Systems and methods for cell and tissue preparation - Patents.com

Abstract

A manufacturing system and method for generating multiple tissue constructs from cells. The system can include a thawing 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 the process can be repeated depending on the specific tissue process being implemented. Multiple tissue types can be combined in multiple bioreactors. The activities of the 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, manage the subsequent process, and alert the user when user action is required.

Description

[Technical field]

[0001] Government Licensing Rights This invention was made with Government support under Contract No. W911NF-17-3-003, F0008, funded by the Advanced Regenerative Manufacturing Institute. 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 (Attorney Docket No. Z69), filed June 17, 2019, and entitled “System and Method for Cell Preparation,” which is incorporated by reference in its entirety. [Background technology]

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

[0004] Manual tissue processing in the laboratory has been attempted for many years. Methods used for processing typically suffer from non-reproducibility or inconsistency from run to run due to the skilled manual nature of the work and the relatively poorly understood biological input material. Current methods are also typically highly labor intensive. However, the building blocks of tissue processing may include processes that can be automated. Among other things, automation can eliminate human error and improve consistency between production runs. Automation must include linking together the heterogeneous processes required to implement an entire processing line and the process start-up timing associated with the biological processes that may underlie tissue generation. Monitoring of specific characteristics indicative of tissue status must also be ongoing 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, can require expansion, concentration, and maturation steps. Typically, expansion occurs in flasks and static bioreactors located in incubators, which require manual media exchange. Typically, monitoring is visual and subjective. Transfer of cells from one stage to the next is typically performed manually.

[0006] If the process begins with frozen cells, proper thawing, resuscitation, and storage of the cells, if necessary, can aid in the successful use of the cells. To thaw the cells, a common manual method is, in part, to submerge a vial of cells in a 37° C. water bath. This manual method cannot meet automated manufacturing requirements, is highly subjective, and creates contamination risks, among other issues. Rapid thawing is essential to prevent ice crystallization during thawing. This is followed by a controlled "quench" of the medium to dilute the cryoprotectant without causing the cells to swell (and burst) too much. Automated systems are able to control rapid thawing better than manual processes. Cell cultures can be lost through accidents, contamination, deterioration or environmentally induced changes, and improper monitoring. When operating in manual mode, the cells will be visually monitored to assess the level of confluence, any changes in medium color, etc.

[0007] What is needed is a manufacturing line that can automatically generate tissues repeatedly and consistently. What is needed is a system where the process that allows for staging is automated and where the movement of cells from one stage to the next is also automated. What is needed is a system where the user does not determine when one stage is finished and another should start, when cells need to be moved, and where the cell transfer is not performed manually. What is needed is a system that provides overall consistent results and avoids the issue of cells / tissues being "ready" late at night or on weekends when the user is not monitoring them. What is needed is a system where monitoring and any feedback loops associated with the results monitored are fully automated. Such a line would be scalable so that the same automated process could be used to generate many tissue constructs. The manufacturing line should be made of modular components and allow for reconfigurability based on the specific tissue process that is needed. The line should also be fluidly closed so that it can be operated in a non-clean space without concerns of contaminating the cells and tissues contained therein. Summary of the Invention [Means for solving the problem]

[0008] The system of the present teachings can include, but is not limited to, a manufacturing line for producing tissue engineered medical products (TEMPs), or more broadly, human cells, tissues, and cell and tissue-based products (HCT / Ps). The manufacturing line can produce consistent results across tissue engineering runs and can address safety and quality concerns since no human intervention is required during the cell maturation and incubation process. The manufacturing line can include, but is not limited to, a station in which cells are thawed, a station in which the thawed cells are expanded, a station in which the expanded cells are concentrated, and a station in which the concentrated cells are incubated into tissue.

[0009] A method of the present teachings for enabling automated production of tissue includes receiving cells into 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 as the cells are transferred to the at least one first bioreactor, automatically generating a first preselected environment to facilitate expansion of the cells in the at least one first bioreactor based on monitoring sensor data of a critical process parameter, automatically harvesting the cells from the microcarrier surface and quenching the harvesting enzyme, and automatically extracting the expanded cells from the at least one first bioreactor. The method may include, but is not limited to, automatically pumping the expanded cells from the at least one first bioreactor to a concentrator when a preselected density is reached; 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 the 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 foster maturation of the concentrated cells into tissue in the at least one second bioreactor.

[0010] The method can optionally include automatically receiving and thawing cells, including 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 automatically controlling, by the thaw controller, removing an insulated container lid of the insulated container using a first gripper. The first gripper can be controlled by the thaw controller. The method can optionally include automatically determining, by the thaw controller, a location of at least one vial in the vial holder, automatically controlling, by the thaw controller, positioning a second gripper at the location, and automatically controlling, by the thaw controller, removing the at least one vial from the insulated container using the second gripper. The second gripper can be controlled by the thaw controller. The method can include automatically controlling, by the thaw controller, returning the insulated container lid to the insulated container using the first gripper. The first gripper can be controlled by the thaw controller. The method may include automatically controlling, by the thaw controller, placing the at least one removed vial in a cell thawing device using a second gripper. The second gripper may be controlled by the thaw controller. The method may include automatically determining when the frozen cells are thawed by a thawing management device controlled by the thaw controller, and automatically controlling, by the thaw controller, removing the at least one vial of thawed cells using a second gripper. The second gripper may be controlled by the thaw controller. The method may include automatically controlling, by the thaw controller, transferring the at least one vial of thawed cells to a third gripper using the second gripper.The second gripper can be controlled by a thaw controller. The method can include automatically controlling, by the thaw controller, a step of moving at least one vial of thawed cells to a first preselected location using an actuator operably coupled to the third gripper, the actuator being controlled by the thaw controller. The method can include automatically controlling, by the thaw controller, a step of decontaminating an exterior of the at least one vial with a decontamination system, the decontamination system being controlled by the thaw controller. The method can include automatically controlling, by the thaw controller, a step of moving at least one vial of thawed cells to a second preselected location for access using an actuator, the actuator being controlled by the thaw controller. The method can include automatically controlling, by the thaw controller, a step of accessing at least one vial with an access system controlled by the thaw controller, the access system including an access device, the access device being continuously energized during the pumping operation. The method may include automatically controlling, by the thaw controller, pumping a solution into at least one vial by a pump controlled by the thaw controller, automatically controlling, by the thaw controller, extracting thawed cells from at least one vial using advection, and automatically controlling, by the thaw controller, discarding at least one vial. The first gripper and the second gripper may optionally comprise a single device. The first gripper, the second gripper, and the third gripper may optionally comprise a single device. The method may optionally include automatically determining, by the first device controlled by the thaw controller, an identity of the at least one vial removed.The decontamination system can optionally include decontaminating an exterior surface of at least one vial. The solution can optionally include a neutralizing agent. The solution can optionally include a medium. Concentrating the expanded cells can optionally include centrifuging the expanded cells.

[0011] A system of the present teachings for enabling automated production of tissues may include, but is not limited to, an expansion subsystem that automatically pumps cells from at least one vial to at least one first bioreactor, where the expansion subsystem automatically closes a valve from the at least one vial to the at least one first bioreactor once the cells are transferred to the at least one first bioreactor, where the expansion subsystem automatically generates a first preselected environment to facilitate expansion of the cells in the at least one first bioreactor, where the expansion subsystem automatically pumps the expanded cells from the at least one first bioreactor to a concentrator once the expanded cells reach a preselected density, where the expansion subsystem is 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 upon the occurrence of a preselected event, and the maturation subsystem automatically generates a second preselected environment based on monitoring sensor data of a critical process parameter to facilitate maturation of the concentrated cells in the at least one second bioreactor.

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

[0013] The thawing subsystem optionally includes a thaw controller for controlling the preparation of the cells, a cooling means for cooling an environment surrounding the frozen cells in the at least one vial, an insulated container station having an insulated container lid for maintaining the frozen cells in a preselected temperature range, an insulated container station having an insulated container lid for retaining the environment, a lid gripper and a vial gripper, the lid gripper moving the insulated container lid under control of the thaw controller, the vial gripper moving the at least one vial under control of the thaw controller, at least one device for locating a position of each of the at least one vial under control of the thaw controller, and a thawing station receiving at least one identified vial from the vial gripper under control of the thaw controller, the thawing station storing the at least one identified vial, the thawing device thawing the cells in the at least one vial, the thawing station including a thawing station controller, the thawing station controller controlling the thawing station. a thawing station, under control of the thawing controller, the thawing station controller providing a status of the cells to the thawing controller; a decontamination station, under control of the thawing controller, receiving at least one vial of thawed cells from a vial gripper, the decontamination station including a means for decontaminating an exterior surface of the at least one vial of thawed cells; a puncturing station, under control of the thawing controller, the puncturing station including at least two needles, the at least two needles puncturing the decontaminated vial under control of the thawing controller, a first needle of the at least two needles having a first length and a second needle of the at least two needles having a second length, the puncturing station, under control of the thawing controller, puncturing the decontaminated at least one vial of thawed cells with the first needle;The system may include a puncturing station, under the control of the thawing management device, puncturing the at least one decontaminated vial of thawed cells with a second needle, the second needle extending a second preselected distance into the at least one decontaminated vial of thawed cells, and a solution pump, under the control of the thawing controller, pumping solution from a solution reservoir, through the solution tubing and the first needle, into the at least one decontaminated vial of thawed cells, where the thawed cells are drawn through the second needle, the thawed cells flow through the cell tubing into the cell reservoir, and the cells are removed from the at least one decontaminated vial of thawed cells, generating at least one waste vial.

[0014] The system may optionally include an identification station that identifies each of the at least one vial under control of a thaw controller. The first length may optionally include a length greater than the second length. The first preselected distance may optionally include a distance less than the second preselected distance. The system may optionally include a gas purge that urges substantially all of the contents of the at least one decontaminated vial out of the at least one decontaminated vial. The decontamination station may optionally include a hood that retains decontamination fluid within a preselected area surrounding the identified at least one vial of thawed cells, a decontamination pump that pumps the decontamination fluid into the preselected area under control of the thaw controller, and a nozzle that directs the decontamination fluid toward the at least one identified vial of thawed cells. The puncture station may optionally include a base gripper that maintains the at least one identified vial in place while the needle controller removes the first and second needles under control of the thaw controller. The system optionally includes a waste system, where the needle controller receives at least one waste vial from the vial gripper under control of the thaw controller after removing the first and second needles, where the waste system discards the at least one waste vial into a waste receptacle. The system optionally includes a hood surrounding the puncturing station, where the hood maintains a controlled clean volume surrounding the puncturing station. The expansion subsystem optionally includes an expansion controller that controls the flow of cells from the thawing subsystem to the expansion subsystem, and a bioreactor controller that monitors and modifies the first preselected environment.The bioreactor controller may optionally include an agitation controller for agitating the cells and encouraging attachment of the cells to the surface, a temperature controller for adjusting the temperature of the at least one first bioreactor based on a preselected desired temperature, a gas mixing processor for adjusting the level of gas in the medium surrounding the cells, the level of gas being based on a preselected desired value of a property of the medium, a monitoring process for sensing a value of the property of the medium, and a pump controller for moving the medium to and from the at least one first bioreactor. The property may optionally include dissolved oxygen and pH. The maturation subsystem may optionally include a media controller for monitoring and modifying the medium prior to introducing the medium into the second at least one bioreactor, and an incubator controller for managing the movement of the at least one second bioreactor, the incubator controller monitoring the properties of the medium in the at least one second bioreactor, and the incubator controller flushing / restoring the medium from / to the at least one second bioreactor. The system may optionally include a media reservoir controller, which may include a media level sensor that monitors the amount of media in the media reservoir and a pump pressure sensor that monitors a pump that moves media from the media reservoir to the media container.

[0015] The cells can be provided in any suitable manner, including, but not limited to, in frozen form. A method of the present teachings for preparing frozen cells for entry into a manufacturing line of a tissue engineering system can include, but is not limited to, 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, which the present system can accommodate. The method can include automatically controlling removal of an insulated container lid of the insulated container using a first gripper. The first gripper can be controlled by a thaw controller. The method can include automatically determining, by the thaw controller, a location of at least one vial in the vial holder, automatically positioning, by the thaw controller, a second gripper at the location, and automatically removing at least one vial from the insulated container using the second gripper. The second gripper can be controlled by the thaw 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 automatically controlling the step of returning the insulated container lid to the insulated container using the first gripper. The method can include automatically determining and automatically controlling the step of placing the removed at least one vial into a cell thawing device using the second gripper by the first device controlled by the thaw controller. The method can include automatically determining, by the thaw controller controlled by the thaw controller, when the frozen cells have thawed a preselected amount, and automatically controlling the step of removing the at least one vial of thawed cells using the second gripper by the thaw controller.The method can include automatically controlling, by the thaw controller, the transfer of at least one vial of thawed cells to a third gripper using a second gripper. The third gripper can be controlled by the thaw 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 thaw controller, the transfer of at least one vial of thawed cells to a first preselected location using an actuator operably coupled to the third gripper. The actuator can be controlled by the thaw controller. The method can include automatically controlling, by the thaw controller, the decontamination of the at least one vial by a decontamination system. The decontamination system can be controlled by the thaw controller and can decontaminate an exterior surface of the vial. The method can include automatically controlling, by the thaw controller, moving at least one vial of thawed cells to a second preselected location for puncturing using the actuator. The method can include automatically controlling, by the thaw controller, puncturing at least one vial with a puncturing system controlled by the thaw controller, and automatically controlling, by the thaw controller, pumping a solution into the at least one vial with a pump controlled by the thaw controller. The method can include automatically controlling, by the thaw controller, the steps and extracting the thawed cells from the at least one vial using advection. The method can include automatically controlling, by the thaw controller, discarding the vial. The solution can optionally include a buffer (PBS) that neutralizes the freezing agent or medium.The method may optionally include the step of identifying at least one vial that was removed.

[0016] A system of the present teachings for preparing cells for entry into a tissue processing system can include, but is not limited to, a thaw controller for controlling the preparation of cells and an insulated container station. The insulated container station can include a cooling means for cooling an 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 thaw controller, and the vial gripper can move the at least one vial under the control of the thaw controller. The system can include at least one device for locating a respective position of the at least one vial under the control of the thaw controller. The system can include a thaw station for receiving at least one identified vial from the vial gripper under the control of the thaw controller. The thaw station can include a thaw device for storing the at least one identified vial. The thaw device can thaw the cells in the at least one vial. The thawing station may include a thawing station controller under the control of the thawing controller. The thawing station controller may provide a status of the cells to the thawing controller. The system may include a decontamination station that receives at least one vial of thawed cells under the control of the thawing controller from the vial gripper. The decontamination station may include means for decontaminating an exterior surface of the at least one vial of thawed cells. The system may include a puncture station that receives at least one decontaminated vial of thawed cells from the vial gripper under the control of the thawing controller. The puncture station may include at least two needles. The at least two needles may puncture the decontaminated vial under the control of the thawing controller.A first needle of the at least two needles can include a first length and a second needle of the at least two needles can include a second length. The puncturing station can include a needle controller that punctures the at least one decontaminated vial of thawed cells with the first needle under the control of the thawing controller. The first needle can extend a first preselected distance into the at least one decontaminated vial of thawed cells. The needle controller can puncture the at least one decontaminated vial of thawed cells with a second needle under the control of the thawing controller. The second needle can extend a second preselected distance into the at least one decontaminated vial of thawed cells. The system can include a solution pump that pumps solution from a solution reservoir through the 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 can be drawn through the second needle and can flow through the cell tubing into the cell reservoir. Cells can be removed from the at least one decontaminated vial of thawed cells to generate at least one waste vial.

[0017] The first needle length can optionally include a length greater than the second needle length. The first preselected distance the needle extends into the vial can optionally include a length less than the second preselected distance the needle extends into the vial. The system can optionally include a gas purge that urges substantially all of the contents of the 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 the identified at least one vial of thawed cells, a decontamination pump that pumps the decontamination fluid into the preselected area under the control of the thaw controller, and a nozzle that directs the decontamination fluid toward the at least one identified vial of thawed cells. The puncture station can optionally include a base gripper that maintains the 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 control of the thaw controller after the needle controller removes the first and second needles. The waste system can optionally discard the 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 the at least one vial under control of the thaw controller. The present invention provides, for example, the following: (Item 1) 1. A method for enabling automated production of tissue, comprising: Receiving the cells in 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 to facilitate expansion of the cells in the at least one first bioreactor based on monitoring sensor data of a critical process parameter; automatically harvesting the cells from the microcarrier surface and quenching 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; and automatically pumping the concentrated cells into the at least one first bioreactor upon the occurrence of a preselected event; automatically pumping the concentrated cells from the at least one first bioreactor to at least one second bioreactor once the concentrated cells are suspended; automatically generating a second preselected environment based on sensor data of critical process parameters to foster maturation of the enriched cells into tissue in the at least one second bioreactor; A method comprising: (Item 2) Automatically receiving and thawing cells, comprising: accessing an insulated container, the insulated container storing at least one vial of the frozen cells, the insulated container having an insulated container lid and a vial holder; automatically controlling, by a thaw controller, removing the insulated container lid of the insulated container using a first gripper, the first gripper being controlled by the thaw controller; automatically determining, by the thawing controller, a location of the at least one vial within the vial holder; automatically controlling, by the defrost controller, the positioning of a second gripper at the location; automatically controlling, by the thaw controller, the removal of the at least one vial from the insulated container using the second gripper, the second gripper being controlled by the thaw controller; automatically controlling, by the thaw controller, returning the insulated container lid to the insulated container using the first gripper, the first gripper being controlled by the thaw controller; automatically controlling, by the thawing controller, placing the removed at least one vial into a cell thawing device using the second gripper, the second gripper being controlled by the thawing controller; automatically determining when the frozen cells have been thawed by a thawing management device controlled by the thawing controller; automatically controlling, by the thaw controller, the removal of the at least one vial of thawed cells using the second gripper, the second gripper being controlled by the thaw controller; automatically controlling, by the thawing controller, the transfer of the at least one vial of thawed cells to a third gripper using the second gripper, the second gripper being controlled by the thawing controller; automatically controlling, by the thaw controller, the movement of the at least one vial of thawed cells to a first preselected location using an actuator operably coupled to the third gripper, the actuator being controlled by the thaw controller; automatically controlling, by the thawing controller, decontamination of the exterior of the at least one vial by a decontamination system, the decontamination system being controlled by the thawing controller; automatically controlling, by the thaw controller, the movement of the at least one vial of thawed cells to a second preselected location for access using the actuator, the actuator being controlled by the thaw controller; automatically controlling, by the thaw controller, accessing the at least one vial by an access system controlled by the thaw controller, the access system including an access device, the access device being 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, the extraction of the thawed cells from the at least one vial using advection; automatically controlling, by the thawing controller, the discarding of the at least one vial; Including The method of claim 1, further comprising: (Item 3) 3. The method of claim 2, wherein the first gripper and the second gripper comprise a single device. (Item 4) 3. The method of claim 2, wherein the first gripper, the second gripper, and the third gripper comprise a single device. (Item 5) 3. The method of claim 2, further comprising automatically determining, by a first device controlled by the thawing controller, an identity of the removed at least one vial. (Item 6) 3. The method of claim 2, wherein the decontamination system includes decontaminating an exterior surface of the at least one vial. (Item 7) 3. The method of claim 2, wherein the solution comprises a neutralizing agent. (Item 8) 3. The method according to claim 2, wherein the solution comprises a medium. (Item 9) 2. The method of claim 1, wherein concentrating the expanded cells comprises centrifuging the expanded cells. (Item 10) 1. A system for enabling automated manufacturing of tissue, comprising: an expansion subsystem, the expansion subsystem configured to automatically pump cells from at least one vial to at least one first bioreactor, the expansion subsystem configured to automatically close a valve from the at least one vial to the at least one first bioreactor once the cells are transferred to the at least one first bioreactor, the expansion subsystem configured to automatically generate a first preselected environment to facilitate expansion of the cells in the at least one first bioreactor, the expansion subsystem configured to automatically pump the expanded cells from the at least one first bioreactor to a concentrator once the expanded cells reach a preselected density, and the expansion subsystem configured to generate resuspended cells from the concentrated cells; a concentration subsystem for automatically concentrating the expanded cells; a maturation subsystem, the maturation subsystem automatically pumping the resuspended cells into at least one second bioreactor upon the occurrence of a preselected event, the maturation subsystem automatically generating a second preselected environment based on monitoring sensor data of a critical process parameter to facilitate maturation of the concentrated cells in the at least one second bioreactor; A system comprising: (Item 11) 11. The system of claim 10, wherein the concentration subsystem comprises a centrifugation device. (Item 12) Item 11. The system of item 10, wherein the preselected event comprises detection of a desired concentration. (Item 13) 11. The system of claim 10, wherein the first preselected environment comprises a first growth medium, and the first growth medium is continuously automatically adjusted based on monitoring sensor data of a critical process parameter to maintain a first preselected level of a growth medium property. (Item 14) 11. The system of claim 10, wherein the second preselected environment comprises a second growth medium, and the second growth medium is continuously automatically adjusted based on monitoring of sensor data of a critical process parameter to maintain a second preselected level of a growth medium property. (Item 15) 11. The system of claim 10, further comprising a thawing subsystem that receives frozen cells in at least one vial and automatically thaws the frozen cells. (Item 16) The decompression subsystem includes: a thawing controller for controlling the preparation of the cells; an insulated container station having a cooling means for cooling an environment surrounding the frozen cells in the at least one vial, an insulated container for maintaining the frozen cells at a preselected temperature range, and an insulated container lid for retaining the environment; a lid gripper and a vial gripper, the lid gripper moving the insulated container lid under control of the thaw controller, and the vial gripper moving the at least one vial under control of the thaw controller; at least one device, wherein the at least one device locates a position of each of the at least one vial under control of the thawing controller; a thawing station, the thawing station receiving the at least one identified vial under control of the thawing controller from the vial gripper, the thawing station including a thawing device that stores the at least one identified vial, the thawing device thawing the cells in the at least one vial, the thawing station including a thawing station controller, the thawing station controller providing a status of the cells to the thawing controller under control of the thawing controller; a decontamination station that receives the at least one vial of thawed cells from the vial gripper under control of the thaw controller, the decontamination station including a means for decontaminating an exterior surface of the at least one vial of thawed cells; a puncturing station, the puncturing station receiving the at least one decontaminated vial of thawed cells from the vial gripper under control of the thawing controller, the puncturing station including at least two needles, the at least two needles puncturing the decontaminated vial under control of the thawing controller, a first needle of the at least two needles having a first length and a second needle of the at least two needles having a second length, the puncturing station including a needle controller puncturing the at least one decontaminated vial of thawed cells with the first needle under control of the thawing controller, the first needle extending a first preselected distance into the at least one decontaminated vial of thawed cells, the needle controller puncturing the at least one decontaminated vial of thawed cells with the second needle under control of a thawing management device, the second needle extending a second preselected distance into the at least one decontaminated vial of thawed cells; a solution pump, under control of the thaw controller, pumping solution from a solution reservoir, through solution tubing and the first needle, and into the at least one decontaminated vial of thawed cells, the thawed cells being drawn through the second needle, the thawed cells flowing through cell tubing and into a cell reservoir, and the cells being removed from the at least one decontaminated vial of thawed cells, generating at least one waste vial; Item 16. The system of item 15, comprising: (Item 17) Item 16. The system of 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) Item 16. The system of item 15, wherein the first length comprises a length greater than the second length. (Item 19) Item 16. The system of item 15, wherein the first preselected distance comprises a distance less than the second preselected distance. (Item 20) Item 16. The system of item 15, comprising a gas purge that forces 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 comprises: a hood, the hood retaining a decontamination fluid within a preselected area surrounding the identified at least one vial of thawed cells; a decontamination pump, the decontamination pump under control of the thaw controller, to pump the decontamination fluid into the preselected area; a nozzle, the nozzle directing the decontamination fluid toward the at least one identified vial of thawed cells; Item 16. The system of item 15, comprising: (Item 22) The puncturing station includes: a base gripper that maintains the at least one identified vial in a fixed position while the needle controller, under control of the thaw controller, removes the first needle and the second needle. Item 16. The system of item 15, comprising: (Item 23) a waste system, the waste system receiving the at least one waste vial from the vial gripper under control of the thaw controller after the needle controller removes the first needle and the second needle, the waste system disposing of the at least one waste vial in a waste receptacle. Item 16. The system of item 15, further comprising: (Item 24) a hood surrounding the lancing station, the hood maintaining a controlled clean volume surrounding the lancing station; Item 16. The system of item 15, further comprising: (Item 25) The expansion subsystem includes: an expansion controller for controlling the flow of cells from the thawing subsystem to the expansion subsystem; a bioreactor controller for monitoring and modifying the first preselected environment; Item 11. The system of item 10, comprising: (Item 26) The bioreactor controller includes: an agitation controller that agitates the cells and promotes attachment of the cells to a surface; a temperature controller that adjusts a temperature of the at least one first bioreactor based on a preselected desired temperature; and a gas mixing processor that adjusts a level of gas in a medium surrounding the cells, the level of gas being based on a preselected desired value of a property of the medium; a monitoring process for sensing a value of a characteristic of said medium; a pump controller, the pump controller moving the medium to and from the at least one first bioreactor; 26. The system of item 25, comprising: (Item 27) 27. The system of claim 26, wherein the characteristics include dissolved oxygen and pH. (Item 28) The maturation subsystem includes: a media controller, the media controller monitoring and modifying the media prior to introducing the media into the second at least one bioreactor; an incubator controller, the incubator controller managing the movement of the at least one second bioreactor, the incubator controller monitoring characteristics of the medium in the at least one second bioreactor, and the incubator controller flushing / restoring medium to the at least one second bioreactor; Item 11. The system of item 10, comprising: (Item 29) a media reservoir controller, a media level sensor for monitoring the amount of media in the media reservoir; a pump pressure sensor monitoring a pump that moves media from the media reservoir to the media container; A media reservoir controller comprising: Item 11. The system of item 10, further comprising: [Brief description of the drawings]

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

[0019] [Figure 1] FIG. 1 is a schematic block diagram of a system of the present teachings.

[0020] [Figure 2A] 2A and 2B are flow diagrams of a process orchestrated by a supervisor in the system of the present teachings. [Figure 2B] 2A and 2B are flow diagrams of a process orchestrated by a supervisor in the system of the present teachings.

[0021] [Diagram 3] FIG. 3 is a schematic block diagram of a human-machine interface under the control of a supervisor of the present teachings.

[0022] [Figure 4] FIG. 4 is a schematic block diagram of a decompression subsystem under the control of a supervisor of the present teachings.

[0023] [Figure 4A] FIG. 4A is a flow chart of the method of the present teachings.

[0024] [Figure 4B] 4B and 4C are schematic block diagrams of an exemplary time progression of the method of the present teachings. [Figure 4C] 4B and 4C are schematic block diagrams of an exemplary time progression of the method of the present teachings.

[0025] [Figure 4D] FIG. 4D is a perspective view of a system of the present teachings.

[0026] [Figure 4E] 4E and 4F are perspective views of the system of FIG. 4D illustrating vial movement of the present teachings. [Figure 4F] 4E and 4F are perspective views of the system of FIG. 4D illustrating vial movement of the present teachings.

[0027] [Figure 4G] FIG. 4G is a perspective view of a decontamination station of the present teachings.

[0028] [Figure 4H] FIG. 4H is a perspective view of a puncturing station of the present teachings with a needle ready to puncture a vial.

[0029] [Figure 4I] FIG. 4I is a perspective view of the piercing station of FIG. 4H with the barb piercing the vial.

[0030] [Figure 4J] FIG. 4J is a perspective view of a lancing station of the present teachings.

[0031] [Figure 4K] FIG. 4K is a perspective view of the solution and cell reservoir connections.

[0032] [Figure 4L] FIG. 4L is a cross-sectional view of a vial piercing hub of the present teachings.

[0033] [Diagram 5] FIG. 5 is a schematic block diagram of an expansion subsystem under the control of a supervisor of the present teachings.

[0034] [Figure 6] FIG. 6 is a schematic block diagram of a concentration subsystem under the control of a supervisor of the present teachings.

[0035] [Figure 7]FIG. 7 is a schematic block diagram of a maturation subsystem under the control of a supervisor of the present teachings.

[0036] [Figure 7A] FIG. 7A is a perspective schematic diagram of a rotation device of the present teachings.

[0037] [Figure 7B] FIG. 7B is an exploded schematic view of the rotation device of FIG. 7A.

[0038] [Figure 7C] FIG. 7C is a cross-sectional view of the rotational device of FIG. 7A coupled with a bioreactor in a first position.

[0039] [Figure 7D] FIG. 7D is a cross-sectional view of the rotational device of FIG. 7A coupled with a bioreactor in a second position.

[0040] [Figure 7E] FIG. 7E is a cross-sectional view of the rotational device of FIG. 7A coupled with a bioreactor in a third position.

[0041] [Figure 8] FIG. 8 is a schematic block diagram of a media supply subsystem under the control of a supervisor of the present teachings.

[0042] [Figure 9A] 9A and 9B are flow charts of the methods of the present teachings. [Figure 9B] 9A and 9B are flow charts of the methods of the present teachings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The system of the present teachings for manufacturing tissues can enable a flexible process for generating tissues from cells. The flexible process can be implemented by a manufacturing production line. The system can remove human components from the process after initial setup and parameter specification, thus ensuring reproducible results. The 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 sterile tubing. The tubing can enable the movement of cells and fluids between stations on the line. Frozen cells can be thawed in a first station and pumped into a bioreactor vessel that contains a nutrient fluid along with a medium and is stored in a controlled environment in a second station. The bioreactor vessel can include microcarriers onto which the cells can settle and attach or adhere as they are introduced into the bioreactor vessel. The microcarriers can be coated with collagen, proteins, or binding domains to encourage cell attachment. Alternatively, they can be chemically modified, electrically charged, or plasma treated to encourage cell attachment. After initial cell seeding, the culture on the microcarriers can be maintained in a bioreactor with continuous sensing of culture parameters such as pH, dissolved oxygen, and temperature. The system can also control automated removal of spent media and introduction of fresh media throughout the culture. Once cell density reaches a preselected level and the microcarriers, if present, are separated from the cells or the cells have sufficiently digested the microcarriers, the proteins, cells, and liquid that attach the cells to the microcarriers, or, if necessary, the surface coating of the microcarriers, can be pumped to a third station, where the cells can be concentrated.In the third station, the cells are separated from the majority of the neutralized digestion solution, cell culture media, and wash buffer, and then the cells are moved to an expansion vessel to prepare them for maturation, in addition to a preselected amount of the previous mixture. After resuspension at a desired concentration, for example, but not limited to, at a preselected cycle time, the concentrated cells can be pumped into a fourth station, which may include an incubator and a media reservoir vessel. Through pumping and tubing, the cells can be fluidly seeded onto a culture surface in the bioreactor for seeding attachment, differentiation, and growth. In some configurations, the bioreactor can include a scaffold surface, on which the cells can attach and grow. In some configurations, the bioreactor can include a horizontal planar growth surface. A first cell type can be placed on the first surface, the first surface can be covered with media from the vessel, 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 the second surface. As the cells grow, features within the bioreactor encourage cells from both surfaces to intermingle, thus achieving the goal of incubating multiple cell types together.

[0044] 1, a 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. In operation, the supervisor 2101 receives instructions from a human-machine interface (HMI) 2133. The instructions can include parameter settings, which can depend, for example, on 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 to receive status messages and provide command messages. In some configurations, the supervisor 2101 and the thawing subsystem 2147 can be enabled by, for example, but not limited to, a ROCKWLL AUTOMATION® COMPACTLOGIX® PLC, and the communication can be through, for example, but not limited to, an Ethernet® connection. The supervisor 2101 can provide commands to the decompression subsystem 2147, the expansion subsystem 2141, the concentration subsystem 2143, and the maturation subsystem 2145 through the network HMI 2103, in the case of the expansion subsystem 2141, using, for example, but not limited to, the Open Platform Unified Architecture protocol (OPC UA) and / or Ethernet® protocols.

[0045] 1, a Supervisor 2101 can manage the operation of the system 2100. The Supervisor 2101 can track the state of subsystems, user inputs, and sensor inputs, and can make predefined codes available to components of the system, which can interpret the codes and act accordingly.

[0046] Continuing with reference to FIG. 1 , the thawing subsystem 2147 can thaw the cells in the vial 2163. After the supervisor 2101 determines that the cells should be thawed, the supervisor 2101 can initiate cell seeding from the thawing subsystem 2147 to the expansion subsystem 2141. The expansion subsystem 2141 can pump the thawed cells from the vial 2163 to the bioreactor vessel 2113, and the supervisor 2101 can allow media to be pumped from the reservoir 6149 (FIG. 8) into the bioreactor vessel 2113. Once 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 the concentration subsystem 2143 where the expanded cells can be concentrated. The supervisor 2101 can optionally receive user input at various points during these processes as desired or necessary. The supervisor 2101 can communicate with the concentrator subsystem through a gateway, which in some configurations may use, but is not limited to, the Profinet protocol. The supervisor 2101 can, for example, monitor the enrichment recipe. The recipe can include a phase value that can be used to trigger a stop command to the enrichment subsystem 2143 and a message to the expansion subsystem 2141 indicating that a subsequent step in the process can proceed. The supervisor 2101 can request a status update from the concentrator subsystem 2143, which can send a status command to the supervisor 2101. Once the cells reach a 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 pump the conditioned media from the vessel 2118 into the bioreactor 2114. The media controller 2121 can regulate the media in the vessel 2118 to prepare the media and properly nourish the cells in the bioreactor 2114.In some configurations, the bioreactor 2114 can include a shallow box with opposing insides. The incubator controller 2123 can control the positioning device to position the bioreactor 2114 so that the first opposing inside faces the ground surface and the second opposing side faces the first opposing side. As the cells are pumped into the bioreactor 2114, the cells can respond to gravity and tend to adhere to the second opposing side. New media can be added to the bioreactor 2114, critical process parameters can be monitored / adjusted, controlled washing can be performed, and media exchanges to aid differentiation can be performed. The incubator controller 2123 can later seed the same or an alternative cell type into the bioreactor 2114. The bioreactor 2114 can be rotated prior to the second seeding to encourage cell adhesion onto the alternative surface. An additional step of seeding can thus be performed. The fluid lines from the media 2118 to the bioreactor 2114 reside within the hollow spindle and are able to maintain their integrity as the bioreactor 2114 is inverted.

[0047] 1, the maturation subsystem 2145 can include a media controller 2121 and an incubator controller 2123. The media controller 2121 can regulate the media, which provides nutrients to the cells in the bioreactor 2114 and can control properties such as, but not limited to, temperature, pH, and dissolved oxygen. The media, which is regulated to have desired properties, can flow through the cells and adhere to either the upper or lower surface of a shallow box in the bioreactor 2114. The media controller 2121 can be connected to the LABOWL 2114. tm The incubator controller 2123 may include, but is not limited to include, a control system such as a ROCKWLL AUTOMATION® COMPACTLOGIX® PLC.

[0048] Continuing with reference to FIG. 1, the supervisor 2101 can receive and process sensed information from the refrigerated media from the load cell 2125 and pressure sensor 2127 using an Ethernet protocol. Processing can include using data from the load cell 2125 to determine the amount of media remaining in the media reservoir area. The supervisor 2101 can alert the user to the need to replenish the media through the process HMI 2133. The supervisor 2101 can send / receive data to / from the process HMI 2133 using, for example, an Ethernet protocol. Messages initiated from the defrost subsystem 2147, the inflate subsystem 2141, the concentrate subsystem 2143, and the mature 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 the subsystems. Message acknowledgments can pass through the reverse path. The supervisor 2101 can communicate with a remote server 2135 (FIG. 3), which can manage a database 2131. Data from operational operation of the system of the present teachings can be stored, possibly remotely, by a logging processor 2129, as desired. Conventional means, such as, for example, but not limited to, ROCKWLL AUTOMATION® FACTORYTALK® Historian software, can be used to receive and catalog the data.

[0049] 1 and 2A, the 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 the supervisor 2101 (FIG. 1) detects that the cells are thawed, the system 2100 (FIG. 1) can include step 1201 (FIG. 2A) of moving the thawed cells from the thawing subsystem 2147 (FIG. 1) to the expansion subsystem 2141 (FIG. 1). When the expansion subsystem 4141 (FIG. 1) detects a desired density of the cells, the system 2100 (FIG. 1) can include step 1203 (FIG. 2A) of moving the cells from the expansion subsystem 2141 (FIG. 1) to the concentrator subsystem 2143 (FIG. 1). When the supervisor 2101 (FIG. 1) detects that a desired cell concentration has been achieved, the system 2100 (FIG. 1) can include step 1205 (FIG. 2A) of moving the cells from the concentrator subsystem 2143 (FIG. 1) to the expansion subsystem 2141 (FIG. 1), and when the cells reach a desired maturity, which may be determined by the passage of a preselected amount of time, the system 2100 (FIG. 1) can include step 1207 (FIG. 2A) of moving the matured tissue out of the maturation subsystem 2145 (FIG. 1). In some configurations, the expansion subsystem 2141 (FIG. 1) can send a message through the supervisor 2101 (FIG. 1) to the process HMI 2133 (FIG. 1), thereby initiating a process enabled by the system 2100 (FIG. 1) by prompting the user for input.The supervisor 2101 (FIG. 1) processes user input, exchanges messages with the expansion subsystem 2141 (FIG. 1), enables flow control, and can instruct the thawing subsystem 2147 (FIG. 1) to (1) check the vials 2163 (FIG. 1), (2) check the position of needles, which may be installed to puncture the vials 2163 (FIG. 1) as described elsewhere herein, (3) determine whether one of the needles has punctured the vials 2163 (FIG. 1), and (4) check whether there are any more vials 2163 (FIG. 1) to process, and then provide the status of these checks to the expansion subsystem 2141 (FIG. 1). In some configurations, the needle position can be assumed and the checks can be reduced. In some configurations, the vial 2163 (FIG. 1) can be uncapped and the contents can be extracted in a variety of ways, including but not limited to inserting an on-board needle into the uncapped mouth of the vial 2163 (FIG. 1). In response, the expansion subsystem 2141 (FIG. 1) can message the supervisor 2101 (FIG. 1). In some configurations, the supervisor 2101 (FIG. 1) can inform the thawing subsystem 2147 (FIG. 1) that flushing of the vial 2163 (FIG. 1) is in progress or that flushing is complete. In some configurations, the following protocol can be executed between the supervisor 2101 (FIG. 1) and the expansion subsystem 2141 (FIG. 1).That is, (1) the expansion subsystem 2141 (FIG. 1) can message the supervisor 2101 (FIG. 1) to initiate the thaw subsystem 2147 (FIG. 1); (2) the supervisor 2101 (FIG. 1) can message the expansion subsystem 2141 (FIG. 1) when the vial 2163 (FIG. 1) is punctured; (3) the expansion subsystem 2141 (FIG. 1) can message the supervisor 2101 (FIG. 1) to set a preselected valve state; and (4) the expansion subsystem 2141 (FIG. 1) can message the supervisor 2101 (FIG. 1) to pump a preselected amount of fluid. (5) the expansion subsystem 2141 (FIG. 1) can message the supervisor 2101 (FIG. 1) when pumping is complete, (6) the supervisor 2101 (FIG. 1) can message the expansion subsystem 2141 (FIG. 1) when gas purging is complete and if there are more vials to process, and (7) the expansion subsystem 2141 (FIG. 1) can message the supervisor 2101 (FIG. 1) to set another preselected valve state based on whether more vials remain to be processed. At this point, the supervisor 2101 (FIG. 1) can message the expansion subsystem 2141 (FIG. 1) to begin the cell expansion process. Once the expansion subsystem 2141 (FIG. 1) determines that expansion is complete, it can message the supervisor 2101 (FIG. 1), which can optionally message the process HMI 2133 (FIG. 1) to prompt the user for further instructions, if necessary. The supervisor 2101 (FIG. 1) can message the concentrator subsystem 2143 (FIG. 1) to start a recipe step, which can run the recipe step. Once the recipe step is completed, the supervisor 2101 (FIG. 1) can message the expansion subsystem 141 to resuspend the cells and return the cells to solution.At this point, the supervisor 2101 (FIG. 1) can message the maturation subsystem 2145 (FIG. 1) to seed the cells into the bioreactor 2114 (FIG. 1). Seeding can occur multiple times, as desired.

[0050] 2A and 2B, the triggers for transitioning from one step to another and when one subsystem takes over control from another, under the control of the supervisor 2101 (FIG. 1), can follow general guidelines, with possible variations within each subsystem depending on the desired outcome. For example, if cells are thawed at 1209 (FIG. 2B), the supervisor 2101 (FIG. 1) can enable step 1201 (FIG. 1) to move the thawed cells into the expansion subsystem 2141 (FIG. 1). In some configurations, the tubing connecting the thawing subsystem 2147 (FIG. 1) and the expansion subsystem 2141 (FIG. 1) can include a means for detecting bubbles in the tubing. If bubbles are detected at 1211 (FIG. 2B), the supervisor 2101 (FIG. 1) can send a message to the expansion subsystem 2141 (FIG. 1) 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 once a predetermined amount of pumping has occurred, the supervisor 2101 (FIG. 1) can message the expansion subsystem 2141 to disable the flow of cells from the thawing subsystem 2147 (FIG. 1). When the cells are in 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 the fluid valves that control the flow of fluid through the expansion subsystem 2141 (FIG. 1). The expansion subsystem 2141 (FIG. 1) can also monitor characteristics of the fluid flowing around the cells, such as, for example, but not limited to, the fluid level in the media container 2116 (FIG. 1), the fluid temperature, the fluid pH, the dissolved oxygen in the fluid, the agitation rate, and the media conductivity. These properties can be adjusted in a variety of 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, liquid can be added to or removed from a reservoir to adjust the liquid level and adjust the liquid properties. 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 cells to be transferred 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 cells to be transferred 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 to adjust the environment of the cells. Monitoring of the media in the vessel 2118 (FIG. 1) can include monitoring properties such as, for example, but not limited to, pH, dissolved oxygen, temperature, and agitation. Monitoring of the media in the tubing connecting the bioreactor and the media reservoir can include in-line monitoring of properties such as, for example, but not limited to, pH, glucose, dissolved oxygen, and air bubbles. Monitoring of the media in the incubator housing the bioreactor 2114 can include 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 bubbles in the tubing between the vessel 2118 and the bioreactor 2114 can also be monitored. The temperature of the media can be adjusted and the media can be agitated. Other actions can also be taken and / or alerts can be generated based on the monitored properties and instrument values.

[0051] 3, user inputs into the system 2100 (FIG. 1) can be provided to the process control 2133 and the network control 2103. In some configurations, inputs to process parameters and inputs to network parameters can be provided and processed in 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 inputs, provide the inputs to the supervisor 2101, and receive messages, including commands, status, and data, from the supervisor 2101. In some configurations, the expansion subsystem 2141, the enrichment 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 development process. In some configurations, a user can change recipes and set points by interacting with the HMIs of the enrichment subsystem 2143, incubator, and 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, in a database 1231 (FIG. 1). The database 2131 (FIG. 1) can store data electronically, including physical long-term storage, cloud storage, short-term storage, or any other method. The system 2100 (FIG. 1) can be controlled remotely, for example, but not limited to, through user input into a wireless device such as a cell phone or tablet, or locally, through a monitor co-located with the physical hardware of the system 2100 (FIG. 1).

[0052] 4, the thawing subsystem 2147 can receive vials of frozen cells and achieve uniform thawing of the cells in a safe and efficient manner, as described elsewhere herein. The supervisor 2101 can coordinate the activities of the thawing subsystem 2147 with the activities of other subsystems. For example, the supervisor 2101 can receive recipe information and emergency stop information from the HMI subsystem 2133 / 2103 (FIG. 3) and provide that information to the components of the thawing subsystem 2147. The safety processor 21471 can monitor the status of safety equipment associated with the thawing subsystem 2147 and provide that information to the supervisor 2101. The supervisor 2101 can message the status information to the HMI subsystem 2133 / 2103 (FIG. 3) for possible display to a user. The user can potentially initiate an emergency stop based on the safety equipment status. In some configurations, the safety processor 21471 can receive an emergency stop message from the supervisor 2101 and initiate a shutdown activity associated with the thawing subsystem 2147, such as the gantry, and thus the movement of the vial 2163. Reasons for an unintended stop can include, but are not limited to, a vial misplacement, a vial defect, a gantry malfunction, or an unknown vial barcode.

[0053] Continuing with reference to FIG. 4, the thawing subsystem 2147 can enable accessing the vial 2163, accessing an identifier associated with the vial 2163, moving the vial 2163 from one station to another, and receiving status information from the processor and controller that operates the vial 2163. The mechanical control / monitoring 2155 can enable picking a vial from a vial bank by controlling a jaw that performs the picking and placement of the vial. The identification process 2157 can access an identification associated with the vial 2163. In some configurations, the identification can include a bar code, and the identification process 2157 can include a bar code reader. The identification process 2157 can provide the vial identifier to the supervisor 2101, and the vial identification can be used to determine processing parameters associated with the vial. In some configurations, the processing parameters can be in the form of a recipe for processing of the cells in the vial. The supervisor 2101 and subsystems can use the recipe to automate the processing of the cells. A recipe can be put together, at least in part, by a user, for example, using the HMI subsystem.

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

[0055] 4A, a method 150 of the present teachings for preparing cells for engineering tissue can generally include, but is not limited to, 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 puncturing station. These processes can be initiated automatically, allowing the method 150 to be used in a manufacturing line. Specifically, the method 150 can include a step 151 of automatically removing a lid of an insulated container station with a lid gripper. All automatic operations can be controlled by a thaw controller. The method 150 can include a 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 vial position and the location where the vial was removed. The sensor can inform the thaw controller that it has located the lid gripper at a location containing a vial. The method 150 can include automatically removing 155 the vial from the insulated container using a vial gripper and replacing the insulated container lid using a lid gripper. Both grippers can be controlled by a thaw controller. The method 150 can optionally include automatically identifying 157 the vial. Identification can be enabled by, for example, but not limited to, an RFID device, a barcode reader, or other type of sensor that can read the labeled vial or query the contents of the vial. The method 150 can include automatically moving the vial to a thaw station and automatically thawing 159 the cells. The thaw station can include a thaw management device that is under the control of and communicates with the thaw controller and can electronically indicate when the cells are thawed.If the cells are not determined to be in a thawed state by the thaw management device and the thaw controller at 161, the method 150 may include continuing with thawing the cells at 159. If the cells are determined to be thawed at 161, the method 150 may include automatically moving the vial to a decontamination station and decontaminating the vial at 163. Decontamination may include, but is not limited to, automatically applying a substance, such as alcohol, to the exterior surface of the vial for a predetermined amount of time. The method 150 may include automatically moving the vial into a controlled clean volume surrounding the puncture station within an enclosure or hood once the thaw controller determines that decontamination is complete. The method 150 may include automatically puncturing the vial with two needles at 165, the needles being of different lengths, once the vial is properly positioned within the puncture station. The method 150 may include a step 167 of penetrating the vial to a first preselected amount with a first of the two needles, and a step 169 of penetrating the vial to a second preselected amount with a second of the two needles. Different needle lengths of the two needles may ensure that the tips of the needles will extend to different levels within the vial. The method 150 may include a step 171 of automatically pumping the solution into the vial through one of the needles, such as the second needle, once the thaw controller determines that the needles are properly positioned within the vial. In some configurations, the first needle may be shorter than the second needle. In some configurations, the needles may include beveled edges that may be brought together with the beveled edges facing in opposite directions. The method 150 may include a step 173 of receiving the cells into the first needle as the cells are moved toward the first needle by the introduction of the solution through the second needle into the vial. The cells are allowed to continue their journey through the tubing, which may connect the first needle to a cell reservoir, until a substantial number of cells have been expelled from the vial.Method 150 can include automatically associating 175 the identified vial with cells in the cell reservoir. Method 150 can optionally include pumping the solution for a preselected amount of time, automatically determining that the vial is free of cells, automatically moving the vial out of the hooded area, and / or automatically removing the vial using a vial gripper. Method 150 can include automatically discarding 177 the vial into a waste container using the vial gripper under control of the thaw controller.

[0056] 4B, the cell preparation process of the present teachings is described herein in terms of a time-related sequence of events. Other designs are possible, for example, multiple vials 112 can be processed at approximately the same time, or the processing of multiple vials 112 can overlap, or various steps in the process can be overlapped, if possible, or the processing timing can include time frames that vary from vial to vial, for example, based on changing processing conditions. For example, the lid gripper 103 can return the lid 107 at the same time that the vial gripper moves the vial 112 from the identification station 113 (if present) to the thawing station 115. A configuration with 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, a frozen vial 112 can be seated. The thaw controller 101 can command the vial gripper 105 to remove the vial 112 from the insulated container 109. At time t=2, the thaw controller 101 can command the lid gripper to return the lid 107 to the insulated container 109. In some configurations, the thaw controller 101 can command the vial gripper 105 to move the vial 112 past an identification station 113 (if present) and can receive vial identification information from the identification station 113 (if present). Other methods of generating a record of the identification of the vial 112 can also be used, such as, for example, but not limited to, reading the identification information from the insulated container 109 or associating the vial 112 with the cells therein later in the cell preparation process of the present teachings. At time t=3, the thaw controller 101 can command the vial gripper 105 to move the vial 112 from its previous location to the thaw station 115.In the thawing station 115, the thawing station controller 117 can signal the thawing controller 101 when the cells in the vial 112 are deemed thawed according to the process performed by the thawing station 115.

[0057] 4C, at time t=4, the thaw controller 101 can command the vial gripper 105 to move the vial 112 from its previous location to the decontamination station 123. Once the thaw controller 101 determines that the vial 112 is correctly positioned in the decontamination station 123, the thaw controller 101 can command the solution pump 119 to provide a decontamination solution through the tube 128 toward the exterior of the vial 112. In some configurations, the decontamination solution can include, for example, antimicrobial and volatile properties. The thaw controller 101 can pump a gaseous substance toward the vial 112 according to a preselected recipe or according to a sensor-associated stimulus. The gaseous substance can include air or another mixture of gases appropriate for the decontamination process. At T=5, the thaw controller 101 can command the vial gripper 105 to move the vial 112 through the flap 127 (FIG. 4D) under the hood 124 and into the puncture station 143. Once the thaw controller 101 determines that the vial 112 is properly positioned relative to the guiding actuator 207 (FIG. 4H) and the needle 145 / 148, the thaw controller 101 can command the needle controller 133 to lower the guiding actuator 207 (FIG. 4H) so that the needle 145 / 148 may puncture the vial 112. Once the needle 145 / 148 is positioned according to a preselected or sensor-based location, the thaw controller 101 can command the solution pump 141 to pump solution from the solution reservoir 139, through the tubing 129 and the needle 148, and into the vial 112. As cells in the vial 112 are displaced by the solution, the cells may travel into the needle 145, out through the tubing 131, and into the cell reservoir 137.At T=6 (FIG. 4B), the thaw controller 101 can determine, by a preselected method or by a sensor-based calculation, that sufficient cells have been displaced from the vial 112 into the cell reservoir 137 and can command the vial gripper 105 to remove the vial 112 from the puncturing station 143 and place it in the waste station 106 (FIG. 4B). The thaw controller 101 can associate the vial 112 with the cells in the cell reservoir 137, as may be dependent upon the method of identifying the vial 112 as described herein.

[0058] 4D-4F, an automated system 100 for performing method 150 (FIG. 4A) can include an insulated container 109 that can maintain the integrity of frozen cells. The insulated container 109 can include loading locations for any number of vials 112A-112E. The insulated container 109 can include any commercial cell insulated container station that can accommodate frozen cells maintained at a predetermined temperature. In some configurations, the insulated container 109 can include materials that can passively maintain the contents of the insulated container 109, e.g., cells, within about a predetermined temperature range. The predetermined temperature range can be based on the requirements of the contents and their subsequent processing. In some configurations, the insulated container 109 can include an active system for maintaining the contents within about a predetermined 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 may include a moving means for moving the vials 112A-112E, for example, but not limited to, one by one, into the thawing station 115. The moving means may be controlled by the controller 101. The thawing station 115 may include, but is not limited to, a commercially available thawing station that can accommodate the thawing requirements described herein, for example, fast thawing with high reproducibility and minimal contamination risk. The thawing station 115 may include, for example, but not limited to, AsteroBio's ThawSTAR® automated cell thawing system, which may be controlled by the controller 101 through a custom interface.

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

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

[0061] 4D-4F, the thaw station controller 117 can include electronics to sense the status of the contents of the vial 112A and, for example, inform the thaw controller 101 when it is time to move the vial 112A, for example, to the vial gripper system 147. The moving device can include, for example, by means described herein, attaching to the vial 112A and moving the vial 112A, for example, from the thaw station 115 to the vial gripper system 147, for example, along 13221. The thaw controller 101 can recognize when the vial 112A is correctly positioned, for example, in the vial gripper system 147, and can, for example, control the tightening of the grippers 149 (FIG. 4H) around the vial 112A. Once it is sensed that the gripper 149 has been tightened by a preselected amount, the thaw controller 101 can command the moving means 129 to move the vial gripper system 147 and the vial 112A into a decontamination station 123, where the exterior of the vial 112A can be decontaminated, for example. In some configurations, the decontamination means 300 (FIG. 4G) can include a sprayer 319 (FIG. 4G) that deposits material from the gas tube 125 (FIG. 4G) and / or the solution tube 128 (FIG. 4G) onto the vial 112A. The material can be pumped through the decontamination tube 323 (FIG. 4G) and through the cavity 123A (FIG. 4G) above the vial 112A. The mist generation manifold 321 (FIG. 4G) can assist in the individual control of the gas tube 125 (FIG. 4G) and the solution tube 128 (FIG. 4G) as the gas pump 121 (FIG. 4G) and the solution pump 331 (FIG. 4G) pump the gas and solution into the decontamination tube 323 (FIG. 4G). The door 123B (FIG. 4G) can optionally include a door that can retain the solution within the decontamination station 123. The door can optionally include, but is not limited to including, a flap and / or bristles. The decontamination means 300 (FIG. 4G) can be controlled and its activation time can be sequenced by the defrost controller 101.Decontamination means 300 (FIG. 4G) can decontaminate the exterior of vial 112A before entering a controlled clean volume surrounding the puncture station environment within hood 124, e.g., through flap 127. In some configurations, hood 124 can include a device that can accommodate laminar airflow, e.g., forced by positive pressure, through an air filter that can filter particles down to about 0.12-0.3 micron size. In some configurations, filtered air can exit through the working area of ​​hood 124. In some configurations, hood 124 can be configured to have a lid that can be lid down to about 10-20 microns, e.g., up to 10-20 microns. 5 = 100,000 particles / cubic meter.

[0062] 4H and 4I, once decontamination is completed, the thaw controller 101 can command the moving means 129 to move the vial gripper system 147 and the vial 112A, for example, through the flap 127 (FIG. 4F) and into the piercing station 143 (FIG. 4J) in the hood 124. The thaw controller 101 can command the moving means 129 to position the vial gripper system 147 and the vial 112A, for example, to ensure that the vial 112A is aligned with the needle 145 / 148 (FIG. 4K) in a preselected manner. The piercing station 143 (FIG. 4K) can flush the vial 112A with a solution, for example, from the solution reservoir 139 through the tube 129 and the connector 202 (FIG. 4K), while collecting the cells displaced from the vial 112A, for example, in the cell reservoir 137. In some configurations, multiple vials 112A-112E can be punctured substantially simultaneously. In some configurations, the connector 202 (FIG. 4K) can include a threaded Luer lock fitting, such as, but not limited to, a 10-32 threaded female Luer lock fitting. In some configurations, larger vials can be accommodated by the system 100 when it is desired to process a larger number of cells. The thaw controller 101 can vertically extend the guiding actuator 207, for example, based on at least the position of the vial 112A. In some configurations, the thaw controller 101 can raise 144 (FIG. 4H) the guiding actuator rod 225 when the thaw controller 101 commands the moving means 129 to position the vial gripper system 147 and the vial 112A, for example, in a preselected position relative to the needle 145 / 148 (FIG. 4K). When vial puncture is desired, the thaw controller 101 can cause the guiding actuator rod 225 to lower 142 (FIG. 4I).

[0063] 4J, the puncture station 143 can include, but is not limited to include, an extrusion bracket 205, a guide actuator 207, a T-bracket 249, a vial puncture hub 201, and a needle 145 / 148 (FIG. 4K). A first end of the needle 145 / 148 (FIG. 4K) can be fitted into a needle cavity (not shown) in the T-bracket 249. A second end of the needle 145 / 148 (FIG. 4K) can include a beveled edge. The guide actuator 207, the T-bracket 249, and the vial puncture hub 201 can be operatively coupled by a fastener that passes from the post connector 247, through the T-bracket 249, and into the cavity 245 in the vial puncture hub 201 (FIG. 4K). The guiding actuator post 225 can advance in and out of the guiding actuator 207 under pressure from a puncture cylinder (not shown), which can be operatively coupled to the extrusion 209 through the extrusion bracket 205. The extrusion 209 and the extrusion bracket 205 can enable flexible horizontal positioning of the guiding actuator 207 and thus the needle 145 / 148. The guiding actuator 207 can include, but is not limited to, a commercially available actuator, such as, for example, but not limited to, the FESTO® guiding actuator DFM-25-80-PA-KF, or any actuator that has high resistance to torque and lateral forces. In some configurations, the guiding piston rod 225A can be secured against rotation by the guide rod 225. The guide rod 225 can also help enable multiple simultaneous and / or semi-simultaneous punctures of multiple vials 112. In some configurations, the puncture cylinder (not shown) can provide a puncture force in the range of 5-7 kgf to properly puncture the vials 112. The guiding piston rod 225A can include a cylinder bore of approximately 10 mm to 20 mm and can deliver a force of approximately 70 N to 220 N at a delivery pressure in the range of approximately 4 to 7 bar. In some configurations, a minimum force of over 60 N can be produced.

[0064] 4K and 4L, the vial piercing hub 201 can include a vial piercing fitting cavity 231 / 233 that can receive the 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 in the vial 112 can force the thawed cells present in the vial 112 into the needle 145, through the vial piercing hub 201, the second vial piercing fitting 202, the cell tube 131, and finally into the cell reservoir 137. In some configurations, the needle 145 / 148 can comprise 316 stainless steel and the beveled edge of the needle 145 / 148 can be aligned in opposing directions ±10°. In some configurations, the vial piercing hole 231 / 233 can comprise dimensions, for example, but not limited to, about 0.0605+0.0015,-0.0008 inches, and the tube 129 / 131 can comprise a bore size within the range of about 0.03-0.06 inches. With respect to the needle 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. A suitable needle wall thickness can ensure that the needle 145 / 148 can pierce the cap of the vial 112 without buckling, for example, in the range of about 0.008 to 0.015 inches. In some configurations, both needles 145 / 148 can include a soldered or brazed joint diameter clearance 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] 4L, vial piercing hub 201 can include a gasket 234 that can cooperate with deformation 236 to prevent cell leakage, i.e., prevent cells being expelled from vial 112 from following a route other than through one of needles 145 / 148. Puncture of vial 112 can puncture gasket 234 and form deformation 236 along with a leak-proof seal.

[0066] 5, after the cells are thawed, if cell thawing is required, the supervisor 2101 can allow for the transfer of cell movement and control 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 valves 2109, which can allow for control of the direction in which 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 a bubble sensor 3145. The cell transfer from the vial 4163 (FIG. 4) to the bioreactor vessel 2113 can be considered complete after a preselected amount or range of time has passed. 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 transferred 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 in the bioreactor 2113. The absence of cell growth facilitators other than the microcarriers 2116, or none at all, is also possible. For example, some cells grow as cluster aggregates. The bioreactor controller 21412 can allow sequencing of gas mixing 3147, agitation 3149, and temperature control 3151 of the contents of the bioreactor 2113. Stepper motors can be used to agitate the bioreactor 2113. The bioreactor controller 21412 can control the movement of media and cells through a pump controller 3155, which can allow pumping of media from the reservoir 6149 to the bioreactor 2113 and from the bioreactor 2113 to a waste location. The bioreactor 2113 can be agitated by an agitation controller 3149, and a temperature controller 3151 can allow for regulation of the temperature of the medium.The gas mixing processor allows for control of the gases in the medium and therefore the properties of the medium. The monitoring process 3153 can monitor the properties of the medium in the bioreactor 2113. Properties of the medium that may be monitored can include, but are not limited to, temperature, dissolved oxygen, and pH. The cell density in the bioreactor 2113 can be monitored as a preselected target range for cell density can trigger the end of cell expansion in a particular bioreactor.

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

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

[0069] 7, once the enrichment cycle is complete, the supervisor 2101 can allow the cells to be moved from the concentrator subsystem 2143 to the expansion subsystem 2141 and then to the maturation subsystem 2145 for enrichment adjustment. The length of the enrichment cycle can be preselected, set by the user, cell type dependent, set in a recipe, and / or dynamically determined based on data collected during the enrichment cycle. The maturation subsystem 2145 can include, but is not limited to including, a media controller 5145 and an incubator controller 5147. In some configurations, the supervisor 2101 can allow the cells to be moved from the concentrator subsystem 2143 to the expansion subsystem 2141 for enrichment adjustment and then transferred to the maturation subsystem under the control of the incubator controller 5147. The media can be moved to the vessel 2118 under the control of the media controller 5145. The media controller 5145 can adjust the properties of the media in the vessel 2118, agitate 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 gas, which may be required, for example, to adjust pH. The pump controller 5153 and the pneumatic valve controller 5155 can allow the movement of media from the reservoir 6149 (FIG. 8) to the vessel 2118 and from the vessel 2118 to the bioreactor 2114. Within the bioreactor 2114 is a surface to which the cells may attach while they are maturing. A first type of cells can be introduced from the vessel 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 and a second cell type can be introduced. In this way, the two cell types can mature simultaneously and the geometry of the bioreactor 2114 can encourage the cells to grow together.The incubator controller 5147 can receive data from an in-line sensor 5163, for example, through a serial interface 5161, and can also receive data from a sensor 5165. Many things can be sensed regarding the maturing cells, such as temperature, pH, and glucose. The supervisor 2101 can generate an alert if the sensed data is outside of an acceptable range. In some configurations, the supervisor 2101 can enable adjustments to the environment of the bioreactor 2114 based on the sensed data.

[0070] Continuing with reference to FIG. 7, after the enriched cells are prepared for maturation, the incubator controller 5147 can exchange messages with a 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 industrial systems, such as, 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 properties of the cells and the fluid surrounding them as they mature. In some configurations, an in-line sensor 5163 can be used to monitor pH, dissolved oxygen, and glucose, possibly through a serial interface. Other sensors can monitor relative humidity, temperature, carbon dioxide enrichment, and incubator alarms.

[0071] 7A and 7B, the rotation of the bioreactor 2114 can be managed by a rotation device 1900. The rotation device 1900 can potentially accommodate bioreactors 2114 of any shape and size by modifying the dimensions of the rotation device 1900 as needed. The rotation device yoke 1909 can provide cantilevered support to the bioreactor 2114, allowing for unobstructed observation of the sample. The rotation device 1900 can allow the cell and media supply and gas vent tubing to remain intact and unentangled throughout the limited operating rotation range of the bioreactor 2114, e.g., + / - 270 degrees. The rotation device 1900 can include, but is not limited to including, a hollow spindle 1901 that can receive and protect the tubing 1919A / B / C as the bioreactor 2114 rotates. The hollow spindle 1901 can be held in place by a spindle cover 1913, which is mounted on a mounting board 1917. The worm wheel and worm screw 1907 can allow the yoke 1909 / bioreactor 2114 combination to rotate while the tubing 1919A / B / C remains within the hollow spindle 1901 and unentangled, reducing inertia concerns during rotation. The motor 1905 can allow the worm screw and worm wheel 1907 to rotate the yoke 1909. The bolt 1912 can house a gear cover for the worm screw 1907, which can cover the worm screw 1907 and protect the device and process from metal wear debris emissions. The remainder of the rotating device 1900 can be washed to be rated to, for example, IEC 60529 IP65 standards.

[0072] 7C, the bioreactor 2114 can be seeded through either tube 1919B or 1919C, depending on the orientation of the bioreactor 2114. In the view shown in FIG. 7C, the bioreactor side 2114A can receive cells and media through tube 1919B, while the pinch valve 1903 associated with tube 1919B can be forced open. The pinch valve 1903 associated with tube 1919A can be closed on tube 1919A. The cells and media can enter the bioreactor interior 1920 through tube 1919B and can be urged by gravity to adhere to the side 2114A of the bioreactor 2114. The tube 1919A can enter the bioreactor 2114, for example, through a cavity 2114C, which is centrally located on the bioreactor side 2114B. Tube 1919B can enter bioreactor 2114, for example, through cavity 2114D, located laterally on bioreactor side 2114A. Entry locations 2114C / D can be located 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] 7C-7E, in operation, after the bioreactor 2114 has been seeded, the worm screw 1907 undergoes a slight tilt, for example up to about 3°, in the yoke 1909 (and bioreactor 2114), followed by a momentary dwell to provide uniform liquid distribution and promote consistent cell seeding density and attachment to the bioreactor side 2114B. The worm gear 1907 can return the yoke 1909 to a horizontal position to allow attachment to occur, and can be re-tilted if desired. Finally, the worm screw 1907 can be rotated so that the yoke 1909 / bioreactor 2114 rests 180° from its starting position. At this point, cells can be seeded through the tube 1919A onto the side 2114A of the bioreactor 2114. 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 gases produced during seeding and maturation can be vented through tube 1919C, which can terminate in a filtered exhaust opening.

[0074] 7C-7E, to harvest tissue, the bioreactor 2114 can be arranged in 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 sanitized between uses. The incubator can house the controller for the motor and can include a network connection. The rotation device 1900 can be configured to operate in pairs, in which the bioreactor 2114 can be cantilevered on one driven side and simply supported on the second side.

[0075] 8, the supervisor 2101 can, for example, control aspects of the media reservoir 6149. In some configurations, the media reservoir 6149 can be located within an environmentally controlled area 6145 to reduce the possibility of degradation of heat sensitive adjuvants in the media.

[0076] 9A and 9B, a method 1850 of the present teachings for manufacturing tissue along a production line can include, but is not limited to, pumping cells from at least one vial to at least one first bioreactor at 1851. When the cells are transferred to the at least one first bioreactor at 1853, the method 1850 can include managing fluid flow between the at least one vial and the at least one bioreactor at 1855 and generating an environment that allows for draining, washing, digesting, and quenching processes and is conducive to cell expansion in the at least one first bioreactor at 1857. The environment can be automatically generated and automatically maintained based on sensor data of critical process parameters. Once a desired cell density is reached in the at least one first bioreactor at 1859 and other processes are completed, the method 1850 can include pumping the expanded cells to a concentrator at 1861 and concentrating the cells at 1863. When the cells are at a desired concentration at 1865, method 1850 can include pumping 1867 the concentrated cells to at least one first bioreactor, resuspending 1869 the concentrated cells, and pumping 1871 the resuspended cells to at least one second bioreactor for seeding and maturation. Method 1850 can include creating 1873 an environment in the at least one second bioreactor to foster maturation based on monitoring sensor data of critical process parameters, among other parameters. After tidal exchange, media regeneration, washing, digestion, and media replacement for differentiation, when the cells reach a desired maturity at 1875, method 1850 can include harvesting 1877 tissue resulting from the matured cells.

[0077] In some configurations, the sequence of steps performed by various components of system 2100 (FIG. 1) under the automated control of supervisor 2101 (FIG. 1) (except where human input is desired in the preparation phase (steps 7, 11, 17)) 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. In addition, although some exemplary configurations of the present disclosure are shown in the drawings and / or discussed herein, it is not intended to limit the present disclosure thereto, and the present disclosure is intended to be read as broadly as the technical field will permit, as the present specification. Therefore, the above description should not be interpreted as limiting, but merely as an illustration of a particular configuration. Also, those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto. Other elements, steps, methods, and techniques that are substantially different from those described above and / or in the appended claims are also intended to be within the scope of the present disclosure.

[0079] The drawings are presented only to demonstrate certain embodiments of the present disclosure. Also, the drawings described are illustrative only and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to a particular scale for illustrative purposes. In addition, elements shown in the drawings having the same number may be the same elements or similar elements, depending on the context.

[0080] When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. When an indefinite or definite article, such as "a", "an", or "the", is used when referring to a singular noun, this also includes the plural of that noun, unless something is specifically stated otherwise. Thus, the term "comprising" should not be interpreted 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 of only components A and B.

[0081] Moreover, the terms "first," "second," "third," and the like, whether used in the description or claims, are provided to distinguish between similar elements and do not necessarily describe a sequential or chronological order. It is to be understood that terms so used are interchangeable under appropriate circumstances (unless expressly disclosed otherwise) and that the exemplary configurations of the disclosure described herein are capable of operation in sequences and / or arrangements other than those described or illustrated herein.

Claims

1. 1. A method for enabling automated production of tissue, comprising: Receiving the cells in 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 conducive to an increase in the number of cells in the at least one first bioreactor based on monitoring sensor data; automatically harvesting the cells from the microcarrier surface and diluting the cryoprotectant; automatically pumping the increased number of cells from the at least one first bioreactor to a concentrator when the increased number of cells reaches a preselected density; automatically concentrating said increased number of cells; and automatically pumping the concentrated cells into the at least one first bioreactor upon the occurrence of a preselected event; resuspending the concentrated cells; automatically pumping the concentrated cells from the at least one first bioreactor to at least one second bioreactor once the concentrated cells are suspended; automatically generating a second preselected environment in the at least one second bioreactor based on the sensor data, the second preselected environment being conducive to maturation of the enriched cells into tissue; The method includes:

2. The method further comprises automatically receiving and thawing the cells; Automatically receiving and thawing the cells includes: a thaw controller automatically controls removing an insulated container lid of an insulated container using a first gripper, the first gripper being controlled by the thaw controller; the thawing controller automatically determining a location of the at least one vial within a vial holder; the defrost controller automatically controls positioning of a second gripper at the location; the thaw controller automatically controls the removal of the at least one vial from the insulated container using the second gripper, the second gripper being controlled by the thaw controller; and the thaw controller automatically controls the return of the insulated container lid to the insulated container using the first gripper, the first gripper being controlled by the thaw controller; and the thawing controller automatically controls placing the removed at least one vial into a cell thawing device using the second gripper, the second gripper being controlled by the thawing controller; a thawing management device controlled by the thawing controller automatically determining when the frozen cells are thawed; the thawing controller automatically controls the removal of the at least one vial of thawed cells using the second gripper, the second gripper being controlled by the thawing controller; the thawing controller automatically controls the transfer of the at least one vial of thawed cells to a third gripper using the second gripper, the second gripper being controlled by the thawing controller; the thawing controller automatically controls the movement of the at least one vial of thawed cells to a first preselected location using an actuator operably coupled to the third gripper, the actuator being controlled by the thawing controller; the thawing controller automatically controls decontamination of the exterior of the at least one vial by a decontamination system, the decontamination system being controlled by the thawing controller; the thawing controller automatically controls the movement of the at least one vial of thawed cells to a second preselected location for access using the actuator, the actuator being controlled by the thawing controller; the thaw controller automatically controlling access to the at least one vial by an access system controlled by the thaw controller, the access system including an access device, the access device being continuously energized during a pumping operation; and said thawing controller automatically controlling pumping of a solution into said at least one vial by a pump controlled by said thawing controller; the thawing controller automatically controls the extraction of the thawed cells from the at least one vial; the thawing controller automatically controls discarding of the at least one vial; The method of claim 1 , comprising:

3. The method of claim 2 , wherein the first gripper and the second gripper are a single device.

4. The method of claim 2 , wherein the first gripper, the second gripper, and the third gripper are a single device.

5. The method of claim 2, further comprising a first device controlled by the thawing controller automatically determining an identity of the at least one vial that was removed.

6. The method of claim 2 , wherein the decontamination system includes decontaminating an exterior surface of the at least one vial.

7. The method of claim 2 , wherein the solution includes a neutralizing agent.

8. The method of claim 2 , wherein the solution comprises a culture medium.

9. 10. The method of claim 1, wherein concentrating the increased number of cells comprises centrifuging the increased number of cells.

10. 1. A system for enabling automated manufacturing of tissue, comprising: 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 the at least one vial to the at least one first bioreactor when the cells are transferred to the at least one first bioreactor, the expansion subsystem automatically creating a first preselected environment conducive to an increase in the number of the 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 to a concentrator when the increased number of cells reaches a preselected density, and the expansion subsystem configured to generate resuspended cells from the concentrated cells; an enrichment subsystem for automatically enriching the increased number of cells; a maturation subsystem that automatically pumps the resuspended cells into at least one second bioreactor upon the occurrence of a preselected event, the maturation subsystem automatically generating a second preselected environment conducive to maturation of the concentrated cells in the at least one second bioreactor based on monitoring sensor data; A system comprising:

11. The system of claim 10 , wherein the concentration subsystem comprises a centrifugation device.

12. The system of claim 10 , wherein the preselected event comprises detecting that the increased number of cells has reached a desired density.

13. 11. The system of claim 10, wherein the first preselected environment comprises a first growth medium, the first growth medium being continuously automatically adjusted based on monitoring the sensor data to maintain a first preselected level of a growth medium characteristic.

14. 11. The system of claim 10, wherein the second preselected environment comprises a second growth medium, the second growth medium being continuously automatically adjusted based on monitoring the sensor data to maintain a second preselected level of a growth medium characteristic.

15. The system of claim 10, further comprising a thawing subsystem, the thawing subsystem receiving frozen cells into at least one vial and automatically thawing the frozen cells.

16. The decompression subsystem includes: a thawing controller for controlling the preparation of the cells; an insulated container station having a cooling means for cooling an environment surrounding the frozen cells in the at least one vial, an insulated container for maintaining the frozen cells at a preselected temperature range, and an insulated container lid for retaining the environment; a lid gripper and a vial gripper, the lid gripper moving the insulated container lid under control of the thaw controller, and the vial gripper moving the at least one vial under control of the thaw controller; at least one device, under control of the thawing controller, for determining a location of each of the at least one vial; a thawing station, the thawing station receiving the at least one identified vial under control of the thawing controller from the vial gripper, the thawing station including a thawing device that stores the at least one identified vial, the thawing device thawing the cells in the at least one vial, the thawing station including a thawing station controller, the thawing station controller providing a status of the cells to the thawing controller under control of the thawing controller; a decontamination station that receives the at least one vial of thawed cells from the vial gripper under control of the thaw controller, the decontamination station including means for decontaminating an exterior surface of the at least one vial of thawed cells; a puncturing station, the puncturing station receiving the at least one decontaminated vial of thawed cells from the vial gripper under control of the thawing controller, the puncturing station including at least two needles, the at least two needles puncturing the decontaminated vial under control of the thawing controller, a first needle of the at least two needles having a first length and a second needle of the at least two needles having a second length, the puncturing station including a needle controller puncturing the at least one decontaminated vial of thawed cells with the first needle under control of the thawing controller, the first needle extending a first preselected distance into the at least one decontaminated vial of thawed cells, the needle controller puncturing the at least one decontaminated vial of thawed cells with the second needle under control of a thawing management device, the second needle extending a second preselected distance into the at least one decontaminated vial of thawed cells; a solution pump, under control of the thaw controller, pumping solution from a solution reservoir, through solution tubing and the first needle, and into the at least one decontaminated vial of thawed cells, the thawed cells being drawn through the second needle, the thawed cells flowing through cell tubing and into the at least one first bioreactor, and the cells being removed from the at least one decontaminated vial of thawed cells, generating at least one waste vial; The system of claim 15 , comprising:

17. The system of claim 16, further comprising an identification station that identifies each of the at least one vial under the control of the thawing controller.

18. The system of claim 16 , wherein the first length comprises a length greater than the second length.

19. 17. The system of claim 16, wherein the first preselected distance comprises a distance less than the second preselected distance.

20. The system of claim 16, wherein the system includes a gas purge that forces substantially all of the contents of the at least one decontaminated vial out of the at least one decontaminated vial.

21. The decontamination station comprises: a hood that retains a decontamination fluid within a preselected area surrounding said identified at least one vial of thawed cells; a decontamination pump, under control of the thaw controller, for pumping the decontamination fluid into the preselected area; a nozzle for directing the decontamination fluid toward the at least one identified vial of thawed cells; The system of claim 17, comprising:

22. The puncturing station includes: a base gripper that maintains the at least one identified vial in a fixed position while the needle controller, under control of the thaw controller, removes the first needle and the second needle. The system of claim 16, comprising:

23. The system of claim 16, further comprising a waste system that receives the at least one waste vial from the vial gripper under control of the thawing controller after the needle controller removes the first needle and the second needle, and the waste system disposes of the at least one waste vial in a waste receptacle.

24. The system further comprising a hood surrounding the puncture station; The system of claim 16 , wherein the hood maintains a controlled clean volume surrounding the lancing station.

25. 16. The system of claim 15, wherein the expansion subsystem comprises a bioreactor controller that monitors and modifies the first preselected environment.

26. The bioreactor controller includes: an agitation controller for agitating the cells to promote attachment of the cells to a 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 a level of gas in a medium surrounding the cells, the level of gas being based on a preselected desired value of a property of the medium; a monitoring process for sensing a value of a characteristic of the medium; a pump controller for moving the medium to the at least one first bioreactor and for moving the medium from the at least one first bioreactor; 26. The system of claim 25, comprising:

27. 27. The system of claim 26, wherein the characteristics include dissolved oxygen and pH.

28. The maturation subsystem includes: a culture medium controller for monitoring and modifying the culture medium prior to introduction of the culture medium into the at least one second bioreactor; an incubator controller managing the movement of the at least one second bioreactor, the incubator controller monitoring a characteristic of the medium in the at least one second bioreactor, the incubator controller flushing and restoring medium from the at least one second bioreactor, and flushing and restoring medium to the at least one second bioreactor; The system of claim 10 comprising:

29. The system further comprising a medium storage controller, The medium storage controller includes: a medium level sensor for monitoring the amount of medium in the medium reservoir; a pump pressure sensor monitoring a pump that moves medium from the medium reservoir to a medium container; The system of claim 10 comprising:

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