawing station controller, and under the control of the thawing controller, the thawing station controller provides the status of the cells to the thawing controller, a decontamination station for receiving at least one vial of thawed cells from the vial gripper under the control of the thawing controller, where the decontamination station includes means for decontaminating the outer surface of at least one vial of thawed cells, a puncturing station for receiving at least one decontaminated vial of thawed cells from the vial gripper under the control of the thawing controller, where the puncturing station includes at least two needles, the at least two needles puncture the decontaminated vial under the control of the thawing controller, the first needle of the at least two needles has a first length, the second needle of the at least two needles has a second length, the puncturing station includes a needle controller for puncturing at least one decontaminated vial of thawed cells using the first needle under the control of the thawing controller, the first needle extends a first preselected distance into at least one decontaminated vial of thawed cells, and the needle controllerUnder the control of the thawing management device, at least one decontaminated vial of thawed cells is punctured using a second needle, the second needle extending a second preselected distance into at least one decontaminated vial of thawed cells, a puncturing station, and under the control of a thawing controller, a solution is pumped from a solution reservoir, through solution tubing and a first needle, into at least one decontaminated vial of thawed cells, a solution pump, wherein the thawed cells are withdrawn through the second needle, the thawed cells flow through cell tubing into a cell reservoir, the cells are removed from at least one decontaminated vial of thawed cells, generating at least one waste vial, and a solution pump.

[0014] The system can optionally include an identification station that identifies each of at least one vial under the control of a thawing controller. The first length can optionally include a length longer than the second length. The first preselected distance can optionally include a distance shorter than the second preselected distance. The system can optionally include a gas purge that presses substantially all of the contents of at least one decontaminated vial out of the at least one decontaminated vial. The decontamination station can optionally include a hood that retains a decontamination fluid within a preselected area surrounding at least one identified vial of thawed cells, a decontamination pump that pumps the decontamination fluid into the preselected area under the control of the thawing controller, and a nozzle that directs the decontamination fluid toward at least one identified vial of thawed cells. The piercing station can optionally include a base gripper that holds at least one identified vial in place while a needle controller removes a first needle and a second needle under the control of the thawing controller. The system can optionally be a waste system that receives at least one waste vial from a vial gripper under the control of a thawing controller after the needle controller removes the first needle and the second needle, the waste system including a waste system that discards at least one waste vial into a waste receptacle. The system can optionally include a hood that surrounds the piercing station, the hood including a hood that maintains a controlled clean volume surrounding the piercing station. The expansion subsystem can optionally include an expansion controller that controls the flow of cells from a thawing subsystem to the expansion subsystem, and a bioreactor controller that monitors and modifies a first preselected environment.The bioreactor controller can optionally include a stirring controller that stirs the cells and promotes cell attachment to the surface, a temperature controller that adjusts the temperature of at least one first bioreactor based on a preselected desired temperature, a gas mixing processor that adjusts the level of gas in the medium surrounding the cells, where the gas level is based on a preselected desired value of the characteristics of the medium, a monitoring process that senses the value of the characteristics of the medium, and a pump controller that moves the medium to and from at least one first bioreactor. The characteristics can optionally include dissolved oxygen and pH. The maturation subsystem can optionally include a medium controller that monitors and modifies the medium before introducing the medium into a second at least one bioreactor, and an incubator controller that manages the movement of at least one second bioreactor, where the incubator controller monitors the characteristics of the medium within at least one second bioreactor and the incubator controller can include an incubator controller that flushes / returns the medium from / to at least one second bioreactor. The system can optionally include a medium storage unit controller that can include a medium level sensor that monitors the amount of medium in the medium reservoir and a pump pressure sensor that monitors a pump that moves the medium from the medium reservoir to the medium container.

[0015] The cells can be provided in any suitable manner, including but not limited to a frozen form. The method of the present disclosure for preparing frozen cells for entry into a tissue engineering system manufacturing line can include, but is not limited to, the step of accessing an insulated container. The insulated container can store at least one vial of frozen cells and can include an insulated container lid and a vial holder. The insulated container can hold a single vial or multiple vials, and the system can accommodate it. The method can include the step of automatically controlling, using a first gripper, the removal of the insulated container lid of the insulated container. The first gripper can be controlled by a thawing controller. The method can include the steps of automatically determining, by the thawing controller, the location of at least one vial within the vial holder, automatically positioning, by the thawing controller, a second gripper at that location, and automatically removing, using the second gripper, at least one vial from the insulated container. The second gripper can be controlled by a thawing controller. In some configurations, the actions of the first gripper and the second gripper can be performed by a single device. The method can include the step of automatically controlling, using the first gripper, the return of the insulated container lid to the insulated container. The method can include the steps of automatically determining and automatically controlling, by a thawing controller and using a first device controlled by the thawing controller, the step of installing, using the second gripper, at least one removed vial into a cell thawing device. The method can include the steps of automatically determining, by the thawing controller, when a preselected amount of frozen cells has been thawed, and automatically controlling, by the thawing controller, the step of removing, using the second gripper, at least one vial of the thawed cells.The method can include automatically controlling, by a thawing controller, a step of transferring, using a second gripper, at least one vial of thawed cells to a third gripper. The third gripper can be controlled by the thawing controller. In some configurations, the actions of the second gripper and the third gripper can be performed by a single device. In some configurations, the actions of the first gripper, the second gripper, and the third gripper can be performed by a single device. The method can include automatically controlling, by the thawing controller, a step of moving, using an actuator operably coupled to the third gripper, at least one vial of thawed cells to a first preselected location. The actuator can be controlled by the thawing controller. The method can include automatically controlling, by the thawing controller, a step of decontaminating at least one vial by a decontamination system. The decontamination system can be controlled by the thawing controller and can decontaminate the outer surface of the vial. The method can include automatically controlling, by the thawing controller, a step of moving, using an actuator, at least one vial of thawed cells to a second preselected location for piercing. The method can include automatically controlling, by the thawing controller, a step of piercing at least one vial by a piercing system controlled by the thawing controller and a step of automatically controlling, by the thawing controller, a step of pumping a solution into at least one vial by a pump controlled by the thawing controller. The method can include automatically controlling, by the thawing controller, a step of extracting thawed cells from at least one vial using convection. The method can include automatically controlling, by the thawing controller, a step of discarding the vial. The solution can optionally include a buffer (PBS) that neutralizes a cryoagent or medium.The method can optionally include a step of identifying at least one vial that has been removed.

[0016] The system of the present teachings for preparing cells for entry into a tissue processing system can include, but is not limited to, a thawing controller that controls the preparation of the cells, and an insulated container station. The insulated container station can include cooling means for cooling the environment surrounding the frozen cells in at least one vial, an insulated container for maintaining the frozen cells within a preselected temperature range, and an insulated container lid for retaining the environment. The system can include a lid gripper and a vial gripper. The lid gripper can move the insulated container lid under the control of the thawing controller, and the vial gripper can move at least one vial under the control of the thawing controller. The system can include at least one device for locating the respective position of at least one vial under the control of the thawing controller. The system can include a thawing station for receiving at least one identified vial from the vial gripper under the control of the thawing controller. The thawing station can include a thawing device for storing at least one identified vial. The thawing device can thaw the cells in at least one vial. The thawing station can include a thawing station controller that is under the control of the thawing controller. The thawing station controller can provide the status of the cells to the thawing controller. The system can include a decontamination station for receiving at least one vial of thawed cells from the vial gripper under the control of the thawing controller. The decontamination station can include means for decontaminating the outer surface of at least one vial of thawed cells. The system can include a puncturing station for receiving at least one decontaminated vial of thawed cells from the vial gripper under the control of the thawing controller. The puncturing station can include at least two needles. The at least two needles can puncture the decontaminated vial under the control of the thawing controller.The first of the at least two needles can include a first length, and the second of the at least two needles can include a second length. The piercing station can include a needle controller that, under the control of the thaw controller, pierces at least one decontaminated vial of thawed cells using the first needle. The first needle can extend into at least one decontaminated vial of thawed cells by a first preselected distance. The needle controller can, under the control of the thaw controller, pierce at least one decontaminated vial of thawed cells using the second needle. The second needle can extend into at least one decontaminated vial of thawed cells by a second preselected distance. The system can include a solution pump that, under the control of the thaw controller, pumps a solution from a solution reservoir, through solution tubing and the first needle, into at least one decontaminated vial of thawed cells. The thawed cells can be withdrawn through the second needle and flow through cell tubing into a cell reservoir. The cells can be removed from at least one decontaminated vial of thawed cells to produce at least one waste vial.

[0017] The first needle length can optionally include a length that is longer than the second needle length. The first preselected distance that the needle extends into the vial can optionally include a distance that is shorter than the second preselected distance that the needle extends into the vial. The system can optionally include a gas purge that presses substantially all of the contents of at least one decontaminated vial out of the at least one decontaminated vial. The decontamination system can optionally include a hood that retains the decontamination fluid within a preselected area surrounding at least one identified vial of thawed cells, a decontamination pump that pumps the decontamination fluid into the preselected area under the control of a thaw controller, and a nozzle that directs the decontamination fluid toward at least one identified vial of thawed cells. The piercing station can optionally include a base gripper that holds at least one identified vial in place while the needle controller removes the first and second needles under the control of the thaw controller. The system can optionally include a waste system that receives at least one waste vial from the vial gripper under the control of the thaw controller after the needle controller removes the first and second needles. The waste system can optionally discard at least one waste vial into a waste receptacle. The system can optionally include a hood surrounding the piercing station that can maintain a controlled clean volume surrounding the piercing station. The system can optionally include an identification station that identifies each of at least one vial under the control of the thaw controller. The present invention provides, for example, the following. (Item 1) A method for enabling the automatic production of tissue, comprising: receiving cells within at least one vial; automatically pumping the cells from the at least one vial into at least one first bioreactor; Automatically controlling fluid delivery from the at least one vial to the at least one first bioreactor when the cells are transferred to the at least one first bioreactor; Automatically generating a first preselected environment based on sensor data of critical process parameters to facilitate expansion of the cells within the at least one first bioreactor; Automatically collecting the cells from the microcarrier surface and arresting the collection enzyme; Automatically pumping the expanded cells from the at least one first bioreactor to a concentrator when the expanded cells reach a preselected density; Automatically concentrating the expanded cells; Automatically pumping the concentrated cells into the at least one first bioreactor when a preselected event occurs; Automatically pumping the cells from the at least one first bioreactor to at least one second bioreactor when the concentrated cells are suspended; Automatically generating a second preselected environment based on sensor data of critical process parameters to facilitate maturation of the concentrated cells into tissue within the at least one second bioreactor A method comprising. (Item 2) Automatically receiving and thawing cells, Accessing a thermally insulated container that stores at least one vial of the frozen cells, the thermally insulated container having a thermally insulated container lid and a vial holder; Automatically controlling, by a thawing controller, the removal of the thermally insulated container lid of the thermally insulated container using a first gripper, the first gripper being controlled by the thawing controller; Automatically determining, by the thawing controller, the location of the at least one vial within the vial holder; Automatically controlling, by the thawing controller, positioning the second gripper at the location; Automatically controlling, by the thawing controller, removing the at least one vial from the insulated container using the second gripper, wherein the second gripper is controlled by the thawing controller; Automatically controlling, by the thawing controller, returning the insulated container lid to the insulated container using the first gripper, wherein the first gripper is controlled by the thawing controller; Automatically controlling, by the thawing controller, placing the removed at least one vial into the cell thawing device using the second gripper, wherein the second gripper is controlled by the thawing controller; Automatically determining, by the thawing management device controlled by the thawing controller, when the frozen cells are thawed; Automatically controlling, by the thawing controller, removing the at least one vial of thawed cells using the second gripper, wherein the second gripper is controlled by the thawing controller; Automatically controlling, by the thawing controller, transferring the at least one vial of thawed cells to a third gripper using the second gripper, wherein the second gripper is controlled by the thawing controller; Automatically controlling, by the thawing controller, moving the at least one vial of thawed cells to a first preselected location using an actuator operably coupled to the third gripper, wherein the actuator is controlled by the thawing controller; Automatically controlling, by the thawing controller, the decontamination system to decontaminate the exterior of the at least one vial, wherein the decontamination system is controlled by the thawing controller; Automatically controlling, by the thawing controller, using the actuator, to move the at least one vial of thawed cells to a second preselected location for accessing, wherein the actuator is controlled by the thawing controller; Automatically controlling, by the thawing controller, accessing the at least one vial by an access system controlled by the thawing controller, wherein the access system includes an access device that is continuously energized during a pumping operation; Automatically controlling, by the thawing controller, pumping a solution into the at least one vial by a pump controlled by the thawing controller; Automatically controlling, by the thawing controller, extracting the thawed cells from the at least one vial using advection; Automatically controlling, by the thawing controller, discarding the at least one vial; including; The method according to item 1, further including. (Item 3) The method according to item 2, wherein the first gripper and the second gripper constitute a single device. (Item 4) The method according to item 2, wherein the first gripper, the second gripper, and the third gripper constitute a single device. (Item 5) The method according to item 2, further including automatically determining the identification of the at least one removed vial by a first device controlled by the thawing controller. (Item 6) The decontamination system according to item 2, including decontamination of the outer surface of the at least one vial. (Item 7) The method according to item 2, wherein the solution contains a neutralizing agent. (Item 8) The method according to item 2, wherein the solution contains a medium. (Item 9) The method according to item 1, wherein concentrating the expanded cells includes centrifuging the expanded cells. (Item 10) A system for enabling automatic production of tissue, An expansion subsystem that automatically pumps cells from at least one vial to at least one first bioreactor, the expansion subsystem automatically closes a valve from the at least one vial to the at least one first bioreactor when the cells are moved to the at least one first bioreactor, the expansion subsystem automatically generates a first preselected environment to facilitate expansion of the cells in the at least one first bioreactor, the expansion subsystem automatically pumps the expanded cells from the at least one first bioreactor to a concentrator when the expanded cells reach a preselected density, and the expansion subsystem is configured to generate resuspended cells from the concentrated cells, an expansion subsystem; A concentration subsystem that automatically concentrates the expanded cells; A maturation subsystem that automatically pumps the resuspended cells into at least one second bioreactor when a preselected event occurs, and the maturation subsystem automatically generates a second preselected environment based on monitoring sensor data of critical process parameters to facilitate maturation of the concentrated cells in the at least one second bioreactor, a maturation subsystem Comprising a system. (Item 11) The concentration subsystem comprises a centrifugal device, the system according to item 10. (Item 12) The system according to item 10, wherein the preselected event includes detection of a desired concentration. (Item 13) The system according to item 10, wherein the first preselected environment comprises a first growth medium, and the first growth medium is continuously and automatically adjusted based on sensor data monitoring critical process parameters to maintain a first preselected level of growth medium characteristics. (Item 14) The system according to item 10, wherein the second preselected environment comprises a second growth medium, and the second growth medium is continuously and automatically adjusted based on monitoring of sensor data of critical process parameters to maintain a second preselected level of growth medium characteristics. (Item 15) The system according to item 10, further comprising a thawing subsystem that receives at least one vial of frozen cells and automatically thaws the frozen cells. (Item 16) The thawing subsystem comprises a thawing controller that controls the preparation of the cells, a thermally insulated container station having a cooling means for cooling the environment surrounding the frozen cells in the at least one vial, a thermally insulated container for maintaining the frozen cells within a preselected temperature range, and a thermally insulated container lid for retaining the environment, a lid gripper and a vial gripper, wherein the lid gripper moves the thermally insulated container lid under the control of the thawing controller, and the vial gripper moves the at least one vial under the control of the thawing controller, At least one device, wherein the at least one device identifies the position of each of the at least one vial under the control of the thawing controller, and the at least one device A thawing station, wherein the thawing station receives the at least one identified vial from the vial gripper under the control of the thawing controller, and the thawing station includes a thawing device for storing the at least one identified vial, and the thawing device thaws the cells in the at least one vial, and the thawing station includes a thawing station controller, and under the control of the thawing controller, the thawing station controller provides the status of the cells to the thawing controller, and the thawing station A decontamination station, wherein the decontamination station receives the at least one vial of thawed cells from the vial gripper under the control of the thawing controller, and the decontamination station includes means for decontaminating the outer surface of the at least one vial of thawed cells, and the decontamination station A piercing station, wherein the piercing station receives, from the vial gripper and under the control of the thawing controller, the at least one decontaminated vial of thawed cells, the piercing station includes at least two needles, the at least two needles pierce the decontaminated vial under the control of the thawing controller, a first needle of the at least two needles has a first length, a second needle of the at least two needles has a second length, the piercing station includes a needle controller that pierces the at least one decontaminated vial of thawed cells with the first needle under the control of the thawing controller, the first needle extends a first preselected distance into the at least one decontaminated vial of thawed cells, the needle controller pierces the at least one decontaminated vial of thawed cells with the second needle under the control of a thawing management device, and the second needle extends a second preselected distance into the at least one decontaminated vial of thawed cells, the piercing station and A solution pump, wherein the solution pump pumps a solution from a solution reservoir, through solution tubing and the first needle, into the at least one decontaminated vial of thawed cells under the control of the thawing controller, the thawed cells are drawn out through the second needle, the thawed cells flow through cell tubing into a cell reservoir, the cells are removed from the at least one decontaminated vial of thawed cells, and at least one waste vial is generated, the solution pump The system according to item 15, comprising. (Item 17) The system according to item 15, further comprising an identification station that identifies each of the at least one vial under the control of the thawing controller. (Item 18) The system according to item 15, wherein the first length is longer than the second length. (Item 19) The system according to item 15, wherein the first pre-selected distance comprises a distance shorter than the second pre-selected distance. (Item 20) The system according to item 15, comprising a gas purge that presses substantially all of the contents of the at least one decontaminated vial out of the at least one decontaminated vial. (Item 21) The decontamination station is a hood, and the hood retains the decontamination fluid within a pre-selected area surrounding the at least one identified vial of thawed cells, a hood, a decontamination pump, and the decontamination pump pumps the decontamination fluid into the pre-selected area under the control of the thawing controller, a decontamination pump, a nozzle, and the nozzle directs the decontamination fluid toward the at least one identified vial of thawed cells, a nozzle The system according to item 15, comprising (Item 22) The piercing station is a base gripper, and the base gripper maintains the at least one identified vial in a fixed position while the needle controller removes the first needle and the second needle under the control of the thawing controller, a base gripper The system according to item 15, comprising (Item 23) a waste system, and the waste system receives the at least one waste vial from the vial gripper under the control of the thawing controller after the needle controller removes the first needle and the second needle, and the waste system discards the at least one waste vial into a waste receptacle, a waste system The system according to item 15, further comprising (Item 24) A hood surrounding the puncture station, the hood maintaining a controlled clean volume surrounding the puncture station The system according to item 15, further comprising (Item 25) The inflation subsystem includes An inflation controller that controls the flow of cells from the thawing subsystem to the inflation subsystem, And a bioreactor controller that monitors and modifies the first preselected environment The system according to item 10, comprising (Item 26) The bioreactor controller is A stirring controller that stirs the cells and promotes adhesion of the cells to the surface, A temperature controller that adjusts the temperature of the at least one first bioreactor based on a preselected desired temperature, A gas mixing processor that adjusts the level of gas in the medium surrounding the cells, the level of gas being based on a preselected desired value of the characteristics of the medium, A monitoring process that senses the value of the characteristics of the medium, A pump controller that moves the medium to and from the at least one first bioreactor, The system according to item 25, comprising (Item 27) The system according to item 26, wherein the characteristics include dissolved oxygen and pH. (Item 28) The maturation subsystem includes A medium controller that monitors and modifies the medium before introducing the medium into the second at least one bioreactor, An incubator controller, wherein the incubator controller manages the movement of the at least one second bioreactor, the incubator controller monitors the characteristics of the medium in the at least one second bioreactor, and the incubator controller flushes / restores the medium from / to the at least one second bioreactor, an incubator controller and The system according to item 10, comprising. (Item 29) A medium storage unit controller, A medium level sensor that monitors the amount of medium in the medium reservoir, A pump pressure sensor that monitors a pump that moves the medium from the medium reservoir to the medium container and A medium storage unit controller comprising. The system according to item 10, further comprising.

Brief Description of Drawings

[0018] The foregoing features of the present disclosure will be more readily understood by reference to the following description in conjunction with reference to the accompanying drawings.

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DETAILED DESCRIPTION OF THE INVENTION

[0043] The system of the present teachings for manufacturing tissue can enable a flexible process for generating tissue from cells. The flexible process can be implemented by a manufacturing production line. The present system can remove human components from the process after initial setup and parameter specification, and thus ensure reproducible results. The present system can enable interchangeability of parts along the production line by including a programmable controller that can follow a plug-and-play paradigm. The stations along the production line can be connected by sterilized tubing. The tubing can enable the movement of cells and fluids between stations on the line. Frozen cells can be thawed within a first station and pumped into a bioreactor vessel that contains a nutrient fluid along with a medium and is stored within a controlled environment within a second station. The bioreactor vessel can contain microcarriers, onto which cells can settle and adhere or attach as they are introduced into the bioreactor vessel. The microcarriers can be coated with collagen, protein, or a binding domain and can facilitate cell attachment. Alternatively, they can be chemically modified, electrically charged, or plasma treated to facilitate cell attachment. After initial cell seeding, the culture on the microcarriers can be maintained within the bioreactor using continuous sensing of culture parameters such as pH, dissolved oxygen, and temperature. The present system can also control the automated removal of used medium and the introduction of fresh medium throughout the culture. When the cell density reaches a preselected level and the microcarriers, if present, are separated from the cells or the cells sufficiently digest the microcarriers, the cells, microcarriers, and liquid attaching the cells to the surface coating of the microcarriers, or proteins as needed, can be pumped to a third station, where the cells can be concentrated.At the third station, the cells are separated from most of the neutralized digestion solution, cell culture medium, and wash buffer, and then the cells are transferred to an expansion vessel for preparing them for maturation, in addition to a preselected amount of the previous mixture. At a preselected cycle time, for example, but not limited to, after resuspension at a desired concentration, the concentrated cells can be pumped into a fourth station that can include an incubator and a media storage vessel. Through pumping and tubing, the cells can be fluidically seeded onto the culture surface within the bioreactor for seeding attachment, differentiation, and growth. In some configurations, the bioreactor can include a scaffold surface on which the cells can adhere and grow. In some configurations, the bioreactor can include a horizontal planar growth surface. A first cell type can be placed on a first surface that can be coated with media from a container and the cells can grow and differentiate. At a preselected or dynamically determined time, the first surface can be rotated so that a second cell type can be introduced onto a second surface. As the cells grow, features within the bioreactor encourage the cells from both surfaces to intermingle and thus can achieve the goal of incubating multiple cell types together.

[0044] Referring now to FIG. 1, the system 2100 of the present teachings for manufacturing tissue can include, but is not limited to, a supervisor 2101 that controls stations along a production line. The stations can include, but are not limited to, a thawing subsystem 2147, an expansion subsystem 2141, a concentration subsystem 2143, and a maturation subsystem 2145. Operationally, the supervisor 2101 receives instructions from a human-machine interface (HMI) 2133. The instructions can include, for example, parameter settings that can depend on, for example, the type of cells being processed and the type of tissue desired at the end of the process. The supervisor 2101 can communicate with the thawing subsystem 2147, receive status messages, and provide command messages. In some configurations, the supervisor 2101 and the thawing subsystem 2147 can be enabled, for example, but not limited to, by a ROCKWLL AUTOMATION® COMPACTLOGIX® PLC, and their communication can be done, for example, but not limited to, through an Ethernet® connection. The supervisor 2101 can use, for example, but not limited to, the Open Platform Unified Architecture Protocol (OPC UA) and / or the Ethernet® protocol to provide commands to the thawing subsystem 2147, the expansion subsystem 2141, the concentration subsystem 2143, and the maturation subsystem 2145, in the case of the expansion subsystem 2141, through the network HMI 2103.

[0045] Continuing to refer to FIG. 1, the supervisor 2101 can manage the operation of the system 2100. The supervisor 2101 can track the state of the subsystems, user input, and sensor input, and can make available predefined code to components of the system that can interpret the code and actions as appropriate.

[0046] Continuing to refer to FIG. 1, the thawing subsystem 2147 can thaw the cells within the vial 2163. After the supervisor 2101 determines that the cells are to be thawed, the supervisor 2101 can initiate seeding of the cells from the thawing subsystem 2147 to the expansion subsystem 2141. The expansion subsystem 2141 can pump the thawed cells from the vial 2163 into the bioreactor vessel 2113, and the supervisor 2101 can enable the medium to be pumped into the bioreactor vessel 2113 from the reservoir 6149 (FIG. 8). When the cells reach a preselected density, the expansion subsystem 2141 can perform tasks such as draining and washing and can notify the supervisor 2101, and the cells can be transferred to a concentration subsystem 2143 where the expanded cells can be concentrated. The supervisor 2101 can optionally receive user input at various times during these processes as desired or necessary. The supervisor 2101 can communicate with the concentrator subsystem through a gateway, which in some configurations can use, but is not limited to using, the Profinet protocol. The supervisor 2101 can, for example, monitor a concentration recipe. The recipe can include phase values that can be used to trigger a stop command to the concentration subsystem 2143 and a message to the expansion subsystem 2141 indicating that the subsequent step in the process can proceed. The supervisor 2101 can request a status update from the concentrator subsystem 2143, and the concentrator subsystem 2143 can send a status command to the supervisor 2101. When the cells reach the desired concentration, the cells can return to the expansion subsystem 2141, and the supervisor 2101 can instruct the maturation subsystem 2145 to pump the cells into the bioreactor 2114 and to pump the conditioned medium from the vessel 2118 into the bioreactor 2114. The medium controller 2121 can condition the medium within the vessel 2118, prepare the medium, and appropriately nourish the cells within the bioreactor 2114.In some configurations, the bioreactor 2114 can include a shallow box having opposing inner sides. The incubator controller 2123 can control a positioning device to position the bioreactor 2114 such that a first opposing inner side faces a ground plane and a second opposing side faces the first opposing side. As cells are pumped into the bioreactor 2114, the cells can tend to adhere to the second opposing side in response to gravity. Fresh media can be added to the bioreactor 2114, critical process parameters can be monitored / regulated, controlled washing can be performed, and media exchange to assist differentiation can be performed. The incubator controller 2123 can later seed the bioreactor 2114 with the same or alternative cell types. The bioreactor 2114 can be rotated prior to the second seeding to encourage cell adhesion on alternative surfaces. Additional steps of seeding can thus be performed. The fluid line from the media 2118 to the bioreactor 2114 resides within a hollow spindle and can maintain its integrity as the bioreactor 2114 is inverted.

[0047] Continuing to refer to FIG. 1, the maturation subsystem 2145 can include a media controller 2121 and an incubator controller 2123. The media controller 2121 can provide nutrients to the cells within the bioreactor 2114 and can condition the media to control characteristics such as, but not limited to, temperature, pH, and dissolved oxygen. The media conditioned to have desired characteristics can flow through the cells and can adhere to either the upper or lower surface of the shallow box within the bioreactor 2114. The media controller 2121 can include a LABOWL tm control system, but is not limited to including the same. The incubator controller 2123 can include, but is not limited to including, a ROCKWLL AUTOMATION® COMPACTLOGIX® PLC.

[0048] Continuing to refer to FIG. 1, the supervisor 2101 can receive and process information sensed from the refrigerated medium from the load cell 2125 and the pressure sensor 2127 using the Ethernet (registered trademark) protocol. The processing can include determining the amount of medium remaining within the medium storage area using data from the load cell 2125. The supervisor 2101 can alert the user about the need to replenish the medium through the process HMI 2133. The supervisor 2101 can send / receive data to / from the process HMI 2133, for example, using the Ethernet (registered trademark) protocol. Messages initiated from the thaw subsystem 2147, the expansion subsystem 2141, the concentration subsystem 2143, and the maturation subsystem 2145 can be directed to the process HMI 2133 through the supervisor 2101. In some configurations, a gateway can be used to enable communication between subsystems. Message acknowledgments can pass through the reverse path. The supervisor 2101 can communicate with a remote server 2135 (FIG. 3) that can manage the database 2131. Data from the operation of the system of the present teachings can be optionally logged and perhaps remotely stored by the logging processor 2129. For example, without limitation, conventional means such as ROCKWLL AUTOMATION (registered trademark) FACTORYTALK (registered trademark) Historian software can be used to receive and catalog the data.

[0049] Referring now to FIGS. 1 and 2A, system 2100 (FIG. 1) can be used to generate tissue from cells by including a supervisor 2101 (FIG. 1) that can respond to triggers presented by each subsystem. For example, when supervisor 2101 (FIG. 1) detects that a cell has been thawed, system 2100 (FIG. 1) can include step 1201 (FIG. 2A) of moving the thawed cell from thaw subsystem 2147 (FIG. 1) to expansion subsystem 2141 (FIG. 1). When expansion subsystem 4141 (FIG. 1) detects a desired density of the cells, system 2100 (FIG. 1) can include step 1203 (FIG. 2A) of moving the cells from expansion subsystem 2141 (FIG. 1) to concentrator subsystem 2143 (FIG. 1). When supervisor 2101 (FIG. 1) detects that a desired cell concentration has been achieved, system 2100 (FIG. 1) can include step 1205 (FIG. 2A) of moving the cells from concentrator subsystem 2143 (FIG. 1) to expansion subsystem 2141 (FIG. 1), and when the cells reach a desired maturity that can be determined by the elapse of a preselected amount of time, system 2100 (FIG. 1) can include steps enabling step 1207 (FIG. 2A) of moving the matured tissue out of maturation subsystem 2145 (FIG. 1). In some configurations, expansion subsystem 2141 (FIG. 1) can start the process enabled by system 2100 (FIG. 1) by sending a message to process HMI 2133 (FIG. 1) through supervisor 2101 (FIG. 1), thereby prompting the user regarding the input.The supervisor 2101 (Figure 1) processes user input, exchanges messages with the inflation subsystem 2141 (Figure 1), enables flow control, and instructs the thawing subsystem 2147 (Figure 1) to (1) check the vial 2163 (Figure 1), (2) check the position of the needle that can be installed to puncture the vial 2163 (Figure 1) as described anywhere in this specification, (3) determine whether one of the needles has punctured the vial 2163 (Figure 1), (4) check whether there are no further vials 2163 (Figure 1) to be processed, and then provide the status of these checks to the inflation subsystem 2141 (Figure 1). In some configurations, the needle position can be assumed and the checks can be reduced. In some configurations, the vial 2163 (Figure 1) can have its cap removed, and the contents can be removed in various ways, including but not limited to inserting the installed needle into the mouth of the vial 2163 (Figure 1) with the cap removed. In response, the inflation subsystem 2141 (Figure 1) can send a message to the supervisor 2101 (Figure 1). In some configurations, the supervisor 2101 (Figure 1) can inform the thawing subsystem 2147 (Figure 1) that the flushing of the vial 2163 (Figure 1) is in progress or has been completed. In some configurations, the following protocol can be executed between the supervisor 2101 (Figure 1) and the inflation subsystem 2141 (Figure 1).That is, (1) the expansion subsystem 2141 (FIG. 1) can send a message to the supervisor 2101 (FIG. 1) to start the thawing subsystem 2147 (FIG. 1), (2) the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 2141 (FIG. 1) when the vial 2163 (FIG. 1) is punctured, (3) the expansion subsystem 2141 (FIG. 1) can send a message to the supervisor 2101 (FIG. 1) to set a preselected valve state, (4) the expansion subsystem 2141 (FIG. 1) can pump a preselected amount of fluid, (5) the expansion subsystem 2141 (FIG. 1) can send a message to the supervisor 2101 (FIG. 1) when the pumping is completed, (6) the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 2141 (FIG. 1) when the gas purge is completed and if there are additional vials to be processed, (7) the expansion subsystem 2141 (FIG. 1) can send a message to the supervisor 2101 (FIG. 1) to set another preselected valve state based on whether there are additional vials to be processed. At this point, the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 2141 (FIG. 1) to start the cell expansion process. When the expansion subsystem 2141 (FIG. 1) determines that the expansion is complete, the expansion subsystem 2141 (FIG. 1) can send a message to the supervisor 2101 (FIG. 1), and the supervisor 2101 (FIG. 1) can optionally send a message to the process HMI 2133 (FIG. 1) to prompt the user for further instructions if necessary. The supervisor 2101 (FIG. 1) can send a message to the concentrator subsystem 2143 (FIG. 1) to start a recipe step, and the concentrator subsystem 2143 (FIG. 1) can execute the recipe step. When the recipe step is completed, the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 141 to resuspend the cells and return the cells into the solution.At this point, the supervisor 2101 (FIG. 1) can send a message to the mature subsystem 2145 (FIG. 1) to seed the cells into the bioreactor 2114 (FIG. 1). Seeding can occur multiple times, if desired.

[0050] Referring now to FIGS. 2A and 2B, a trigger for transitioning from one step to another and for one subsystem to take over control from another subsystem under the control of the supervisor 2101 (FIG. 1) can follow general guidelines with variations that may occur within each subsystem depending on the desired outcome. For example, in 1209 (FIG. 2B), when the cells are thawed, the supervisor 2101 (FIG. 1) can enable step 1201 (FIG. 1) of moving the thawed cells into the expansion subsystem 2141 (FIG. 1). In some configurations, the tube connecting the thawing subsystem 2147 (FIG. 1) and the expansion subsystem 2141 (FIG. 1) can include means for detecting bubbles within the tube. In 1211 (FIG. 2B), when a bubble is detected, the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 2141 that priming is complete. In some configurations, the amount of pumping required to move the cells into the expansion subsystem 2141 (FIG. 1) can be predetermined, and when a predetermined amount of pumping occurs, the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 2141 to disable the flow of cells from the thawing subsystem 2147 (FIG. 1). When the cells are within the expansion subsystem 2141, various parameters can be monitored 1213A (FIG. 2B), and some can potentially be adjusted 1213B (FIG. 2B) based on the monitored values. The supervisor 2101 can monitor, for example, but not limited to, the valve state of a fluid valve that controls the flow of fluid through the expansion subsystem 2141 (FIG. 1). The expansion subsystem 2141 (FIG. 1) can also monitor, for example, but not limited to, characteristics of the fluid flowing around the cells such as the fluid level, fluid temperature, fluid pH, dissolved oxygen in the fluid, agitation rate, and media conductivity within the media container 2116 (FIG. 1). These characteristics can be adjusted in various ways.For example, carbon dioxide can be added to the liquid to adjust the pH, oxygen or nitrogen can be added to the liquid to adjust the dissolved oxygen, the liquid can be added to or removed from the reservoir to adjust the liquid level and can adjust the properties of the liquid. The cell density can also be monitored. In 1215A (FIG. 2B), when the cell density reaches a predetermined amount, the supervisor 1201 (FIG. 1) can enable the step of moving the cells to the concentrator subsystem 2143 (FIG. 1). In 1215B (FIG. 2B), when the desired cell concentration is reached, the supervisor 1201 (FIG. 1) can enable the step of moving the cells to the maturation subsystem 2145 (FIG. 1). The maturation subsystem 2145 (FIG. 1) can monitor 1217A (FIG. 2B) and adjust 1217B (FIG. 2B) the medium and can adjust the cell environment. Monitoring of the medium in the container 2118 (FIG. 1) can include steps of monitoring properties such as, for example, but not limited to, pH, dissolved oxygen, temperature, and agitation. Monitoring of the medium in the pipes connecting the bioreactor and the medium storage can include, for example, but not limited to, in-line monitoring of properties such as pH, glucose, dissolved oxygen, and bubbles. Monitoring of the medium in the incubator storing the bioreactor 2114 can include steps of monitoring properties such as, for example, but not limited to, carbon dioxide, temperature, and relative humidity. The status of the pumping system and the leading edge of the bubbles in the pipes between the container 2118 and the bioreactor 2114 can also be monitored. The temperature of the medium can be adjusted and the medium can be agitated. Other measures can also be taken and / or alerts can be generated based on the monitored properties and the values of the equipment.

[0051] Referring now to FIG. 3, user input into the system 2100 (FIG. 1) can be provided to process control 2133 and network control 2103. In some configurations, input for process parameters and input for network parameters can be provided and processed on a single computer. In some configurations, multiple monitors (process HMI monitor 2133A and network HMI monitor 2139) and / or processors (process computer 2135 and network computer 2141) can receive and process user input, provide that input to supervisor 2101, and receive messages from supervisor 2101, including commands, status, and data. In some configurations, the expansion subsystem 2141, the concentration subsystem 2143, the maturation subsystem 2145, and the incubator can each include an HMI. In some configurations, user interaction with the expansion subsystem 2141 can initiate the tissue generation process. In some configurations, the user can change recipes and set points by interacting with the HMIs of the concentration subsystem 2143, the incubator, and the maturation subsystem 2145. In some configurations, the process computer 1135 can host a logging processor 2129 (FIG. 1) that can collect time series information and store it, for example, within a database 1231 (FIG. 1). The database 2131 (FIG. 1) can include physical long-term memory, cloud memory, short-term memory, or any other method and can store data electronically. The system 2100 (FIG. 1) can be controlled remotely, for example, through user input into a wireless device such as, but not limited to, a cell phone or tablet, or locally through a monitor co-located with the physical hardware of the system 2100 (FIG. 1).

[0052] Referring now to FIG. 4, thaw subsystem 2147 can receive a vial of frozen cells and achieve uniform thawing of the cells safely and efficiently, as described elsewhere in this specification. Supervisor 2101 can coordinate the activities of thaw subsystem 2147 with the activities of other subsystems. For example, supervisor 2101 can receive recipe information and emergency stop information from HMI subsystem 2133 / 2103 (FIG. 3) and supply that information to the components of thaw subsystem 2147. Safety processor 21471 can monitor the status of safety devices associated with thaw subsystem 2147 and provide that information to supervisor 2101. Supervisor 2101 can message status information to HMI subsystem 2133 / 2103 (FIG. 3) for possible display to the user. The user can initiate an emergency stop, possibly based on the safety device status. In some configurations, safety processor 21471 can receive an emergency stop message from supervisor 2101 and initiate shutdown activities associated with thaw subsystem 2147, such as movement of gantry, and thus movement of vial 2163. Reasons for an unintentional stop can include, but are not limited to, misplacement of the vial, vial defect, gantry malfunction, or unknown vial barcode.

[0053] Continuing to refer to FIG. 4, the thaw subsystem 2147 can access the vial 2163, access the identifier associated with the vial 2163, move the vial 2163 from one station to another, and receive status information from the processor and controller that operate the vial 2163. Machine control / monitoring 2155 can enable the step of selecting a vial from the vial bank by controlling Joe, who performs vial sorting and placement. The identification process 2157 can access the identification associated with the vial 2163. In some configurations, the identification can include a barcode, and the identification process 2157 can include a barcode reader. The identification process 2157 can provide the vial identifier to the supervisor 2101, and the vial identification can be used to determine the processing parameters associated with the vial. In some configurations, the processing parameters can be in the form of a recipe for processing the cells within the vial. The supervisor 2101 and the subsystem can automate the processing of the cells using the recipe. The recipe can be compiled, at least in part, by the user, for example, using the HMI subsystem.

[0054] Continuing to refer to FIG. 4, the gantry controllers (X / Y gantry controller 2151 and A / Z gantry controller 2153) can enable the vial to move along the vial rail from station to station. In some configurations, for example, but not limited to, a stepper motor controller module such as a FESTO® CMXH-ST2 controller can control the X / Y axis motors. In some configurations, for example, but not limited to, a fieldbus module such as a FESTO® bus node CTEU-EP can provide an interface between the A / Z axis motor and the thawing controller 21491. The gantry controllers can provide the gantry position and gantry status to the thawing controller 21491. The status processor 2161 can monitor other components of the thawing subsystem 2147 and report that information to the thawing controller 21491. The thawing controller 21491 can enable power to the thawing subsystem components through a power controller 2159 that can supply power status to the thawing controller 21491. The thawing controller 21491 can exchange information with the vial rail, sorting and placement equipment, joe valves, pressure switches, and refrigerator alarms through a manifold such as, for example, but not limited to, a FESTO® manifold.

[0055] Referring now to FIG. 4A, method 150 of the present teachings for preparing cells for engineering tissue generally can include, but is not limited to, the steps of thawing a vial of cells, decontaminating the thawed vial, puncturing the decontaminated thawed vial, and removing the cells from the vial, where the removal occurs within a controlled clean volume surrounding the puncture station. These processes can be automatically initiated and can enable method 150 to be used in a manufacturing line. Specifically, method 150 can include step 151 of automatically removing the lid of an insulated container station using a lid gripper. All automated operations can be controlled by a thaw controller. Method 150 can include step 153 of automatically selecting a vial for removal from the insulated container. The vial selection can be based on a preselected vial location, or a sensor can determine the location and position of the vial from which the vial was removed. The sensor can inform the thaw controller of the location of the lid gripper at the location containing the vial. Method 150 can include step 155 of automatically removing the vial from the insulated container using a vial gripper and step of returning the insulated container lid using the lid gripper. Both grippers can be controlled by a thaw controller. Method 150 can optionally include step 157 of automatically identifying the vial. The identification can be enabled, for example, but not limited to, by an RFID device, a barcode reader, or other types of sensors that can read a labeled vial or query the contents of the vial. Method 150 can include the steps of automatically moving the vial to a thaw station and automatically thawing the cells 159. The thaw station can include a thaw management device under the control of and in communication with the thaw controller, which can electronically indicate when the cells have been thawed.At 161, if the cell is not determined to be in a thawed state by the thaw management device and the thaw controller, method 150 can include the step of continuing step 159 of thawing the cell. At 161, if the cell is determined to be thawed, method 150 can include the step of automatically moving the vial to the decontamination station and step 163 of decontaminating the vial. Decontamination can include, but is not limited to, the step of automatically applying a substance such as alcohol to the outer surface of the vial over a predetermined amount of time. Method 150 can include the step of automatically moving the vial into a controlled clean volume surrounding the piercing station within the enclosure or hood when the thaw controller determines that decontamination is complete. Method 150 can include step 165 of automatically piercing the vial using two needles when the vial is correctly positioned within the piercing station, and the needles are of different lengths. Method 150 can include step 167 of piercing the vial to a first preselected amount using the first of the two needles and step 169 of piercing the vial to a second preselected amount using the second of the two needles. The different needle lengths of the two needles can ensure that the needle tips will extend to different levels within the vial. Method 150 can include step 171 of automatically pumping a solution into the vial through one of the needles, such as the second needle, when the thaw controller determines that the needles are properly positioned within the vial. In some configurations, the first needle can be shorter than the second needle. In some configurations, the needles can include bevels that can be aligned with the bevel edges facing in opposite directions. Method 150 can include step 173 of receiving the cells into the first needle as the cells are moved towards the first needle by the introduction of the solution through the second needle into the vial. The cells can continue their progression through tubing that can connect the first needle to the cell reservoir until a substantial number of cells are discharged from the vial.Method 150 can include step 175 of automatically associating the identified vial with the cells in the cell reservoir. Method 150 can optionally include steps of pumping a solution over a preselected amount of time, automatically determining that a vial is cell-free, automatically moving the vial outside of the hooded area, and / or automatically removing the vial using a vial gripper. Method 150 can include step 177 of automatically discarding the vial into a waste container using a vial gripper under the control of a thaw controller.

[0056] Referring to FIG. 4B, the process of cell preparation of the present disclosure is described herein from the perspective of a temporally related sequence of events. Other designs are also conceivable. For example, multiple vials 112 can be processed substantially simultaneously, or the processing of multiple vials 112 can be overlapping, or various steps in the process can be overlapping if possible, or the processing timing can include a time frame that varies for each vial, for example, based on varying processing conditions. For example, the lid gripper 103 can return the lid 107 at the same time the vial gripper moves the vial 112 from the identification station 113 (when present) to the thawing station 115. A configuration involving sequential time-based processing can include that at time t = 1, the thawing controller 101 can command the lid gripper 103 to lift the lid 107 from the insulated container 109 whose temperature can be maintained by the cooling means 111. Below the lid 107, the frozen vial 112 can be seated. The thawing controller 101 can command the vial gripper 105 to remove the vial 112 from the insulated container 109. At time t = 2, the thawing controller 101 can command the lid gripper to return the lid 107 to the insulated container 109. In some configurations, the thawing controller 101 can command the vial gripper 105 to move the vial 112 past the identification station 113 (when present) and can receive vial identification information from the identification station 113 (when present). For example, but not limited to, other methods of identifying the vial 112 such as reading the identification information from the insulated container 109 or generating a record of the identification of the vial 112 such as associating the cells in the vial 112 with the vial 112 later in the cell preparation process of the present disclosure can also be used. At time t = 3, the thawing controller 101 can command the vial gripper 105 to move the vial 112 from its previous location to the thawing station 115.At thawing station 115, thawing station controller 117 can signal thawing controller 101 when the cells in vial 112 are deemed to be thawed according to the process executed by thawing station 115.

[0057] Referring now to FIG. 4C, at time t = 4, the thawing controller 101 can instruct the vial gripper 105 to move the vial 112 from its previous location to the decontamination station 123. When the thawing controller 101 determines that the vial 112 is correctly positioned within the decontamination station 123, the thawing controller 101 can instruct the solution pump 119 to provide the decontamination solution through the tube 128 towards the exterior of the vial 112. In some configurations, the decontamination solution can include, for example, antibacterial and volatile properties. The thawing controller 101 can pump a gaseous substance towards the vial 112 according to a preselected recipe or sensor-related stimuli. The gaseous substance can include air or another mixture of gases appropriate for the decontamination process. At T = 5, the thawing controller 101 can instruct the vial gripper 105 to move the vial 112 through the flap 127 (FIG. 4D) under the hood 124 into the puncture station 143. When the thawing controller 101 determines that the vial 112 is properly positioned with respect to the induction actuator 207 (FIG. 4H) and the needles 145 / 148, the thawing controller 101 can instruct the needle controller 133 to lower the induction actuator 207 (FIG. 4H) so that the needles 145 / 148 can puncture the vial 112. When the needles 145 / 148 are positioned according to a preselected location or sensor-based location, the thawing controller 101 can instruct the solution pump 141 to pump the solution from the solution reservoir 139 through the tubes 129 and the needles 148 into the vial 112. As the cells within the vial 112 are displaced by the solution, the cells can proceed through the tube 131 into the needle 145 and out towards the cell reservoir 137.At T = 6 (Figure 4B), the thaw controller 101 can determine that sufficient cells have been displaced from the vial 112 into the cell reservoir 137, either by a preselected method or by sensor-based calculations, and can instruct the vial gripper 105 to remove the vial 112 from the piercing station 143 and place it into the waste station 106 (Figure 4B). The thaw controller 101 can associate the cells within the cell reservoir 137 with the vial 112 such that it can depend on the method of identifying the vial 112 as described herein.

[0058] Referring now to FIGS. 4D - 4F, an automated system 100 for implementing method 150 (FIG. 4A) can include a thermally insulated container 109 that can maintain the integrity of frozen cells. The thermally insulated container 109 can include a mounting location for any number of vials 112A - 112E. The thermally insulated container 109 can include any commercial cell thermal insulation container station that can house frozen cells maintained at a predetermined temperature. In some configurations, the thermally insulated container 109 can include a material that can passively maintain the contents of the thermally insulated container 109, e.g., the cells, within a desired temperature range. The desired temperature range can be based on the contents and the requirements of their subsequent processing. In some configurations, the thermally insulated container 109 can include an active system for maintaining the contents within a desired temperature range. The active system can include a freezer, and the temperature within the freezer can be controlled by a controller 101. The controller 101 can include wired or wireless communication with the systems being controlled and can manage the sequencing of events as described herein. The system 100 can include moving means for moving the vials 112A - 112E, one by one, for example, but not limited to, into a thawing station 115. The moving means can be controlled by the controller 101. The thawing station 115 can include, but is not limited to, a commercially available thawing station that can be adapted for high - speed thawing with thawing requirements as described herein, e.g., high reproducibility and minimal risk of contamination. The thawing station 115 can be controlled by the controller 101 through a custom interface and can include, for example, but not limited to, the AsteroBio ThawSTAR® automated cell thawing system.

[0059] Continuing to refer to FIGS. 4D - 4F, in some configurations, thaw controller 101 can manage an apparatus (not shown) that can move vial 112A, for example, from insulated container 109 to thaw station 115, for example, along path 213. The moving apparatus can include a mechanism for gripping and releasing vial 112A using, for example, controlled suction, controlled crimping, controlled gripping, or any other suitable means. Thaw controller 101 can include steps of communicating control information, using a control interface, with thaw station controller 117, vial transport means 129, and the gripping system, as well as decontamination means (not shown) associated with decontamination station 123, puncture station 143 (FIG. 4J), and optionally insulated container 109. Thaw station controller 117 can provide an interface between thaw controller 101 and thaw station 115.

[0060] Continuing to refer to FIGS. 4D - 4F, vial gripper system 147 can include any commercially available vial gripper such as, but not limited to, a FESTO® 3 - point gripper, or similar gripping force. Vial gripper system 147 can be mounted, for example, on plate 211, which can allow it to be mounted, for example, on moving means 129. Moving means 129 can include a commercially available moving assembly that can move vial gripper system 147 from decontamination station 123 to puncture station 143 (FIG. 4J), but is not limited to including it.

[0061] Continuing to refer to FIGS. 4D - 4F, the thaw station controller 117 can include electronics for sensing the state of the contents of vial 112A and notifying the thaw controller 101, for example, when it is time to move vial 112A, for example, to vial gripper system 147. The moving device can include, for example, by means described herein, steps of attaching to vial 112A and moving vial 112A, for example, from thaw station 115 to vial gripper system 147, for example, along 13221. The thaw controller 101 can recognize when vial 112A is correctly positioned within, for example, vial gripper system 147 and can control, for example, the tightening of gripping portion 149 (FIG. 4H) around vial 112A. When it is sensed that gripping portion 149 is tightened by a pre - selected amount, the thaw controller 101 can command the moving means 129 to move vial gripper system 147 and vial 112A into, for example, decontamination station 123, where the exterior of vial 112A can be decontaminated. In some configurations, the decontamination means 300 (FIG. 4G) can include a nebulizer 319 (FIG. 4G) that deposits material from gas line 125 (FIG. 4G) and / or solution line 128 (FIG. 4G) onto vial 112A. The material can be pumped through decontamination tube 323 (FIG. 4G) and through cavity 123A (FIG. 4G) on vial 112A. The misting manifold 321 (FIG. 4G) can assist in the individual control of gas line 125 (FIG. 4G) and solution line 128 (FIG. 4G) as gas pump 121 (FIG. 4G) and solution pump 331 (FIG. 4G) pump gas and solution into decontamination tube 323 (FIG. 4G). The access port 123B (FIG. 4G) can optionally include a door that can retain solution within decontamination station 123. The door can optionally include, but is not limited to, a flap and / or bristles. The decontamination means 300 (FIG. 4G) can be controlled and its activation time can be sequenced by the thaw controller 101.The decontamination means 300 (FIG. 4G) can decontaminate the exterior of the vial 112A, for example, before entering the controlled clean volume surrounding the piercing station environment in the hood 124 through the flap 127. In some configurations, the hood 124 can include a device that can contain a laminar airflow, for example, pushed in by positive pressure, through an air filter that can filter particles to a size of about 0.12 to 0.3 microns. In some configurations, the filtered air can exit through the working area of the hood 124. In some configurations, the hood 124 can include, for example, a maximum of 10. 5 = It can include an ISO class 5 clean room having 100,000 particles per cubic meter.

[0062] Referring now to FIGS. 4H and 4I, when decontamination is complete, thaw controller 101 can instruct moving means 129 to move vial gripper system 147 and vial 112A into puncture station 143 (FIG. 4J) within hood 124, for example, through flap 127 (FIG. 4F). Thaw controller 101 can instruct moving means 129 to position vial gripper system 147 and vial 112A so as to ensure that vial 112A is aligned with needles 145 / 148 (FIG. 4K) in a preselected manner. Puncture station 143 (FIG. 4K) can wash displaced cells from vial 112A, for example, into cell reservoir 137, while collecting the cells, using a solution from vial 112A, for example, solution reservoir 139, through tube 129 and connector 202 (FIG. 4K). In some configurations, a plurality of vials 112A-112E can be punctured substantially simultaneously. In some configurations, connector 202 (FIG. 4K) can include a threaded luer locking fitting, for example, but not limited to, a female luer locking fitting with 10-32 threads. In some configurations, larger vials can be accommodated by system 100 when it is desired to process a greater number of cells. Thaw controller 101 can extend guide actuator 207 vertically, for example, based at least on the position of vial 112A. In some configurations, when thaw controller 101 instructs moving means 129 to position vial gripper system 147 and vial 112A at a preselected position relative to needles 145 / 148 (FIG. 4K), thaw controller 101 can raise guide actuator rod 225 by 144 (FIG. 4H). When vial puncture is desired, thaw controller 101 can lower guide actuator rod 225 by 142 (FIG. 4I).

[0063] Referring now to FIG. 4J, the piercing station 143 can include, but is not limited to, an extrusion bracket 205, an induction actuator 207, a T-bracket 249, a vial piercing hub 201, and a needle 145 / 148 (FIG. 4K). The first end of the needle 145 / 148 (FIG. 4K) can be fitted into a needle cavity (not shown) within the T-bracket 249. The second end of the needle 145 / 148 (FIG. 4K) can include a bevel edge. The induction actuator 207, the T-bracket 249, and the vial piercing hub 201 can be operably coupled by a fastener that passes from the post connector 247, through the T-bracket 249, to a cavity 245 (FIG. 4K) within the vial piercing hub 201. The induction actuator post 225 can travel inside and outside of the induction actuator 207 under pressure by a piercing cylinder (not shown) that can be operably coupled to the extrusion portion 209 through the extrusion bracket 205. The extrusion portion 209 and the extrusion bracket 205 can enable flexible horizontal positioning of the induction actuator 207 and thus the needle 145 / 148. The induction actuator 207 can include, for example, but is not limited to, a commercially available actuator such as a FESTO® induction actuator DFM-25-80-P-A-KF, or any actuator that includes high resistance to torque and lateral forces. In some configurations, the induction piston rod 225A can be fixed against rotation by the guide rod 225. The guide rod 225 can also assist in enabling simultaneous and / or semi-simultaneous piercing of multiple vials 112. In some configurations, to properly pierce the vial 112, the piercing cylinder (not shown) can provide a piercing force within the range of 5 to 7 kgf. The induction piston rod 225A can include a cylinder bore of about 10 mm to 20 mm and can supply a force of about 70 N to 220 N at a supply pressure within the range of about 4 to 7 bar. In some configurations, a minimum force greater than 60 N can be produced.

[0064] Referring now to FIGS. 4K and 4L, the vial piercing hub 201 can include vial piercing fitting cavities 231 / 233 that can receive a vial piercing fitting 202. The first vial piercing fitting 202 and the solution tube 129 can provide an interface between the solution reservoir 139 and the needle 148. The second vial piercing fitting 202 and the cell tube 131 can provide an interface between the cell reservoir 137 and the needle 145. In some configurations, the thaw controller 101 can command a pump (not shown) to move solution from the solution reservoir 139, through the solution tube 129 and the first vial piercing fitting 202, through the vial piercing hub 201, and into the needle 148. The solution within the vial 112 can finally push the thawed cells present within the vial 112, through the vial piercing hub 201, the second vial piercing fitting 202, the cell tube 131, and into the cell reservoir 137 through the needle 145. In some configurations, the needles 145 / 148 can include 316 stainless steel, and the bevel edges of the needles 145 / 148 can be aligned in opposing directions of ±10°. In some configurations, the vial piercing holes 231 / 233 can include dimensions, for example, but not limited to, about 0.0605 +0.0015, -0.0008 inches, and the tubes 129 / 131 can include bore sizes within the range of about 0.03 - 0.06 inches. With respect to the needles 145 / 148, the inner diameter can be selected to be large enough to achieve a reasonable flow rate and avoid excessive shear on the cells and / or long pumping times. An appropriate needle wall thickness can ensure that the needles 145 / 148 can pierce the cap of the vial 112 without buckling, for example, within the range of about 0.008 - 0.015 inches. In some configurations, both needles 145 / 148 can include a soldered or brazed joint diameter gap with respect to the vial piercing hub 201 of about 0.0035 inches. In some configurations, the vial piercing hub 201 can include 316 stainless steel.

[0065] Referring now to FIG. 4L, the vial piercing hub 201 can include a gasket 234 that, in conjunction with the deformation portion 236, prevents cell leakage, i.e., prevents cells being discharged from the vial 112 from following a route through other than one of the needles 145 / 148. Piercing of the vial 112 can pierce the gasket 234 and, together with the leak prevention seal, form the deformation portion 236.

[0066] Referring now to FIG. 5, after the cells are thawed, if cell thawing is necessary, the supervisor 2101 can enable the transfer and control of the cells from the thawing subsystem 2147 (FIG. 4) to the expansion subsystem 2141. The expansion subsystem 2141 can include an expansion controller 21411 and a bioreactor controller 21412. The expansion controller 21411 can control the fluid and pneumatic valve 2109, which can enable the control of the direction in which the fluid is pumped from one location to another. The expansion controller 21411 can monitor the priming and leading edge of the fluid in the tubing between the vial 2163 (FIG. 4) and the bioreactor vessel 2113 through the bubble sensor 3145. The transfer of cells from the vial 4163 (FIG. 4) to the bioreactor vessel 2113 can be considered complete when a preselected amount or range of time has elapsed. In some configurations, part of the transfer can be done by pumping and part can be done using a gas purge. The cells can be moved from the vial 4163 (FIG. 4) to the bioreactor vessel 2113, where they can attach, for example, but not limited to, to the microcarriers 2116 within the bioreactor 2113. It is also possible that there are no cell growth promoting factors other than the microcarriers 2116, or none at all. For example, some cells grow as cluster aggregates. The bioreactor controller 21412 can enable the sequencing of the gas mixing 3147, stirring 3149, and temperature control 3151 of the contents of the bioreactor 2113. A stepper motor can be used to stir the bioreactor 2113. The bioreactor controller 21412 can control the movement of the medium and cells through the pump controller 3155, which can enable the pumping of the medium from the reservoir 6149 to the bioreactor 2113 and from the bioreactor 2113 to the disposal location. The bioreactor 2113 can be stirred by the stirring controller 3149, and the temperature controller 3151 can enable the adjustment of the temperature of the medium.The gas mixing processor enables control of the gas in the medium and thus can control the properties of the medium. The monitoring process 3153 can monitor the properties of the medium within the bioreactor 2113. The properties of the medium that can be monitored can include, but are not limited to, temperature, dissolved oxygen, and pH. The cell density within the bioreactor 2113 can be monitored as the preselected target range regarding the cell density can trigger the end of cell expansion within a particular bioreactor.

[0067] Continuing to refer to FIG. 5, the expansion controller 21411 can control the pneumatic valve 2109 and the foam sensor 2145 through the FESTO (registered trademark) valve terminals and can include, for example, but not limited to, a ROCKWLL AUTOMATION (registered trademark) COMPACTLOGIX (registered trademark) controller. The bioreactor controller 21412 can include an EPPENDORF (registered trademark) DASGIP (registered trademark) controller, or any type of bioreactor controller that can operate multiple bioreactors in parallel, but is not limited to including it. The bioreactor controller 21412 can control and monitor the bioreactor temperature and agitation. Temperature control can be enabled, for example, but not limited to, by EPPENDORF (registered trademark) DASGIP (registered trademark) Bioblock control. The media pH, dissolved oxygen, liquid level, and cell density can be monitored by a monitoring module that reports the data to the bioreactor controller 21412. The state of the gas mixing valve, which can control changes in carbon dioxide and ultimately changes in pH, can be controlled by a gas mixing processor 3137 that responds to the bioreactor controller 21412. In some configurations, the %CO2, %O2, and gas flow rate out of the headspace of the bioreactor vessel 2114 can be measured and the oxygen consumption rate can be determined. Media addition / removal can be monitored and controlled by a peristaltic pump at a pump rate that can be adjusted by the bioreactor controller 21412 based on data monitored by the bioreactor controller 21412.

[0068] Referring now to FIG. 6, when the cell density reaches the desired level, possibly as described in the recipe, the resulting expanded cells can be concentrated at another station along the production line, namely, the concentration station 2143. The supervisor 2101 can detect the cell density from the data monitored by the expansion subsystem 2141 (FIG. 5) and enable the step of moving the cells into the concentration subsystem 2143, where the expanded cells can possibly be concentrated by centrifugation. In some configurations, the expanded cells can be pumped into the concentration subsystem 2143, concentrated, and moved back to the expansion subsystem 2141 (FIG. 7). In some configurations, the concentration subsystem 2143 can provide, but is not limited to providing, cell concentration and media classification through separation of live cells from dead cells and debris. In some configurations, the concentration subsystem 2145 can include, but is not limited to including, CARR™ CENTRITECH™ LAB III. In some configurations, the supervisor 2101 can interface with the concentration subsystem 2143 through the concentrator interface 4143. In some configurations, the concentrator interface 4143 can provide a protocol specific to the industrial system that communicates with control devices such as the concentrator 4145 within the industrial system via Industrial Ethernet™. In some configurations, the protocol can include Profinet, an industrial technical standard for data communication.

[0069] Referring now to FIG. 7, upon completion of the concentration cycle, the supervisor 2101 can enable the cells to move from the concentrator subsystem 2143 to the expansion subsystem 2141 and then to the maturation subsystem 2145 for concentration regulation. The length of the concentration cycle can be determined dynamically based on a pre-selected, user-set, cell type-dependent, recipe-set, and / or data collected during the concentration cycle. The maturation subsystem 2145 can include, but is not limited to, a media controller 5145 and an incubator controller 5147. In some configurations, the supervisor 2101 can enable the cells to move from the concentrator subsystem 2143 to the expansion subsystem 2141 for concentration regulation and then transfer them to the maturation subsystem under the control of the incubator controller 5147. The media can be moved to the container 2118 under the control of the media controller 5145. The media controller 5145 can adjust the properties of the media in the container 2118, stir the media, and pump the media to the bioreactor 2114. The property controller 5151 can adjust the properties of the media, such as, but not limited to, temperature, pH, and dissolved oxygen. The gas mixing valve 5149 can provide a gas that may be required, for example, to adjust the pH. The pump controller 5153 and the pneumatic valve controller 5155 can enable the movement of the media from the reservoir 6149 (FIG. 8) to the container 2118 and from the container 2118 to the bioreactor 2114. Inside the bioreactor 2114, there is a surface to which the cells can attach while they are maturing. The first type of cells can be introduced from the container 2118 and they can adhere to one side of the surface. The incubator controller 5147 can command the motor driver 5157 to enable the motor 5159 to rotate the surface so that the second cell type can be introduced. In this way, two cell types can mature simultaneously, and the geometry of the bioreactor 2114 can promote the growth of the cells together.The incubator controller 5147 can receive data from the in-line sensor 5163, for example, through the serial interface 5161, and can also receive data from the sensor 5165. For example, many things such as temperature, pH, and glucose can be sensed regarding the cells during maturation. The supervisor 2101 can issue an alert if the sensed data is outside the acceptable range. In some configurations, the supervisor 2101 can be enabled to adjust the environment of the bioreactor 2114 based on the sensed data.

[0070] Continuing to refer to FIG. 7, after the concentrated cells are prepared for maturation, the incubator controller 5147 can exchange messages with the motor driver that controls the movement of the bioreactor 2114. The incubator controller 5147 can include, but is not limited to, a ROCKWLL AUTOMATION® COMPACTLOGIX® controller. The motor driver 5157 can include a stepper motor 5159 for an industrial system, for example, but not limited to, an ANG1 AnyNET-I / O integrated stepper motor controller / driver. In the maturation subsystem 2145, the incubator controller 5147 can monitor the cells and the properties of the fluid surrounding them as they mature. In some configurations, the in-line sensor 5163 can potentially be used to monitor pH, dissolved oxygen, and glucose through the serial interface. Other sensors can monitor relative humidity, temperature, carbon dioxide concentration, and incubator alarms.

[0071] Referring now to FIGS. 7A and 7B, the rotation of the bioreactor 2114 can be managed by the rotation device 1900. The rotation device 1900 can potentially accommodate bioreactors 2114 of any shape and size, possibly by modifying the dimensions of the rotation device 1900 as needed. The rotation device yoke 1909 can provide a cantilevered support to the bioreactor 2114 and enable unobstructed observation of the sample. The rotation device 1900 can enable the cell and media supply as well as the gas vent tubes to remain intact and unentangled throughout a limited operating rotation range of the bioreactor 2114, e.g., the entire + / - 270 degrees. The rotation device 1900 can include, but is not limited to, a hollow spindle 1901 that can receive and protect the tubes 1919A / B / C as the bioreactor 2114 rotates. The hollow spindle 1901 can be held in place by a spindle cover 1913 mounted on the mounting board 1917. The worm wheel and worm screw 1907 can enable the rotation of the combination of the yoke 1909 / bioreactor 2114 while keeping the tubes 1919A / B / C within the hollow spindle 1901 and unentangled, and can reduce inertia concerns during rotation. The motor 1905 can enable the worm screw and worm wheel 1907 to rotate the yoke 1909. The bolt 1912 can accommodate a gear cover for the worm screw 1907 that can cover the worm screw 1907 and protect the device and process from metal wear powder release. The remainder of the rotation device 1900 can be cleaned, for example, to be rated to the IEC60529 IP65 standard.

[0072] Referring now to FIG. 7C, bioreactor 2114 can be seeded through either tube 1919B or 1919C, depending on the orientation of bioreactor 2114. In the view shown in FIG. 7C, bioreactor side 2114A can receive cells and media through tube 1919B, while pinch valve 1903, associated with tube 1919B, can be forced open. Pinch valve 1903, associated with tube 1919A, can be closed on tube 1919A. Cells and media can enter into bioreactor interior 1920 through tube 1919B and can be urged by gravity to adhere to side 2114A of bioreactor 2114. Tube 1919A can enter bioreactor 2114, for example, through cavity 2114C, which is located centrally on bioreactor side 2114B. Tube 1919B can enter bioreactor 2114, for example, through cavity 2114D, which is located laterally on bioreactor side 2114A. The entry locations 2114C / D can be positioned to encourage one type of cell to grow around another type of cell. For example, a first cell type can be seeded through cavity 2114D and a second cell type can be seeded through cavity 2114C, thus encouraging the first cell type to grow around the second cell type.

[0073] Referring now to FIGS. 7C - 7E, operationally, after seeding, the worm screw 1907 undergoes a slight inclination within the yoke 1909 (and bioreactor 2114), for example, up to approximately 3°, followed by a period of temporary residence, which can provide a uniform liquid distribution and promote consistent cell seeding density and adhesion to the bioreactor side 2114B. The worm gear 1907 can return the yoke 1909 to a horizontal position, enabling adhesion to occur, and can be re - inclined as desired. Finally, the worm screw 1907 can rotate such that the yoke 1909 / bioreactor 2114 is stationary at 180° from its starting position. At this point, cells can be seeded onto the side 2114A of the bioreactor 2114 through the tube 1919A. The amount of time required for each step depends on the type of tissue desired, the process for forming that tissue, and other considerations. Any gas produced during seeding and maturation can be vented through the tube 1919C, which can terminate within a filtered exhaust opening.

[0074] Continuing to refer to FIGS. 7C - 7E, for tissue harvesting, the bioreactor 2114 can be arranged within a rack, and the entire rack can be removed from the incubator. The incubator can include, but is not limited to, a welded stainless - steel assembly that can be sterilized between uses. The incubator can house a controller for the motor and can include a network connection. The rotary device 1900 can be configured to operate in pairs, in which the bioreactor 2114 can be cantilever - supported on one driven side and simply supported on the second side.

[0075] Referring now to FIG. 8, the supervisor 2101 can control, for example, the side of the media reservoir 6149. In some configurations, the media reservoir 6149 is located within the environmentally controlled area 6145, which can reduce the potential for degradation of heat - sensitive adjuvants in the media.

[0076] Referring now to FIGS. 9A and 9B, the method 1850 of the present teachings for manufacturing tissue along a production line can include, but is not limited to including, a step 1851 of pumping cells from at least one vial to at least one first bioreactor. In 1853, when the cells are moved to at least one first bioreactor, the method 1850 can include a step 1855 of managing fluid flow between at least one vial and at least one bioreactor, and a step 1857 of generating an environment that enables a discharge, wash, digestion, and reaction stop process and promotes cell expansion within at least one first bioreactor. The environment can be automatically generated and automatically maintained based on sensor data of critical process parameters. In 1859, when a desired cell density is reached within at least one first bioreactor and other processes are completed, the method 1850 can include a step 1861 of pumping the expanded cells to a concentrator, and a step 1863 of concentrating the cells. In 1865, when the cells are at a desired concentration, the method 1850 can include a step 1867 of pumping the concentrated cells to at least one first bioreactor, a step 1869 of resuspending the concentrated cells, and a step 1871 of pumping the resuspended cells to at least one second bioreactor for seeding and maturation. The method 1850 can include a step 1873 of generating an environment within at least one second bioreactor to promote maturation based on monitoring of sensor data of critical process parameters, among other parameters. After tidal exchange, media regeneration, wash, digestion, and media replacement for differentiation, in 1875, when the cells reach a desired maturity, the method 1850 can include a step 1877 of collecting tissue resulting from the matured cells.

[0077] In some configurations, under the automatic control of the supervisor 2101 (FIG. 1) (except when human input is desired at the preparation stage (steps 7, 11, 17)), the sequence of steps performed by various components of the system 2100 (FIG. 1) can include the following list. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0078] 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. Additionally, although 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, and the present disclosure is as broad as would be acceptable in the art, and this specification is likewise intended to be read. Accordingly, the above description should be construed as illustrative rather than limiting, merely as an example of a particular configuration. Also, those skilled in the art will envision other modifications within the scope and spirit of the appended claims herein. Other elements, steps, methods, and techniques substantially different from those described in the above and / or appended claims are also intended to be within the scope of the present disclosure.

[0079] The drawings are presented only to demonstrate an example of the present disclosure. Also, the drawings described are merely illustrative and non-limiting. In the drawings, for illustrative purposes, the sizes of some of the elements may be exaggerated and may not be drawn to a particular scale. Additionally, elements shown within the drawings having the same number may, depending on the context, be the same element or similar elements.

[0080] When the term "comprising" is used in this specification and claims, this does not exclude other elements or steps. When referring to a singular noun, if an indefinite or definite article, such as "a", "an", or "the", is used, this includes the plural form of that noun unless something else is specifically stated otherwise. Therefore, the term "comprising" should not be construed as being limited to the items listed thereafter. That is, 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 only of components A and B.

[0081] Furthermore, the terms "first", "second", "third", and equivalents are provided to distinguish similar elements whether used in the description or claims and are not necessarily for describing a sequential or chronological order. Such terms are interchangeable under appropriate circumstances (unless it is clearly disclosed otherwise), and it should be understood that the exemplary configurations of the present disclosure described herein are capable of operating in sequences and / or arrangements other than those described or illustrated herein.

Claims

1. A system for manufacturing tissue, the system comprising: An expansion subsystem that automatically pumps cells from at least one vial into at least one first bioreactor, the expansion subsystem automatically generating a first preselected environment that promotes an increase in the number of cells in the at least one first bioreactor, the expansion subsystem automatically pumping the increased number of cells from the at least one first bioreactor into a concentrator; A concentration subsystem that forms resuspended cells by automatically concentrating the increased number of cells; A maturation subsystem that automatically pumps the resuspended cells into at least one second bioreactor, the maturation subsystem automatically generating a second preselected environment that promotes the maturation of the concentrated cells in the at least one second bioreactor based on monitoring sensor data; The system comprising.

2. The system according to claim 1, wherein the concentration subsystem comprises a centrifugation device.

3. The system according to claim 1, wherein the first preselected environment comprises a first growth medium, and the first growth medium is continuously and automatically adjusted based on monitoring the sensor data to maintain a first preselected level of growth medium characteristics.

4. The system according to claim 1, wherein the second preselected environment comprises a second growth medium, and the second growth medium is continuously and automatically adjusted based on monitoring the sensor data to maintain a second preselected level of growth medium characteristics.

5. The system according to claim 1, further comprising a thawing subsystem, the thawing subsystem receiving frozen cells in at least one vial and automatically thawing the frozen cells.

6. The thawing subsystem comprises A thawing controller that controls the preparation of the cells; An insulated container station, wherein the insulated container station has cooling means for cooling the environment surrounding the frozen cells in the at least one vial, and an insulated container for maintaining the frozen cells within a preselected temperature range, an insulated container station. A vial gripper, wherein the vial gripper moves the at least one vial under the control of the thaw controller, a vial gripper. At least one device, wherein the at least one device identifies the respective positions of the at least one vial under the control of the thaw controller, at least one device. A thawing station, wherein the thawing station receives the at least one identified vial, the thawing device thaws the cells in the at least one vial, the thawing station includes a thawing station controller, and under the control of the thaw controller, the thawing station controller provides the status of the cells to the thaw controller, a thawing station. A decontamination station, wherein the decontamination station receives the at least one vial of thawed cells, and the decontamination station includes means for decontaminating the outer surface of the at least one vial of thawed cells, a decontamination station. A piercing station, wherein the piercing station receives the at least one decontaminated vial of thawed cells, the piercing station includes at least two needles, the at least two needles pierce the decontaminated vial, a first needle of the at least two needles has a first length, a second needle of the at least two needles has a second length, the piercing station includes a needle controller that pierces the at least one decontaminated vial of thawed cells using the first needle, the first needle extends into the at least one decontaminated vial of thawed cells by a first preselected distance, the needle controller pierces the at least one decontaminated vial of thawed cells using the second needle, and the second needle extends into the at least one decontaminated vial of thawed cells by a second preselected distance, the piercing station A solution pump, wherein the solution pump pumps a solution from a solution reservoir, through the first needle, into the at least one decontaminated vial of thawed cells, and the thawed cells are drawn through the second needle into the at least one first bioreactor, the solution pump The system according to claim 5, comprising.

7. The system according to claim 6, further comprising an identification station for identifying each of the at least one vial.

8. The system according to claim 6, wherein the first length is longer than the second length.

9. The system according to claim 6, wherein the first preselected distance is shorter than the second preselected distance.

10. The system according to claim 6, further comprising a gas purge for pushing the contents of the at least one decontaminated vial out of the at least one decontaminated vial.

11. The decontamination station is A hood for retaining the decontamination fluid, A decontamination pump for pumping the decontamination fluid around the at least one identified vial, A nozzle for directing the decontamination fluid towards the at least one identified vial The system according to claim 7, comprising. **Claim 12**: The system according to claim 6, wherein the piercing station comprises a base gripper that maintains the at least one identified vial in a fixed position while the needle controller removes the first needle and the second needle. **Claim 13**: The system further comprises a hood surrounding the piercing station, The hood maintains a controlled clean volume surrounding the piercing station, the system according to claim 6. **Claim 14**: The system according to claim 5, wherein the inflation subsystem comprises a bioreactor controller that monitors and modifies the first preselected environment. **Claim 15**: The bioreactor controller, A stirring controller that promotes adhesion of the cells to the surface of the cells by stirring the cells, A temperature controller that adjusts the temperature of the at least one first bioreactor based on a preselected desired temperature, A gas mixing processor that adjusts the level of gas in the medium surrounding the cells, wherein the level of the gas is based on a preselected desired value of the characteristics of the medium, a gas mixing processor, A monitoring process that senses the value of the characteristics of the medium, A pump controller that moves the medium to the at least one first bioreactor and moves the medium from the at least one first bioreactor The system according to claim 14, comprising. **Claim 16**: The system according to claim 15, wherein the characteristics include dissolved oxygen and pH. **Claim 17**: The maturation subsystem, A medium controller that monitors and modifies the medium before introducing the medium into the at least one second bioreactor, An incubator controller that manages the movement of the at least one second bioreactor, the incubator controller monitoring the characteristics of the medium in the at least one second bioreactor, the incubator controller flushing and restoring the medium from the at least one second bioreactor and flushing and restoring the medium to the at least one second bioreactor, an incubator controller The system according to claim 1, comprising. **Claim 18**: The system further comprises a medium storage unit controller, The medium storage unit controller, A medium level sensor that monitors the amount of medium in the medium reservoir, A pump pressure sensor that monitors a pump for moving a medium from the medium reservoir to the medium container The system according to claim 1, comprising:

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