Systems, devices, and methods for conditional execution of automated cell processing

Abstract

This disclosure relates to systems, devices, and methods for conditional execution within a cell processing system. In embodiments, this disclosure relates to a method for automatically executing a pre-programmed workflow that defines a set of cell processing steps, determining whether a condition is met based on at least one measured parameter, and, based on the determination, continuing, modifying, or stopping the workflow.

Description

Technical Field

[0001] Cross-Reference to Related Applications This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 520,313 filed on August 17, 2023 and U.S. Provisional Patent Application No. 63 / 520,312 filed on August 17, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present disclosure relates to systems, devices, and methods for conditional execution of cell processing, e.g., automated cell processing.

Background Art

[0003] Cell therapy involves collecting cells from an individual, processing the cells, and utilizing the processed cells to achieve a clinical response in the same or a different individual. Cell processing, which may include growing or culturing cells, is a complex workflow comprising multiple steps, each of which may take multiple days to complete and often requires intermittent measurement steps to assess the status of a given cell processing step. Many current manufacturing processes are performed manually, which is operationally inefficient and labor-intensive. Even platforms described as automated cell processing in closed systems generally rely on preconfigured instrumentation that limits operational flexibility and does not reliably carry out successful cell processing without operator assistance throughout the process. The lack of operational flexibility often precludes the ability to modify or continue a workflow based on measurements obtained during measurement steps. For example, some cell processing workflows intended for growing therapeutic cells can be significantly delayed or entirely aborted when measured values do not meet quality control metrics and the closed system has insufficient flexibility to correct the issue without human intervention. The problem is compounded when multiple cell processing workflows are executed in parallel with staggered timing. Accordingly, additional systems and methods for automatically executing cell processing workflows are desirable. [Overview of the Initiative]

[0004] This disclosure generally relates to systems, devices, and methods for the conditional execution of cell processes within automated cell processing systems. Generally, a method for cell processing may include automatically executing a pre-programmed workflow that defines a set of cell processing steps. Execution of at least one of the cell processing steps may include measuring at least one parameter based on a set of pre-programmed conditions for that step and the corresponding results. The method may further include determining whether the conditions have been met based on at least one measured parameter, and the workflow may continue, modify, or stop based on the determination. The set of cell processing steps may include one or more of concentration, selection, activation, proliferation, perfusion, sampling, and harvesting. In some variations, two or more cell processing steps may be executed simultaneously. The parameters may include one or more of total cell count value, target cell count value, cell concentration value, cell recovery value, cell diameter value, cell viability value, glucose value, lactate value, dissolved oxygen value, pH value, chimeric antigen receptor expression value, and transgenic T cell receptor expression value. The parameters may be measured at predetermined time intervals.

[0005] The conditions may define thresholds for parameters. If the measured parameters are determined to meet the conditions, a cell processing step may be executed. In some variations, if the measured parameters are determined to meet the conditions, the cells may be transferred from the first module to the second module. If the measured parameters are determined not to meet the conditions, a notification may be generated to the user, the workflow may be stopped, and / or at least one cell processing step may be repeated.

[0006] In some variations, a method for automated cell processing may include receiving a workflow, automatically executing the workflow, automatically measuring at least one parameter in a cartridge within a work cell, comparing at least one measured parameter to a predefined condition, and automatically continuing, modifying, or stopping the workflow based on the comparison. In some variations, if the comparison indicates that the measured parameter does not meet the predefined condition, an alert may be generated, the workflow may be stopped, and / or at least one cell processing step may be repeated. Continuing the workflow may include ending a first cell processing step and executing a second cell processing step. The cell processing steps may be selected from the group consisting of cell washing, isolation, purification, concentration, dilution, and growth. At least one parameter may be selected from the group consisting of cell count, cell diameter, cell concentration, cell viability, glucose, lactate, cell recovery, dissolved oxygen, and pH.

[0007] The workflow can be performed automatically by a robot. Each workflow and / or predefined condition can be pre-programmed into the controller. Measuring at least one parameter can be performed by an analytical instrument. In some variations, a sample of cells in a cartridge can be transferred to the analytical instrument.

[0008] In some variations, a method for automated cell processing may include: performing a first cell processing step on cells in a cartridge; measuring a first parameter based on the first cell processing step; comparing the measured first parameter with a first set of pre-programmed conditions to determine whether a first condition has been met; performing a second cell processing step on cells in the cartridge after determining that the first condition has been met; measuring a second parameter based on the second cell processing step; and comparing the measured second parameter with a second set of pre-programmed conditions to determine whether a second condition has been met. The first and second cell processing steps may include concentration, selection, activation, or proliferation. In some variations, each of the first and second cell processing steps may further include cell perfusion, cell sampling, or cell harvesting. Each of the first and second cell processing steps may be performed automatically by a robot. In some variations, the first cell processing step may be performed by a first instrument, the second cell processing step may be performed by a second instrument, and the first and second instruments may be placed side by side within the work cell. The first and second cell processing steps may each be performed at a predefined time interval.

[0009] In some variations, each of the first and second conditions may include total cell count, target cell count, cell concentration, cell recovery, cell diameter, cell viability, glucose, lactate, dissolved oxygen, pH, chimeric antigen receptor expression, or transgenic T cell receptor expression. The user can pre-program each of the first and second conditions using the controller. If either the first or second condition is not met, a user alert may be generated.

[0010] The systems described herein may include a controller for the conditional execution of an automated cell processing method, comprising at least one pre-programmed workflow defining a set of cell processing steps, and a set of pre-programmed conditions and corresponding results for use when modifying at least one of the cell processing steps. At least one of the pre-programmed conditions may be selectable by the user from a pre-programmed set. The controller may be configured to receive input from the user to pre-program one or more workflows, conditions, and results. The controller may be further configured to generate an alert to the user if at least one of the pre-programmed conditions is not met. The controller may be configured to control a robot configured to automatically execute the pre-programmed workflow. In some variations, the controller may communicate with a sterile liquid transfer device. The system may further include a cell sampling system configured to perform one or more measurements of a cell solution, and / or one or more devices configured to perform one or more cell processing steps.

[0011] This specification also describes a method for automatically growing cells. A method for automated cell growth may include delivering a cartridge to a first instrument bay of a cell processing work cell. The cartridge may contain cells and may comprise at least a first module and a second module. The work cell may comprise a robot, a sterile liquid transfer device, and a controller having at least one pre-programmed workflow defining at least a first cell processing step and a second cell processing step. The method may further include performing at least one of the first and second cell processing steps in at least one of the first and second modules. Each of the first and second cell processing steps may include measuring parameters and determining whether pre-selected conditions are met by the measured parameters. At least one of the first and second cell processing steps may include automatically transferring the cartridge between the first instrument bay and the sterile liquid transfer device by the robot according to a pre-programmed workflow. The method may further include automatically continuing, modifying, or stopping the workflow based on at least one determination.

[0012] The pre-selected conditions may include cell concentration values, which may include CD4+ concentration values ​​greater than approximately 90%. The pre-selected conditions may include cell concentration values, which may include CD8+ concentration values ​​greater than approximately 90%. The pre-selected conditions may include cell recovery values, which may include CD4+ recovery values ​​greater than approximately 30%. The pre-selected conditions may include cell recovery values, which may include CD8+ recovery values ​​greater than approximately 30%. The pre-selected conditions may include cell diameter values, which may be greater than approximately 10 micrometers. The pre-selected conditions may include cell viability values, which may be greater than approximately 70%. The pre-selected conditions may include glucose values, which may be greater than approximately 2 g / L. The pre-selected conditions may include lactate values, which may be less than approximately 2 g / L. The pre-selected conditions may include chimeric antigen receptor expression values, which may be greater than approximately 10%.

[0013] In some variations, a method for growing cells in a closed, automated work cell may include providing a first cartridge containing a first bioreactor for containing and culturing cells; automatically culturing cells in the first bioreactor; automatically transferring a sample of cells from the first bioreactor to an analytical instrument in a sampling system in an automated work cell; automatically using the analytical instrument to determine the parameters of the cells in the first bioreactor; and, when it is determined that the parameters have reached a threshold, automatically transferring the cells from the first bioreactor to a second bioreactor to grow additional cells. The second bioreactor may be located within the first cartridge. In some variations, the second bioreactor may be located within a second cartridge. Transferring a sample of cells from the first bioreactor to the analytical instrument in the sampling system may be carried out by using a sterile liquid transfer device and a robot. The analytical instrument may determine the parameters at predetermined time intervals. Culturing cells in a first bioreactor may include one or more of the following: cell enrichment, cell washing, cell incubation, cell selection, cell activation, cell transfection, and cell transfection. The method may further include retaining the cells in the first bioreactor if it is determined that the parameters have not reached a threshold.

[0014] In some variations, a method for optimizing cell growth in a closed, automated work cell may include providing a first cartridge containing a first bioreactor for containing and culturing cells; culturing cells in the first bioreactor; transferring a sample of cells from the first bioreactor to an analytical instrument for measuring parameters; and, based on the measured parameters, (i) maintaining existing conditions in the first bioreactor; (ii) performing perfusion with a medium change of 25% or more; or (iii) transferring cells from the first bioreactor to a second bioreactor.

[0015] In some variations, a method for culturing cells in a closed, automated work cell may include providing a first cartridge containing a first bioreactor for containing and culturing cells. The method may further include providing a transfection reagent to cells via a first sterile liquid transfer device for transfecting cells in the first bioreactor of the first cartridge, and providing a transduction reagent to cells via a second sterile liquid transfer device for transducing cells in the first bioreactor of the first cartridge. Providing the transfection reagent may be done at a first predetermined time interval. Providing the transduction reagent may be done at a second predetermined time interval. The method may also include culturing cells in the first bioreactor, transferring a sample of cells from the first bioreactor to a sampling system in an automated work cell, using the sampling system to determine parameters in the first bioreactor, and, when it is determined that the parameters have reached a threshold, transferring the cells from the first bioreactor to a second bioreactor to spawn additional cells. The transfer of cell samples may be performed at a third predetermined time interval.

[0016] Further embodiments, features, and advantages of the present invention will become apparent from the following detailed description and through the implementation of the present invention. [Brief explanation of the drawing]

[0017] [Figure 1A] This is a block diagram of an exemplary modified example of a cell processing system. [Figure 1B] Figure 1A is a block diagram of a cartridge that may be provided to the cell processing system. [Figure 2A] This is a rendering of an exemplary variant of a cell processing system. [Figure 2B] Figure 2A is a perspective view of the cell processing system. [Figure 2C]It is a perspective view of the cell processing system and cartridge of Fig. 2A. [Figure 2D] It is a rendering of an alternative modified example of the cell processing system. [Figure 2E] It is a rendering of still another modified example of the cell processing system. [Figure 3] It is a rendering of an alternative modified example of the cell processing system. [Figure 4A] It is a flowchart of an exemplary modified example of the conditional execution method. [Figure 4B] It is a flowchart of an alternative modified example of the conditional execution method. [Figure 5A] It is a flowchart of an alternative modified example of the conditional execution method. [Figure 5B] It is a flowchart of still another modified example of the conditional execution method. [Figure 5C] It is a flowchart of an exemplary modified example of the cell spawning method. [Figure 5D] It is a flowchart of an alternative modified example of the cell spawning method. [Figure 5E] It is a flowchart of still another modified example of the cell spawning method. DETAILED DESCRIPTION OF THE INVENTION

[0018] Disclosed herein are devices, systems, and methods for conditional execution of cell processing workflows in automated cell processing systems. In general, the systems and devices described herein can implement methods of automatically executing cell processing workflows, which can increase operational efficiency (e.g., reduce delay), reduce and / or eliminate manual intervention (e.g., labor), optimize workspace usage, and / or increase throughput of cell processing byproducts. Execution of a cell processing workflow can be conditional such that measurements can be compared against predefined conditions to determine whether the predefined conditions are satisfied, and the workflow can be continued or modified accordingly. Conditional execution of the workflow can be performed automatically, that is, no human intervention may be required at any step of the workflow. The workflow can include cell processing steps required to manufacture and / or evaluate cells. For example, cell processing steps can include concentration, incubation, selection (e.g., isolation), activation, purification, expansion, perfusion (e.g., dilution), washing, transduction, transfection, sampling, analysis, and harvesting. A workcell can perform two or more workflows at a given time, which can further increase the operational efficiency of the cell processing methods described herein. For example, two or more cartridges can be juxtaposed within a workcell such that the workcell can independently perform cell processing steps associated with the cell solution contained in each cartridge. A robot can be configured to move each cartridge to facilitate the workflows described herein. In this way, multiple workflows can be automatically performed by the workcell, which can reduce operator intervention and increase throughput of cell product manufacturing.

[0019] I. Cell Processing System The cell processing systems described herein may be configured to perform one or more cell processing steps within a work cell. The work cell may include a closed, automated environment which may be configured to maintain a sterile environment. The work cell may receive a cartridge and perform one or more cell processing steps on the cells in the cell solution contained within the cartridge. For example, a cell processing system may comprise a work cell with a number of instruments, each of which may be configured to independently perform one or more cell processing steps on cells and / or cell solutions, and a robot capable of moving cartridges within the work cell (e.g., between one or more instruments). The robot and / or instruments may be configured to operate automatically so that operator assistance is not required at any point in the workflow. For example, the robot may receive a cartridge and move it between locations within the work cell (e.g., instruments, bays, storage, feedthroughs) according to a pre-programmed workflow, each location of which may be associated with one or more cell processing steps. After performing one or more cell processing steps of the pre-programmed workflow, the work cell may be configured to transfer the cartridge out of the work cell (e.g., via the robot). Additionally or alternatively, at least a portion of the cell solution may be transferred to a second cartridge (for example, via a fluid transfer container).

[0020] The cell solutions described herein may contain cells that can be processed for subsequent use in cell therapy. The cell solutions may contain cells (e.g., allogeneic cells) in a fluid such as a culture medium (e.g., cell culture medium). The cell solutions may contain cells from the same or different donors. Cells from the same donor may be divided among one or more cartridges so that separate cell processing steps may be performed on each of the cartridges, thereby increasing the overall throughput of the cell processing system described herein. The cell solutions may be transferred to the cartridges, for example, by an operator, before the cartridges are loaded into the work cells. In some modifications, the cartridges may be empty when loaded into the work cells so that the work cells can transfer the cell solutions into the cartridges. In some modifications, cells from two or more cartridges may be combined according to a predetermined ratio that can correspond to the intended therapeutic procedure for a patient.

[0021] An exemplary cell processing system for use in automated devices, systems, and methods is shown in Figure 1A. A block diagram of a cell processing system 100 comprising a work cell 110 and a controller 120 is shown. The work cell 110 may comprise one or more of the following: an instrument 112, a robot 116 (e.g., a robotic arm), a reagent storage unit 118, a sterile liquid transfer port 132, a sterilizer source 129, a fluid source 136, a pump 138, and a sensor 151. Cartridges 114 and fluid devices 142, which may be provided outside the work cell 110 and used within the work cell 110, are illustrated by dashed lines. In some modifications, the fluid device 142 is a sterile liquid transfer device (SLTD). However, it should be understood that the fluid device 142 may be configured to transfer any fluid (including liquids), whether sterile or not. The controller 120 may include one or more of the following: a processor 122, memory 124, communication device 126, input device 128, and display 130.

[0022] The work cell 110 may include a fully or at least partially sealed housing within which one or more cell processing steps may be carried out in a fully or at least partially automated process. The cartridge 114 may be moved using a robot 116 to reduce manual labor in the cell processing steps, and the transfer of fluids in and out of the cartridge 114 may also be carried out in a fully or partially automated process, as described in detail herein. For example, one or more fluids may be stored in a fluid device 142 so that one or more fluids may be transferred to and / or removed from the cartridge 114 via the fluid device 142. In some modifications, the fluid device 114 may be moved within the system 100 by a robot 116. Thus, the work cell 110 described herein advantageously enables the transfer of fluids in an automated and metered manner for automating the production of cell therapy.

[0023] The work cell 110 can facilitate fluid transfer and / or cartridge transfer. For example, in some modifications, a robot 116 may be configured to move two or more cartridges 114 between different instruments to perform a predetermined sequence of cell processing steps (e.g., a workflow). In this way, multiple cartridges 114 can be processed in parallel, so that different steps of the cell processing workflow can be performed on different cartridges simultaneously. In another embodiment, a sterile fluid transfer port 132 may be coupled between two or more cartridges 114 to transfer cell products and / or fluids between the cartridges 114. Furthermore, the sterile fluid transfer port 132 may be coupled between any set of fluid-carrying components of the system 100 (e.g., cartridges 114, reagent storage unit 118, fluid source 136, fluid device 142, etc.). For example, a first sterile fluid transfer port may be coupled between a first cartridge and the corresponding sterile fluid transfer port of a fluid device.

[0024] In some modifications, the reagent storage unit 118 (or more reagent storage units) may be used to store reagents including, but not limited to, cell culture media, buffers, cytokines, proteins, enzymes, polynucleotides, reagents (e.g., transfection reagents, transduction reagents, microbeads), non-viral vectors, viral vectors, antibiotics, nutrients, cryoprotective substances, solvents, cytomaterials, and pharmaceutically acceptable excipients. Additionally or alternatively, waste may be stored in the reagent storage unit or in a fluid device within the reagent storage unit. In some modifications, samples being processed, extracted from one or more cartridges, may be stored in the reagent storage unit or in a fluid device within the reagent storage unit. The reagent storage unit may comprise one or more controlled temperature compartments (e.g., a freezer, a cooler, a water bath, a heating chamber, or others, e.g., approximately -80°C, approximately -20°C, approximately 4°C, approximately 25°C, approximately 30°C, approximately 37°C, and approximately 42°C). The temperatures within these compartments may be varied during the cell manufacturing process to heat or cool the reagents.

[0025] In some modifications, reagents, waste, and / or extracted samples during processing may be stored in a fluid device 142 within a reagent storage unit 118. For this purpose, the fluid device 142 may be transferred to a cartridge in a work cell, or the cartridge may be moved to the reagent storage unit 118 by a robot 116 (or manually by an operator). The reagent storage unit 118 may interface with one or more sterile fluid transfer ports on the cartridge, and reagents or substances may be transferred from the fluid device 142 in the reagent storage unit into the cartridge. Optionally, fluid may be added to or removed from the cartridge before, during, or after the addition or removal of reagents or substances. In some modifications, the equipment 112 of the work cell 110 may include a sterile fluid transfer device similarly configured to transfer fluid in and out of a cartridge in an automated manner. The sterile fluid transfer device may store reagents, for example, by a robot 116 that moves the fluid device 142 containing reagents from a feedthrough of the work cell or elsewhere to the sterile fluid transfer device. In some variations, the robot 116 moves the fluid device 142 from the reagent storage unit 118 to the sterile fluid transfer unit. The reagent storage unit 118 may have an automated door to allow the robot 116 to access the fluid device 142 stored therein.

[0026] In some variations, the sensor 151 of the work cell 110 may be an optical sensor positioned close to the side of the sterile fluid transfer equipment. The sensor 151 may be used during automated fluid transfer procedures to assist in the controlled flow of fluid from one fluid device to another fluid device or cartridge. In particular, the optical sensor may be positioned to have a field of view of the window of the fluid device in order to detect the presence or absence of fluid in the fluid conduit of the fluid device. In this way, the controller 120 can deliver a metered amount of fluid from one fluid device to an adjacent fluid device or cartridge.

[0027] As illustrated in Figure 1B, the cartridge 114 may be configured to contain (e.g., house) a cell solution. Thus, the cartridge 114 may comprise one or more of the following, as will be described in more detail herein: a bioreactor 150, a cell separation system 152, an electroporation module 160, a fluid transfer bus 162, a sensor 164, and a sterile fluid transfer port 166. The bioreactor 150 may be configured to contain a cell solution. The bioreactor 150 may further comprise a mixing chamber in which the cell solution may be mixed with one or more reagents. In some modifications, the cartridge 114 may comprise a first bioreactor and a second bioreactor. The first and second bioreactors may be fluidly isolated from each other so that the cell solution may be contained in each bioreactor without mixing. The bioreactor 150 may comprise an impeller configured to rotate and thus increase the pressure and / or flow of the cell solution contained therein.

[0028] Cartridge 114 may be configured to perform one or more cell processing steps. As shown, cartridge 114 may comprise a cell separation system 152, which may be configured to perform cell processing steps including cell separation. Thus, the cell separation system 152 may comprise one or more of the rotor 154, the flow cell 156, and the magnet 158. In some modifications, the magnet 158 ​​may comprise one or more magnets and / or magnet arrays. For example, the cell separation system 152 may comprise a first magnet configured to magnetically rotate the rotor 154 and a second magnet (e.g., a magnet array) which may each be configured to magnetically separate cells in the flow cell 156. In some modifications, cartridge 114 may have a reduced set of modules, which may facilitate faster and / or cheaper manufacturing of cartridge 114. For example, cartridge 114 may have only one or more modules corresponding to cell processing steps associated only with cell culture and / or sampling. In other words, in some modifications, the cartridge 114 may include a bioreactor 150 and a fluid transfer bus 162. In another modification, the cartridge 114 may further include an elutriation module (not shown).

[0029] Cartridge 114 may be portable to facilitate automated sterile cell processing. For example, the cartridge may be configured to be moved by a user from a location outside the work cell 110 to its feedthrough. In another embodiment, cartridge 114 may be moved by a robot from the feedthrough of the work cell 110 to any equipment within it. Generally, each piece of equipment in the work cell 110 may interface with its respective module on the cartridge. For example, the electroporation module 160 on cartridge 114 may interface with electroporation equipment to perform an electroporation step on cell products and may also interface with common components such as components of a fluid bus line (e.g., pumps, valves, sensors, etc.). In some modifications, multiple cartridges may be used to process a single cell product by transferring the cell product from one cartridge to another cartridge of the same or different type, and / or by dividing the cell product into more cartridges, and / or by pooling multiple cell products into fewer cartridges.

[0030] Various materials may be used to constitute the cartridge 114, including metal, plastic, rubber, and / or glass, or combinations thereof. The cartridge, its components, and its housing may be manufactured by molding, machining, extrusion, 3D printing, or any combination thereof. The outer housing of the cartridge may contain a material (e.g., an impermeable material) suitable for establishing an additional boundary that can further protect the sterility of the cell products.

[0031] Other suitable electroporation systems and devices are provided, for example, in U.S. Patent Application No. 63 / 453,730, which is incorporated herein by reference.

[0032] Figure 2A shows an exemplary cell processing system for use in the devices, systems, and methods described herein. A work cell 202 is shown. The work cell may be divided into an internal zone 204 having access to a feedthrough 206 and quality control (QC) equipment 212. An air filtration inlet (not shown) may provide high-efficiency particulate air (HEPA) filtration to provide ISO 7 or better air quality in the internal zone 204. In some embodiments, this air filtration may maintain sterile cell processing in an ISO 8 or ISO 9 manufacturing environment. The work cell 201 may also have an air filter at the air outlet to maintain the ISO grade of the room. Similar to the work cell described above with reference to Figure 1A, the work cell 201 may further comprise, inside the internal zone 104, a bioreactor instrument 214, a cell selection instrument 216 (e.g., a magnetic separation instrument), an electroporation instrument 220, a counterflow centrifugation elutriation (CCE) instrument 222, a sterile fluid transfer instrument 224 (e.g., to facilitate automated fluid transfer), a reagent storage unit 226, and a sterilization system 260. The reagent storage unit 226 may be accessible to the user through a sample pickup port 228. A robot 230 (e.g., a support arm, a robotic arm, etc.) may be configured to move one or more cartridges 250 from any instrument to any other instrument, move one or more cartridges 250 to and from the reagent storage unit 226, and / or move one or more fluid devices between the reagent storage unit 226 and the sterile fluid transfer instrument 224. In some variations, the work cell 201 may be equipped with one or more movable barriers 213 (e.g., access doors) configured to facilitate access to one or more of the instruments in the work cell 201. Figure 2B is a perspective view of the work cell 202 of the cell processing system. Figure 2C is a perspective view of the cell processing system showing cartridges 250 introduced into the work cell 202. Multiple cartridges may be inserted into the work cell 202 and undergo one or more cell processing steps in parallel.

[0033] Figure 2D is a schematic example of an embodiment of work cell 203. Work cell 203 may comprise an enclosure 202 having four walls, a base, and a roof. Work cell 203 may be divided into an internal zone 204 with access to a feedthrough 206, a biosafety cabinet (BSC) 208, a computing server rack 210 (e.g., controller 120), and quality control (QC) equipment 212. An air filtration inlet (not shown) may provide high-efficiency particulate air (HEPA) filtration to provide ISO 7 or better air quality in the internal zone 204. Work cell 203 may also have an air filter at the air outlet to maintain an ISO rating for the room. Similar to the work cell 202 described above with reference to Figures 2A-2C, the work cell 203 may further include, within the internal zone 204, instruments 211 (e.g., located in a general-purpose instrument bay), bioreactor instrument 214, cell selection instrument 216 (e.g., magnetic separation instrument, cell selection system), cell sorting instrument 218 (e.g., FACS), electroporation instrument 220, countercurrent centrifugation elutriation (CCE) instrument 222, and sterilization fluid transfer instrument 224, along with a reagent storage unit 226 and a sterilization system 260 comprising one or more of a sterilization agent source, a fluid source, and a pump. As described below, the sterilization system 260 may be connectable to a fluid device to sterilize the sterilization fluid transfer port during an automated fluid transfer process. The reagent storage unit 226 may be accessible through a sample pickup port 228. The robot 230 (e.g., support arm, robot arm) may be configured to move one or more cartridges 250 from any device to any other device, move one or more cartridges 250 to and from the reagent storage unit 226, and / or move one or more fluid devices between the reagent storage unit 226 and the sterile fluid transfer device 224.

[0034] In some variations, a human operator may load one or more cartridges 250 into the feedthrough 206. The cartridges 250 may be pre-sterilized, or the feedthrough 206 may sterilize the cartridges 250 using ultraviolet (UV) light or a chemical sterilizer provided as a spray or washing solution. The feedthrough 206 chamber may optionally be configured to automatically spray, wash, irradiate, or otherwise treat the cartridges (e.g., with an ethanol and / or isopropyl alcohol solution) to maintain the sterility of the internal zone 204 (e.g., ISO 7 or higher) or the biosafety cabinet 208 (e.g., ISO 5 or higher). The cartridges 250 may be passed to a biosafety cabinet 208 into which input cell products are provided and into which the cartridges 250 are loaded. The user may then return the cartridges 250 to the feedthrough 206 and initiate automated cell processing using a computer processor (e.g., controller 120) in a computer server rack 210. The robot 230 may be configured to move the cartridge 250 to multiple devices and stations in a predefined sequence using components of a work cell 203 controlled by a computer processor in a computer server rack 210.

[0035] Figure 2E shows an exemplary cell processing system for use in the devices, systems, and methods described herein. In some variations, the work cell 204 includes a robot 230 for the material handling system of the work cell 204. In some variations, a human operator may load one or more cartridges into the feedthrough 206. The work cell 204 may be divided into an internal zone with feedthrough 206 access, a control cabinet, a reagent vault system (RVS) control, just-in-time (JIT) decontamination, and an analytical instrument system (AIS). Similar to the work cells described above, the work cell 204 may further include several instruments (e.g., located in a general-purpose instrument bay), such as bioreactor instruments, cell selection instruments (e.g., magnetic separation instruments, cell selection systems), cell sorting instruments (e.g., FACS), electroporation instruments, countercurrent centrifugation elutriation (CCE) instruments, and sterile fluid transfer instruments, along with a reagent storage unit 226 and a sterilization system comprising one or more of a sterilizer source, a fluid source, and a pump. The sterilization system may be connectable to a fluid device to sterilize the sterile fluid transfer port during an automated fluid transfer process. The reagent storage unit 226 may be accessible through a sample pickup port during loading and / or unloading. The robot 230 (e.g., support arm, robot arm) may be configured to move one or more cartridges from any device to any other device, move one or more cartridges to and from the reagent storage unit 226, move one or more cartridges to and from the sterile liquid transfer device 224, and / or move one or more fluid devices between the reagent storage unit 226 and the sterile liquid transfer device 224.

[0036] Other suitable reagent storage systems are provided, for example, in U.S. Patent Application No. 63 / 470,381, which is incorporated herein by reference. Other suitable sterile liquid transfer equipment and systems are provided, for example, in U.S. Patent Application No. 63 / 524,596, which is incorporated herein by reference. Other suitable sterile liquid transfer devices are provided, for example, in U.S. Patent Application No. 63 / 456,388, which is incorporated herein by reference.

[0037] Figure 3 shows an exemplary cell processing system comprising one or more instrument bays, each of which may be configured to perform one or more cell processing steps. As shown, the work cell 300 may comprise a plurality of instrument bays 310a–310j. Each of the instrument bays 310a–310j may comprise one or more of the following: bioreactor equipment, cell selection equipment (e.g., magnetic separation equipment, cell selection system), cell sorting equipment (e.g., FACS), electroporation equipment, spinoculation equipment, and countercurrent centrifugation elutriation (CCE) equipment. Thus, each of the instrument bays 310a–310j may be referred to as a multifunctional instrument bay. In some modifications, instrument bay 310a may be referred to as the first instrument bay, instrument bay 310b as the second instrument bay, and so on. Each of the instrument bays 310a–310j may be configured to receive a cartridge (not shown). Cartridges can be loaded into each instrument bay via a robot similar to the robot described with reference to Figures 2A to 2E. The work cell 300 may further comprise a plurality of sterile liquid transfer devices 320a to 320b. Each of the sterile liquid transfer devices 320a to 320b may be configured to receive a cartridge (not shown) comprising one or more modules that engage or interact with different instruments in the instrument bay. The sterile liquid transfer devices 320a to 320b may function similarly to the description provided earlier with reference to element 224 shown in Figures 2A to 2E. In this way, cartridges can be transferred between any instrument bays 310a to 310j and any sterile liquid transfer devices 320a and 320b according to a pre-programmed workflow and one or more cell processing steps thereof.

[0038] The function of the work cell 300 can be controlled by one or more controllers. As shown, the work cell 300 may comprise controllers 330a to 330b. Each controller 330a to 330b may be coupled to the outer surface of the work cell 300 so that the user can utilize each controller 330a to 330b without entering the internal zone of the work cell 300. In this way, one or more workflows can continue without interruption, and / or the sterile environment within them can be maintained. Controller 330a may comprise a first controller and may be coupled to the outer surface of the proximal portion of the work cell 300. Controller 330b may comprise a second controller and may be coupled to the outer surface of the distal portion of the work cell 300. Controllers 330a to 330b may be positioned so that the user can minimize movement and thus increase the operational efficiency of the cell processing described herein. Controllers 330 to 330b may be used simultaneously. In this way, multiple users may engage with the work cell 300 to coordinate and / or observe one or more workflows performed therein. For example, each of the controllers 330a to 330b may be used to pre-program one or more of the cell processing steps, conditions, and workflows.

[0039] Other preferred cell processing systems and embodiments thereof are provided, for example, in U.S. Patent Application No. 17 / 198,134 and U.S. Patent Application No. 29 / 898,923, published as U.S. Patent Application Publication No. 2021 / 0283565, respectively, which are incorporated herein by reference.

[0040] A. Workcell i. Controller A work cell as described herein may include a controller configured to control one or more functions of the work cell, such as a robot configured to automatically execute a pre-programmed workflow. The controller may be configured for user use so that the user can pre-program one or more of the cell processing steps, conditions, and / or workflows. In this way, the controller may be configured for the conditional execution of at least one automated cell processing workflow. The controller may communicate with one or more instruments configured to perform one or more cell processing systems. The controller may also communicate with a cell sampling system configured to perform one or more measurements of a cell solution. The controller may also be configured to display status associated with the workflow, including one or more measurements associated with one or more cell processing steps, the location of any cartridges and / or fluid devices, the location of any robot, and environmental conditions (e.g., temperature, pressure, humidity). The controller may be further configured to generate alerts to the user, which may occur if at least one or more conditions are not met by one or more measurements. The alerts may be alarms and / or notifications on the controller's display.

[0041] Referring back to Figure 1, the cell processing system described herein may include a controller 130 (e.g., a computing device). The controller 130 may include one or more of the following: a processor 132, memory 134, communication device 136, input device 138, and display 140. The controller 130 may be configured to control (e.g., operate) any component or setting within the work cell 102. The controller 130 may include multiple devices. For example, the work cell 102 may house one or more components of the controller 130 (e.g., the processor 132, memory 134, and communication device 136), while one or more components of the controller 130 may be provided remotely to the work cell 102 (e.g., the input device 138 and display 140).

[0042] A processor as described herein (e.g., processor 132) may process data and / or other signals to control one or more components of a system. The processor may be configured to receive, process, compile, compute, store, access, read, write, and / or transmit data and / or other signals. Additionally, or alternatively, the processor may be configured to control one or more components of a device (e.g., a console, touchscreen, personal computer, laptop, tablet, or server). In some variations, the processor 132 may be configured to access or receive data and / or other signals from one or more of the work cell 102, the server, the controller 130, and storage media (e.g., memory, flash drive, memory card, or database). The processor 132 may be any suitable processing device configured to operate and / or execute a set of instructions or code, and may include one or more data processors, image processors, graphics processing units (GPUs), physical processing units, digital signal processors (DSPs), analog signal processors, mixed signal processors, machine learning processors, deep learning processors, finite state machines (FSMs), compression processors (e.g., data compression to reduce data rate and / or memory requirements), cryptography processors (e.g., for secure wireless data transfer), and / or central processing units (CPUs). The processor may be, for example, a general-purpose processor, a field programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a processor board, and / or similar. The processor may be configured to operate and / or execute application processes, and / or other modules, processes, and / or functions associated with the system.The underlying device technologies can be provided by various component types, such as metal-oxide semiconductor field-effect transistor (MOSFET) technologies like complementary metal-oxide semiconductor (CMOS), bipolar technologies like emitter-coupled logic (ECL), polymer technologies (e.g., silicone-conjugated polymers, and metal-conjugated polymer-metal structures), and analog and digital hybrid technologies.

[0043] The systems, devices, and / or methods described herein may be implemented by software (running on hardware), hardware, or a combination thereof. Hardware modules may include, for example, general-purpose processors (or microprocessors or microcontrollers), field-programmable gate arrays (FPGAs), and / or application-specific integrated circuits (ASICs). Software modules (running on hardware) may be expressed in various software languages ​​(e.g., computer code), including structured text, TypeScript, C, C++, C#, Java®, Python, Ruby, Visual Basic®, and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, files containing microcode or microinstructions, machine instructions such as those generated by a compiler, code used to generate web services, and high-level instructions executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0044] The cell processing systems and devices described herein may include a memory (e.g., memory 134) configured to store data and / or information. In some variations, the memory may include one or more of the following: random access memory (RAM), static RAM (SRAM), dynamic RAM (DRAM), memory buffer, erasable programmable read-only memory (EPROM), electrically erasable read-only memory (EEPROM), read-only memory (ROM), flash memory, volatile memory, non-volatile memory, or combinations thereof. In some variations, the memory may store instructions causing a processor to execute modules, processes, and / or functions associated with the device, such as image processing, image display, sensor data, data and / or signal transmission, data and / or signal reception, and / or communication. In some variations, computer memory products are used that have a non-temporary computer-readable medium (sometimes also called a non-temporary processor-readable medium) containing instructions or computer code for performing various computer operations. In these variations, the computer-readable medium (or processor-readable medium) is non-temporary in the sense that it does not contain transient propagating signals themselves (e.g., propagating electromagnetic waves that carry information on a transmission medium such as space or a cable). Computer code (which may also be called code or algorithms) may be designed and constructed for a specific purpose. Memory may be configured to store any received data, and / or data generated by the controller and / or work cells. In some variations, memory may be configured to store data temporarily or permanently.

[0045] In some variations, the input device 138 may, for example, include a display or be connected to a display. The input device may be any suitable device capable of receiving input from a user, such as a keyboard, buttons, or a touchscreen. The input device may include at least one switch configured to generate user input. For example, the input device may include a touch surface for the user to provide input (e.g., finger contact to the touch surface) corresponding to user input. An input device including a touch surface may be configured to detect contact and movement on the touch surface using any of a plurality of touch sensitivity techniques, including capacitive, resistive, infrared, optical imaging, dispersed signaling, acoustic pulse recognition, and surface acoustic wave techniques. In embodiments of an input device including at least one switch, the switch may have at least one of the following: a button (e.g., a hard key, a soft key), a touch surface, a keyboard, an analog stick (e.g., a joystick), a directional pad, a mouse, a rail ball, a jog dial, a step switch, a rocker switch, a pointer device (e.g., a stylus), a motion sensor, an image sensor, and a microphone. The motion sensor may receive user movement data from an optical sensor and classify the user's gestures as user input. The microphone can receive audio data and recognize the user's voice as user input.

[0046] In some variations, the cell processing system may optionally include one or more output devices in addition to a display, such as an audio device and / or a haptic device. The audio device may output any system data, alarms, and / or notifications audibly. For example, the audio device may output an audible alarm when a malfunction is detected. In some variations, the audio device may include at least one of a speaker, a piezoelectric audio device, a magnetostrictive speaker, and / or a digital speaker. In some variations, the user may communicate with other users using the audio device and communication channels. For example, the user may form an audio communication channel (e.g., a VoIP call). The haptic device may be configured to provide the user with additional sensory output (e.g., force feedback). For example, the haptic device may generate a haptic response (e.g., vibration) to confirm user input to an input device (e.g., a touch surface). As another example, haptic feedback may notify the user that user input is being invalidated by the processor.

[0047] In some variations, the controller may include a communication device (e.g., communication device 136) configured to communicate with another controller and one or more databases. The communication device may be configured to connect the controller to another system (e.g., the Internet, a remote server, a database, a work cell) via a wired or wireless connection. The system may communicate with other devices via one or more wired and / or wireless networks. In some variations, the communication device may include a radio frequency receiver, transmitter, and / or optical (e.g., infrared) receiver and transmitter configured to communicate with one or more devices and / or networks. The communication device may communicate by wire and / or wirelessly.

[0048] Image data may be output onto a display of the cell processing system (e.g., display 140). The display may include at least one of the following: light-emitting diodes (LEDs), liquid crystal displays (LCDs), electroluminescent displays (ELDs), plasma display panels (PDPs), thin-film transistors (TFTs), organic light-emitting diodes (OLEDs), electronic paper / electronic ink displays, laser displays, and / or holographic displays.

[0049] In some variations, as shown above, the GUI may be configured for designing processes and monitoring their output. For example, the GUI may be a process design homepage. The GUI may indicate that no process is selected or loaded. A create icon (e.g., "Create Process") may be available for the user to initiate the process design process. In some variations, one or more of the GUIs described herein may include a search bar.

[0050] ii.Analytical equipment The cell processing systems described herein may comprise one or more analytical instruments configured to measure one or more parameters associated with a cell solution. In some modifications, the analytical instruments may be integrated into the work cell described herein (e.g., online) and / or located outside the work cell (e.g., offline). Either online or offline analytical instruments may be configured to perform one or more measurements associated with any cell processing step described herein. Measurements obtained via the analytical instruments (e.g., measured parameters) may be communicated to a controller described herein. The controller may compare the measured parameters to predefined (e.g., pre-programmed, pre-selected) conditions so that the workflow may continue, modify, or stop based on a comparison (e.g., a decision).

[0051] The cell processing systems described herein may be configured to sample a portion of the cell solution so that analysis can be performed on the sample before, during, and / or after one or more cell processing steps may be performed on the remaining cell solution. For example, a cartridge may be transferred (e.g., from an instrument bay or feedthrough) to a sampling instrument (e.g., a sterile fluid transfer instrument), where a sample of the cell solution contained in the cartridge (e.g., in its bioreactor) may be transferred to the sampling instrument. Fluid transfer may be performed via one or more fluid paths (e.g., pipes, piping, conduits) between the cartridge and the sampling instrument. The transferred sample of the cell solution may correspond to a volume (e.g., about 0.1 L to about 1 L) sufficient to accurately quantify at least one parameter associated with the cell solution. The sampling instrument may then transfer the sample of the cell solution to a portable fluid device (e.g., an SLTD). The portable fluid device may be moved by a robot as described above to facilitate offline or online analysis. In some variations, a sampling device (e.g., a sterile liquid transfer device) and an analytical device (e.g., one or more online and offline analytical devices) can define a sampling system. In some variations, the sampling system may be configured to perform one or more measurements without sampling a portion of the cell solution. For example, the sampling system may include an analytical device configured to measure the cell solution while the cell solution is being contained in a cartridge. In such variations, the analytical device may include one or more of an optical sensor, a pressure sensor, a balance, and a temperature probe.

[0052] Online sampling and analysis of cell solutions can be performed automatically, which can facilitate continuous (e.g., 24 hours a day, 7 days a week) cell processing. For example, an SLTD containing a sample of cell solution can be moved via a robot to an internal (e.g., integrated) analytical instrument within the work cell, such as an analytical instrument system (AIS). The AIS may be configured to receive the cell solution sample from the SLTD and measure one or more parameters of the cell solution. In another modification, online analysis may be performed on the cell solution contained within a cartridge, so that the cartridge does not need to be moved between the instrument bay and a sterile liquid transfer device for sampling. In this way, cartridge movement can be reduced or eliminated, and the operational efficiency of the work cell can be increased accordingly. That is, one or more cell processing steps may be paused for a shorter period of time to allow measurements to be taken, or in some modifications, measurements may be taken while the cell processing steps are being performed, and may not be paused at all.

[0053] Offline sampling of cell solutions may be performed separately from online sampling so that the overall throughput of the cell processing system can be increased through simultaneous operation. For example, an SLTD containing a sample of cell solution may be moved through the work cell feedthrough via a robot so that a user can remove the SLTD from the feedthrough and transfer it to an external analytical instrument (e.g., a flow cytometer). In this way, the cartridge may remain in the work cell for further cell processing while the external analytical instrument measures one or more parameters. The external analytical instrument may be configured to measure the same parameters previously provided to the internal analytical instrument. In cases where the internal and external analytical instruments measure parameters associated with a sample from the same cell solution, the measured parameters may be cross-referenced for quality control purposes. In some modifications, the external analytical instrument may be configured to measure one or more parameters different from those of the internal analytical instrument, including parameters indicating genetic modification such as chimeric antigen receptor expression values ​​and transgenic T cell receptor expression values. In some modifications, both online and offline sampling may be performed substantially simultaneously for samples from the same or different cell solutions. In this way, the throughput of the work cell can be increased through the use of both online and offline sampling instruments.

[0054] The workflow can be pre-programmed to measure one or more parameters associated with one or more cell processing steps at predetermined time intervals. In some modifications, the measured parameters may be associated with cells in a cell solution, such as one or more of the following: total cell count, target cell count, cell concentration, cell recovery, cell diameter, and cell viability. In further modifications, the measured parameters may be associated with the culture medium of the cell solution, such as one or more of the following: glucose value, lactate value, dissolved oxygen value, and pH value.

[0055] The total cell count may correspond, for example, to a proliferation step. The total cell count may represent the number of substantially all cells (e.g., one or more cell types) in the cell solution. The number of cells in the cell solution may increase as cell growth (e.g., spawning) occurs. Therefore, the total cell count may be useful in determining whether some cells should be added to or removed from the cell solution before proceeding to subsequent cell processing steps.

[0056] The target cell count value may correspond, for example, to a cell selection step. The measured target cell count value may indicate the number of target cells (e.g., cells intended to bind to a reagent) in the cell solution. The number of target cells in the cell solution may change as the cell solution is processed (e.g., incubated, sorted, perfused, harvested). Therefore, the target cell count value may be useful in determining the amount of reagent to be added to the cell solution.

[0057] Cell concentration values ​​(also referred to as volumetric cell density values ​​or cell purity values) may correspond to steps such as concentration, proliferation, depletion, perfusion, and / or selection. Cell concentration values ​​may represent the number of cells of a given type (e.g., white blood cells (WBCs), CD3+ cells, CD4+ cells, CD8+ cells) in a cell solution divided by the total volume of the cell solution. The total volume of the cell solution may be predetermined (e.g., based on the maximum fluid volume of a fluid device) and / or measured by an analytical instrument. The concentration of a given type of cell may change as the cell solution is processed (e.g., sorted, perfused, harvested). Therefore, cell concentration values ​​may be useful, for example, in determining the amount of reagent to be added to the cell solution, the suitability of cells for cell harvesting, the suitability of cells for seeding, or a combination thereof.

[0058] The cell recovery value may correspond, for example, to a cell selection step. The measured cell recovery value may represent the ratio of the number of cells of a given type (e.g., T cells) at the end of the cell processing step to the number of cells of a given type at the beginning of the cell processing step. Thus, in some modifications, determining the cell recovery value may involve performing at least two measurements, at least one of which is performed at the beginning of the cell processing step and at least one of which is performed at the end of the cell processing step. Some cells may be removed and / or damaged during one or more cell processing steps. Thus, the cell recovery value may be useful, for example, in determining whether a sufficient number of cells remain after a cell processing step to proceed to a subsequent cell processing step.

[0059] The cell diameter value may correspond, for example, to an activation step. The cell diameter value may correspond to the dimensions (e.g., diameter) of a given cell. Cell dimensions, such as cell diameter, may increase as the cell grows in size in preparation for division. The measured cell diameter value may be an averaged value across multiple cells, or, in some variations, a value for a single cell. Therefore, the measured cell diameter value may be useful in determining whether a cell is metabolically active (e.g., growing and / or dividing).

[0060] The cell viability value may correspond, for example, to an activation step. The cell viability value may represent the number of cells that are suitable for subsequent cell growth. For example, the cell viability value may be calculated using the number of viable cells divided by the total number of cells (e.g., both live and dead cells). Therefore, the cell viability value may indicate the proportion of viable cells and thus be suitable for continued processing. Similar to the cell diameter value, the measured cell viability value may be useful, for example, in determining whether cells are metabolically active.

[0061] The glucose value may correspond, for example, to a perfusion step. The glucose value may represent the amount of glucose in the cell solution. Glucose can be a nutrient consumed by cells during the cell growth process. Therefore, the glucose value can be useful in determining whether additional glucose (e.g., via perfusion with fresh medium) should be added to the cell solution to provide the nutrients necessary for cell growth.

[0062] The lactate level may correspond, for example, to a perfusion step. The lactate level may represent the amount of lactate in the cell solution. Lactate can be produced (e.g., excreted) by cells during the cell growth process. Therefore, the lactate level may be useful in determining, for example, whether any lactate can be removed from the cell solution (e.g., by perfusion with fresh medium) to minimize the amount of lactate in the cell solution and maximize the amount of glucose.

[0063] The dissolved oxygen (DO) value may correspond, for example, to a perfusion step. The DO value can represent the amount of oxygen dissolved in the cell solution. Dissolved oxygen can be consumed by cells during cell growth. Therefore, the DO value may be useful, for example, in determining whether additional DO can be added to the cell solution to enable a desired cell growth rate.

[0064] pH values ​​can correspond, for example, to growth, depletion, and / or perfusion. A pH value can represent the average pH of a cell solution. The average pH of a cell solution can correspond to the growth rate of cells within it. For example, the cell growth rate may be relatively low if the average pH is substantially alkaline or substantially acidic. In exemplary variations, a stable pH of about 7 to about 8, for example, about 7.4, may correspond to an optimized cell growth rate. Therefore, pH values ​​can be useful, for example, when evaluating the expected cell growth rate in a cell solution.

[0065] Chimeric antigen receptor expression levels may correspond, for example, to transduction, electroporation, and / or harvesting steps. Chimeric antigen receptor expression may indicate the number of cells in a cell solution that can be genetically modified, for example, by knocking out T cell receptors and replacing them with chimeric antigen receptors (CARs). In some modifications, a given cell may contain two or more chimeric antigen receptors. Therefore, chimeric antigen receptor expression levels may correspond to the total number of chimeric antigen receptors. Chimeric antigen receptor expression levels may be useful, for example, in determining the number of cells suitable for use in cell therapy and / or seeding.

[0066] Transgenic T cell receptor expression levels may correspond, for example, to transduction, electroporation, and / or harvesting steps. Transgenic T cell receptor expression levels may indicate the number of cells in a cell solution that can be genetically modified, such as by knocking out T cell receptors and replacing them with transgenic T cell receptors that can be configured to target specific diseases (e.g., cancer). In some modifications, a given cell may contain two or more transgenic T cell receptors. Therefore, transgenic T cell receptor expression levels may correspond to the total number of chimeric antigen receptors. Transgenic T cell receptor expression levels may be useful, for example, in determining the quantity of cells suitable for use in cell therapy and / or seeding.

[0067] The parameters included herein may be described in relation to specific cell processing steps, but any of the parameters may be used in any of the cell processing steps, including the workflow variations described in detail below.

[0068] II. Methods for conditional execution In general, the systems and devices described herein may implement one or more methods for automatically executing cell processing workflows that can increase operational efficiency (e.g., reduce latency), reduce and / or eliminate manual intervention (e.g., labor), optimize workspace utilization, and / or increase the throughput of cell processing byproducts. The execution of a cell processing workflow may be conditional such that a measurement may be compared to a predefined condition to determine whether the condition has been met, and the workflow may be continued, stopped, or modified accordingly. Such conditional execution of a workflow may be performed automatically, i.e., no human intervention may be required at any step in the workflow. Alternatively, the conditional execution of a workflow may be modified by human intervention at various steps in the workflow, at predetermined time intervals, etc. In general, the work cells described herein may measure a first parameter associated with a cell product, compare the first parameter to a first predefined condition, and if the first predefined condition is met, the work cell may continue a predefined response according to a preprogrammed workflow. If the first predefined condition is not met by the first parameter, the work cell may continue with a different predefined response according to a preprogrammed workflow.

[0069] Predefined responses may include continuing, stopping, and / or modifying the workflow. Predefined responses may be pre-programmed by the user using the controller. Predefined responses may correspond to cell processing steps in the workflow. Cell processing steps that may be performed by the work cells described herein may include any processes required to produce and / or evaluate cells. For example, cell processing steps may include sampling, concentration, incubation, activation, growth, selection (e.g., isolation), purification, proliferation, perfusion (e.g., dilution), washing, transduction, transfection, and harvesting.

[0070] Other suitable automated cell processing systems and methods are provided, for example, in U.S. Patent Application No. 63 / 427,720, which is incorporated herein by reference.

[0071] The workflows described herein may include one or more cell processing steps associated with sampling, such that a portion of the cell solution can be transferred to an analytical instrument for analysis of at least one parameter associated with the cell solution. Sampling (e.g., via a sterile liquid transfer device) and / or analysis (e.g., via online and / or offline analytical instruments) may be performed before, during, and / or after any cell processing step described herein. Thus, the analysis may evaluate the parameter associated with a given cell processing step, which may determine whether the cell processing step in the workflow can be continued, stopped, or modified. For example, at least one measured parameter may be compared to a predefined (i.e., pre-selected, predetermined, pre-programmed) condition that may represent a threshold for a given parameter. The result of the comparison may determine the outcome. In some variations, a determination that the measured parameter meets or exceeds a condition may result in the continuation of the workflow. A determination that the measured parameter does not meet or exceeds a condition may result in the repetition of at least one cell processing step. The workflow (e.g., a sequence of one or more cell processing steps), predefined conditions, and / or results can be pre-programmed by the user using the controller.

[0072] Other suitable sampling systems and devices are provided, for example, in U.S. Patent Application No. 63 / 465,129, which is incorporated herein by reference.

[0073] The workflows described herein may include one or more cell processing steps associated with cell concentration (e.g., purification) that allow target cells to be separated from a starting material so that the target cells reach a concentration level suitable for further cell processing. The starting material (e.g., LeucoPak) may include a cell solution containing one or more types of cells (e.g., allogeneic cells) in a culture medium. The cell solution may be liquid (e.g., fresh) or solid (e.g., frozen). Concentration may be carried out by elutriation equipment such as a countercurrent centrifuge elutriator. In some modifications, the elutriation equipment may be configured to separate leukocytes from other substances (e.g., red blood cells, platelets) in the starting material. The leukocytes may then be transferred to one or more modules to carry out subsequent cell processing steps (e.g., incubation, selection). In exemplary modifications, the starting material may be loaded into a cartridge, which may then be loaded into the work cells described herein. The starting material may then be transferred to the elutriation module of the cartridge. In some variations, the elutriation module may be located outside the cartridge so that the starting material can be transferred from the cartridge to the elutriation module (e.g., elutriation equipment) via a sterile liquid transfer device.

[0074] One or more parameters may be measured before, during, and / or after cell enrichment, and may include cell concentration values. In some variations, the measured cell concentration values ​​may include leukocyte concentration values. The measured leukocyte concentration values ​​may be compared to a predefined condition. In some variations, the predefined condition may be a leukocyte concentration value of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The comparison may show that the measured cell concentration value may be less than, equal to, or greater than the predefined cell concentration value. The results of the comparison may include stopping, continuing, or modifying the cell enrichment process.

[0075] Other suitable elutriation systems and devices are provided, for example, in U.S. Patent Application No. 63 / 464,386, which is incorporated herein by reference.

[0076] The workflows described herein may include one or more cell processing steps associated with cell incubation, which may facilitate cell selection by binding one or more reagents to cells in a cell solution. In exemplary modifications, the cell solution may be contained in a mixing chamber of a cartridge so that one or more reagents can be transferred thereto. The reagents may be transferred to the mixing chamber, for example, via an SLTD and a robot. The reagents may be configured to adhere to a particular cell type, such as CD4+ and / or CD8+ cells, according to a pre-programmed workflow. In some modifications, incubation may be carried out with an activating reagent, a transfection reagent, a transduction reagent, or a combination thereof (each of which is described in further detail below).

[0077] The cell incubation process may be carried out over a predetermined duration so that the predetermined efficacy can be achieved. For example, the predetermined duration may be about 10 minutes to about 12 hours, about 15 minutes to about 2 hours, or about 30 minutes to about 1 hour, for example, about 30 minutes. The predetermined duration may correspond to the cell concentration, the volume of the cell solution, the environmental conditions of the cartridge and / or mixing chamber, and / or a combination thereof. The incubation process may be carried out when the cell solution can be maintained at a predetermined temperature that corresponds to the type of reagent added to the cell solution. For example, a reagent configured to target CD8+ cells (e.g., CD8 microbeads) may optimally bind to CD8+ cells at a predetermined temperature of about 4°C. In another embodiment, a reagent containing liquid nanoparticles (LNPs) may optimally bind to target cells at a predetermined temperature of about 37°C. Therefore, the specified temperature can be approximately 0°C to approximately 40°C, approximately 35°C to approximately 39°C (including approximately 37°C), or approximately 2°C to approximately 8°C (including approximately 4°C).

[0078] One or more parameters may be measured before, during, and / or after cell incubation, including the target cell count value. In some variations, the measured target cell count value may correspond to the number of CD4+ and / or CD8+ cells in the cell solution. The measured target cell count value may be compared to a predefined condition. In some variations, the predefined condition may be a target cell count value of approximately 500E5 to 500E9 cells, including approximately 500E6 cells, approximately 600E6 cells, approximately 700E6 cells, approximately 800E6 cells, or approximately 900E6 cells. The comparison may show that the measured target cell count value is less than, equal to, or greater than the predefined target cell count value. The results of the comparison may include stopping, continuing, or modifying the cell incubation process.

[0079] The workflows described herein may include one or more cell processing steps associated with genetic modification, which may alter the phylogenetic composition of cells in a cell solution to provide a therapeutic effect. Genetic modification may include transduction, which may include introducing a transduction reagent (e.g., a lentiviral vector and / or virus) into a cell solution. The transduction reagent may be configured to target a specific cell type. Thus, the amount (e.g., volume) of the transduction reagent may correspond to the cell concentration value in the cell solution. For example, a relatively large amount of transduction reagent may be introduced into a cell solution with a relatively high cell concentration, and a relatively small amount of transduction reagent may be introduced into a cell solution with a relatively low cell concentration. In this way, the amount of transduction reagent may be approximately proportional to the concentration of target cells in the cell solution. The transduction reagent may be used, for example, to introduce a chimeric antigen receptor (CAR) into cells in a cell solution. In another embodiment, a lentiviral vector containing Lenti-CD19 CAR(scFv-41BB-CD3ζ,CTL019) may be configured to target CD19+ cells.

[0080] In some variations, genetic modification may involve electroporation. Electroporation may be configured to introduce gene-editing molecules into cells to manipulate their gene expression and / or cellular function. Electroporation may be combined with different types of gene-editing reagents to achieve different desired results.

[0081] In some variations, electroporation may be performed on cell solution to knock out T cell receptors from cells in the cell solution; otherwise, the T cell receptors may be recognized as foreign by the patient's cells and thus rejected. For example, electroporating cells with a plasmid containing the Cas9 enzyme and a guide RNA molecule targeting the T cell receptor gene may result in the knockout of endogenous T cell receptor protein, which may be necessary in reducing off-tumor graft-versus-host disease for allogeneic cell therapy. In another embodiment, electroporation may be combined with viral transduction. Electroporation may initially knock out the T cell receptors so that transduction can subsequently introduce chimeric antigen receptors. Other reagents with similar gene-editing capabilities when combined with electroporation include plasmid DNA, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0082] Further modifications may include transfection, which involves introducing a transfection reagent (e.g., nucleic acid) by a non-viral method. Transfection may be configured to knock out certain cell types that may be associated with inducing an immune response (e.g., graft-versus-host disease) in subsequent patients. That is, transfection may reduce the likelihood that a cell therapy recipient will reject cells developed by the cell treatment described herein. Transfection may be carried out by adding another transfection reagent to a bioreactor containing a cell solution so that the cells can be modified by the transfection reagent. The transfection reagent may include LNPs. In some modifications, LNPs may be provided in combination with an activating reagent (e.g., via CRISPR).

[0083] One or more parameters may be measured before, during, and / or after genetic modification, and include one or more of the following: cell concentration values, chimeric antigen receptor expression values, and transgenic T cell receptor expression values. In some modifications, cell concentration values ​​may include CD3+ (which may include CD4+, CD8+, and / or CD19+ cells) concentration values. The measured parameter values ​​may be compared to predefined conditions. In some modifications, the predefined conditions may be CD3+ concentration values ​​of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In further modifications, the predefined conditions may be that the chimeric antigen receptor expression values ​​are about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. Further variations may include predefined conditions where the transgenic T cell receptor expression level is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Comparisons may show that the measured parameter value may be smaller than, equal to, or larger than the predefined parameter value. The results of the comparison may include stopping, continuing, or modifying the gene modification process.

[0084] The workflows described herein may include one or more cell processing steps associated with cell activation, which may increase the size of individual cells in a cell solution so that the cells can divide after reaching an appropriate size. The activation process may include adding one or more activating reagents configured to bind to cellular proteins associated with cell growth. For example, the activating reagents may include antibodies. The antibodies may be configured to bind to the major T cell receptor and one or more proteins of T cells (e.g., CD28), which may activate T cells. In some variations, the activating reagents may be magnetically bound beads. In other variations, the activating reagents may be reversibly bound reagents with low binding affinity. Low binding affinity may be useful in avoiding T cell exhaustion. For example, sustained and / or chronic exposure to an activating reagent and subsequent stimulation may result in T cell exhaustion. T cell exhaustion may be a state of dysfunction characterized by a gradual loss of effector function, decreased cytokine production, reduced proliferation, and / or an overall inability to effectively perform the normal immune response tasks of the cell. Therefore, reversible activating reagents may prevent T cell exhaustion.

[0085] One or more parameters may be measured before, during, and / or after cell activation, and include one or more of the following: cell diameter value and cell viability value. The measured parameter values ​​may be compared to predefined conditions. In some variations, the predefined conditions may be cell diameter values ​​from about 1 micrometer to about 20 micrometers, including about 5 micrometers, about 10 micrometers, or about 15 micrometers. In further variations, the predefined conditions may be cell viability values ​​of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The comparison may show that the measured parameter value may be smaller than, equal to, or larger than the predefined parameter value. The results of the comparison may include stopping, continuing, or modifying the cell activation process.

[0086] After adding one or more reagents to a cell solution, the workflow described herein may include one or more cell processing steps associated with cell washing, which may remove unbound reagents from the cell solution. For example, the cell solution may be washed so that any unbound CD4+ microbeads, CD8+ microbeads, and / or LNPs can be removed from the cell solution. In this way, the cell solution may mainly consist of cell-reagent binding pairs, which may undergo further cell processing.

[0087] The workflows described herein may include one or more cell processing steps associated with cell selection (e.g., isolation), which may separate a particular cell type from other cells. For example, cell selection may be carried out via magnetic separation using one or more reagents. Magnetic selection may be configured to select cells bound to a particular reagent (e.g., microbeads). In an exemplary modification, the magnetic selection process may select a binding pair of CD4+ cells with CD4+ microbeads. In another modification, the magnetic selection process may select a binding pair of CD8+ cells with CD8+ microbeads. The selection process may select two or more types of cells, such as both CD4+ and CD8+ cells. The selected cells may be configured to undergo further cell processing, or, in some modifications, may be removed from the work cell for use in cell therapy.

[0088] One or more parameters may be measured before, during, and / or after cell selection, and include one or more of the following: cell concentration values, cell recovery values, and target cell count values. In some variations, cell concentration values ​​may include CD4+ and / or CD8+ concentration values. In further variations, cell recovery values ​​may include CD4+ and / or CD8+ recovery values. The measured parameter values ​​may be compared to predefined conditions. In some variations, the predefined conditions may be CD4+ and / or CD8+ concentration values ​​of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In further variations, the predefined conditions may be CD4+ and / or CD8+ recovery values ​​of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. Further variations may include predefined conditions where the target cell count ranges from approximately 500E5 to 500E9 cells, including approximately 500E6 cells, 600E6 cells, 700E6 cells, 800E6 cells, or 900E6 cells. The comparison may show that the measured parameter value may be smaller than, equal to, or larger than the predefined parameter value. The results of the comparison may include stopping, continuing, or modifying the cell selection process.

[0089] The workflows described herein may include one or more cell processing steps associated with cell proliferation, which may increase the number of cells in a cell solution. Cell proliferation may be carried out in a cartridge bioreactor described herein. For example, the bioreactor may include an impeller configured to mix the cell solution contained therein. The rotation speed of the impeller may be adjusted to increase or decrease the effectiveness of the mixing. For example, increasing the rotation speed may correspond to a larger increase in the number of cells. Conversely, decreasing the rotation speed may correspond to a smaller increase in the number of cells. Cell proliferation may be carried out over a predetermined duration, which may correspond to a target cell count. For example, a relatively low target cell count may correspond to a relatively short predetermined duration (e.g., about 30 minutes to about 72 hours), and a relatively high target cell count may correspond to a relatively long predetermined duration (e.g., about 72 hours to about 14 days). In some variations, cell proliferation may be combined with one or more cell processing steps. For example, perfusion can be combined with cell proliferation so that a volume of cell solution can be exchanged while the impeller is rotating.

[0090] One or more parameters may be measured before, during, and / or after cell proliferation, and include one or more of the cell concentration values ​​and total cell count values. In some modifications, the cell concentration values ​​may include CD4+ and / or CD8+ concentration values, as described above. The measured parameter values ​​may be compared to a predefined condition. In some modifications, the predefined condition may be the CD4+ and / or CD8+ concentration values, as described above. In further modifications, the total cell count value may be approximately 500E6 cells to approximately 500E9 cells, including approximately 500E6 cells, approximately 500E7 cells, approximately 500E8 cells, approximately 20E9 cells, or approximately 500E9 cells. The comparison may show that the measured parameter value may be smaller than, equal to, or larger than the predefined parameter value. The results of the comparison may include stopping, continuing, or modifying the cell proliferation process.

[0091] The workflows described herein may include one or more cell processing steps associated with perfusion (e.g., dilution, partial depletion), which may replace a predetermined volume of culture medium in the cell solution. Perfusion may be useful in maintaining the amount of nutrients in the cell solution associated with a desired cell growth rate. For example, since cells may consume glucose during the cell growth process, low levels of glucose may reduce or inhibit cell growth. Conversely, since cells may excrete (e.g., secrete) lactate during the cell growth process, excess lactate may inhibit cell growth. Therefore, the perfusion process may ensure that the amount of glucose in the cell solution remains above a level sufficient to support desired cell growth, and / or that the amount of lactate in the cell solution remains below a level sufficient to prevent inhibition of cell growth. In some modifications, perfusion may be performed continuously, such that a certain volume of culture medium may be continuously removed and new (e.g., fresh) culture medium may be continuously added. In further modifications, perfusion may be performed at discrete time intervals, which may correspond to a predetermined workflow. For example, the time interval may correspond to measured parameters that satisfy or exceed predefined conditions. Cells in the cell solution may be retained in the bioreactor (e.g., via a filter) so that only the medium can be replaced. In some modifications, the predetermined volume of medium to be replaced (e.g., removed and replaced) may be about 10% to about 100%, including about 25%, about 50%, and about 75% of the original cell solution. The predetermined volume may be based on the cell concentration value and / or a predefined workflow. In some modifications, perfusion may be performed in the first bioreactor of the cartridge. In other modifications, at least a portion of the cell solution may be transferred to a second bioreactor so that perfusion may be performed in the second bioreactor.

[0092] One or more parameters may be measured before, during, and / or after perfusion, and include one or more of the following: lactate level, glucose level, dissolved oxygen level, and pH level. The measured parameter values ​​may be compared to predefined conditions. In some variations, the predefined conditions may be lactate levels of approximately 1 g / L to 5 g / L, including approximately 2 g / L. In further variations, the predefined conditions may be glucose levels of approximately 1 g / L to 5 g / L, including approximately 2 g / L. In even further variations, the predefined conditions may be dissolved oxygen levels of approximately 1 mg / L to 15 mg / L, approximately 3 mg / L to 13 mg / L, or approximately 6 mg / L to 11 mg / L. For example, in some variations, the dissolved oxygen level may be approximately 1 mg / L, 3 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, or 11 mg / L. Further variations may include predefined conditions where the pH values ​​range from approximately 6 to approximately 8, including approximately 7, approximately 7.3, approximately 7.4, approximately 7.5, or approximately 7.75. Comparisons may show that the measured parameter values ​​may be smaller than, equal to, or larger than the predefined parameter values. The results of the comparison may include stopping, continuing, or modifying the perfusion process.

[0093] The workflow described herein may include one or more cell processing steps associated with cell depletion, which may remove unmodified cells from a cell solution. Cell depletion may be configured to separate cells in a cell solution using one or more depletion reagents. For example, a depletion reagent (e.g., depletion microbeads) may be added to a cell solution, which may be stored in a bioreactor, so that the cells can be incubated with the depletion reagent. That is, the depletion reagent may be configured to bind to certain cells (e.g., target cells), which may mark unbound cells for subsequent removal. In an exemplary modification, the depletion reagent may bind to CD3+ cells (which may include CD4+ and CD8+ cells) so that bound CD3+ can be separated from unmodified T cells. Thus, unmodified T cells can be removed from the cell solution.

[0094] Cell depletion may be carried out at a predetermined temperature, which may correspond to a desired efficiency (e.g., the percentage of CD3+ cells successfully bound to the microbead reagent). For example, a relatively higher predetermined temperature may correspond to a higher efficiency. In some modifications, the predetermined temperature may be approximately 30°C to 40°C, including approximately 37°C. In some modifications, cell depletion may be configured to filter the cell solution via bulk depletion or microdepletion, which may refer to a depletion period. For example, in bulk depletion, a certain amount of depletion reagent may be incubated with the cell solution over a bulk depletion period, which may be approximately 2 to 4 hours. In microdepletion, a certain amount of depletion reagent may be incubated with the cell solution over a microdepletion period, which may be approximately 2 to 6 hours. Thus, in some modifications, the microdepletion period may be longer than the bulk depletion period so that a larger portion of the depletion reagent binds to the targeted cells, thereby increasing the efficiency of the depletion process.

[0095] One or more parameters may be measured before, during, and / or after cell depletion, and include one or more of the cell concentration values ​​and pH values. In some modifications, the cell concentration values ​​may include CD4+ and / or CD8+ concentration values, as described above. The measured parameter values ​​may be compared to predefined conditions. The predefined conditions for the cell concentration values ​​and / or pH values ​​may be as described above. The comparison may show that the measured parameter values ​​are less than, equal to, or greater than the predefined parameter values. The results of the comparison may include stopping, continuing, or modifying the cell depletion process.

[0096] The workflow described herein may include one or more cell processing steps associated with harvesting, which may yield cells suitable for cell therapy in one or more patients. Harvesting may include removing cells from a cell solution (e.g., separating target cells from a culture medium) and transferring the cells to one or more containers (e.g., SLTDs, cryopreservation bags). The harvested cells may be configured for a predetermined cell therapy in one or more patients. In some modifications, the harvested cells may be placed in a bioreactor in a cartridge (e.g., seeding) so that a cell spawning process described herein may be carried out to generate additional cells. In further modifications, the harvested cells may be stored (e.g., via cryopreservation) for later use in one or more of the cell growth processes and cell therapies.

[0097] One or more parameters may be measured before, during, and / or after collection, and include one or more of the following: total cell count, cell concentration, and chimeric antigen receptor expression. The total cell count, cell concentration, and chimeric antigen receptor expression may be as described previously. The measured parameter values ​​may be compared to predefined conditions. In some modifications, the predefined conditions may be CD4+ and / or CD8+ concentration values, as described previously. In further modifications, the total cell count may be approximately 500E8 to 500E9 cells, including approximately 4E9 cells. The comparison may show that the measured parameter value may be smaller than, equal to, or larger than the predefined parameter value. The results of the comparison may include stopping, continuing, or modifying the collection process.

[0098] The workflows described herein may include one or more cell processing steps associated with cryopreservation, which may preserve the cell solution for subsequent processing at a later point in time. The cell solution to be preserved may undergo one or more cell processing steps. Thus, the cell solutions described herein may undergo cryopreservation at any point in the workflow. Cryopreservation may be configured to preserve the cells in the cell solution without damaging and / or killing the cells within it. Cryopreservation may be carried out by removing any volume of cell solution from the cartridge and / or work cell and transferring the cell solution to a container configured to withstand the environmental conditions (e.g., temperature, pressure, humidity) associated with cryopreservation. In exemplary modifications, the container may comprise a bag made from plastic suitable for cryogenic temperatures (e.g., below about -190°C). Any cell solution that has previously undergone cryopreservation may be thawed and transferred back to the cartridge and / or work cell. For example, a cell solution that has undergone cell separation before cryopreservation, which may contain separated CD4+ and / or CD8+ cells, may be transferred to a bioreactor in the cartridge and then used for further cell processing.

[0099] The workflow can be configured for any cell process, including but not limited to the production of cells. In some variations, there may be multiple workflows running simultaneously within a work cell, each of which may have simultaneous or alternating timing with respect to the others. In this way, the work cell can be configured to optimize the total throughput of cell products (e.g., harvested cells) by efficiently executing each part of the multiple workflows.

[0100] Figures 4A and 4B provide flowcharts of exemplary methods for conditional execution of a workflow in a cell processing system as described herein. As shown in Figure 4A, method 401 may include receiving a workflow 410 that defines one or more cell processing steps to be performed on cells in a cartridge. The one or more cell processing steps may include one or more of concentration, selection, activation, proliferation, perfusion, sampling, and harvesting. The workflow may be pre-programmed by a user using a controller. The workflow may be associated with one or more cell processes, such as producing cells. The method may further include automatically executing the pre-programmed workflow 420. The workflow may be executed by a work cell, which may comprise one or more of a robot, instrument, feedthrough, and storage unit. At least one parameter in the cartridge may be measured automatically 430. The parameter may be measured by online and / or offline analytical instruments. The measured parameter may be communicated to a controller. At least one measured parameter may be compared to a predefined condition 440. The controller may be configured to perform the comparison. Predefined conditions can be predefined by the user using the controller. The workflow can be continued, modified, or stopped based on comparisons.450 For example, a comparison indicating that a measured parameter meets or exceeds a predefined condition may result in the workflow continuing, so that subsequent cell processing steps can be performed. A comparison indicating that a measured parameter does not meet a predefined condition may result in the workflow being modified, such as by repeating one or more cell processing steps or stopping the workflow, so that an alert may be generated by the controller.

[0101] As shown in Figure 4B, Method 402 may encompass performing two or more cell processing steps and measuring two or more parameters. As illustrated, Method 402 may include performing a first cell processing step on cells in a cartridge 460. The first cell processing step may include any of the cell processing steps described herein. For example, the first cell processing step may include concentration, which may be configured to separate target cells from other substances in the cell solution. Method 402 may further include measuring a first parameter based on the first cell processing step 462. The first parameter may be measured by online and / or offline analytical instruments. For example, the first parameter may include cell concentration values, such as leukocyte concentration values. The measured first parameter may be compared to a first set of pre-programmed conditions to determine whether a first condition has been met 464. For example, the first set of pre-programmed conditions may include a threshold for leukocyte concentration values. The comparison may be performed by a controller. If the comparison indicates that the measured parameter does not meet a threshold, the first cell processing step may be repeated. Method 402 may include performing a second cell processing step on the cells in the cartridge 470 when it is determined that the first condition has been met. The second cell processing step may include any of the cell processing steps described herein. For example, the second cell processing step may include cell activation. Cell activation may be performed by adding one or more reagents to the cell solution and mixing the one or more reagents with the cell solution for a predetermined duration. A second parameter may be measured based on the second cell processing step 472. The second parameter may be measured by online and / or offline analytical instruments. For example, the second parameter may include a cell diameter value. The measured second parameter may be compared to a second set of pre-programmed conditions to determine whether the second condition has been met 474. In some variations, a user alert may be generated if either the first or second condition is not met. The workflow can be continued, modified, or stopped based on the comparison.In this way, the workflow can be executed conditionally based on a comparison between measured parameters and pre-programmed conditions.

[0102] Conditional execution may be useful for producing cells for use in cell therapy. Accordingly, Figures 5A–5D provide flowcharts of exemplary methods for producing cells (e.g., cell spawning) via conditional execution of a workflow in a cell processing system as described herein. As shown in Figure 5A, method 501 may include delivering a cartridge to a first instrument bay of a cell processing work cell 510. The first instrument bay may comprise one or more instruments configured to perform one or more cell processing steps. The cartridge may be delivered to the first instrument bay via a robot. The cartridge may comprise at least a first module and a second module, and may contain cells in one or more of the first and second modules. The work cell may comprise a robot, a sterile fluid transfer device, and a controller comprising at least one pre-programmed workflow defining at least a first cell processing step and a second cell processing step. The method may further include performing at least one of the first and second cell processing steps in at least one of the first and second modules 512. The first and second cell processing steps can be independently selected from the group consisting of concentration, selection, activation, proliferation, perfusion, sampling, and harvesting.

[0103] Each of the first and second cell processing steps may include measuring a parameter and determining whether a pre-selected condition is met based on the measured parameter. The measured parameter and associated pre-selected condition may include one or more of the following: total cell count, target cell count, cell concentration, cell recovery, cell diameter, cell viability, glucose, lactate, and chimeric antigen receptor expression. For example, the pre-selected condition may include the total cell count, and the total cell count may be greater than the pre-selected value. The pre-selected condition may include the target cell count, and the target cell count may be greater than the pre-selected value. The pre-selected condition may include the cell concentration, and the cell concentration may be a leukocyte concentration of over 80%. The pre-selected condition may include the cell concentration, and the cell concentration may be a CD4+ concentration of over 90%. The pre-selected condition may include the cell concentration, and the cell concentration may be a CD8+ concentration of over 90%. The pre-selected conditions may include cell recovery values, which may include more than approximately 30% CD4+ recovery. The pre-selected conditions may include cell recovery values, which may include more than approximately 30% CD8+ recovery. The pre-selected conditions may include cell diameter values, which may be greater than approximately 10 micrometers. The pre-selected conditions may include cell viability values, which may be more than approximately 70%. The pre-selected conditions may include glucose values, which may be greater than approximately 2 g / L. The pre-selected conditions may include lactate values, which may be less than approximately 2 g / L. The pre-selected conditions may include chimeric antigen receptor expression values, which may be greater than approximately 10%.

[0104] At least one of the first and second cell processing steps may include, by means of a robot, automatically transferring a cartridge between a first instrument bay and a sterile fluid transfer device according to a pre-programmed workflow. The sterile fluid transfer device may be configured to transfer a sample of the cell solution to a fluid device, such as a sterile fluid transfer device. The fluid device may be transferred via the robot to at least one analytical instrument. In some variations, the measured parameters may be measured by the analytical instrument in the first instrument bay. The method may further include automatically continuing, modifying, or stopping the workflow based on at least one determination.514 For example, the first cell processing step may be repeated if it is determined that the measured parameters do not meet pre-selected conditions.

[0105] As shown in Figure 5B, Method 502 may relate to transferring cells between bioreactors for growing additional cells. As illustrated, Method 502 may relate to providing a first cartridge 520 which may include a first bioreactor for containing and culturing cells. The cells may include allogeneic cells. Method 502 may further relate to automatically culturing cells in the first bioreactor 522. Culturing cells in the first bioreactor may relate to one or more of the following: cell enrichment, cell washing, cell incubation, cell selection, cell activation, cell transduction, and cell transfection. A sample of cells from the first bioreactor may be automatically transferred to an analytical instrument in a sampling system in an automated work cell 524. Transferring a cell sample from the first bioreactor to an analytical instrument in a sampling system may be carried out by using a sterile liquid transfer device and a robot. Method 502 may also relate to automatically using an analytical instrument to determine parameters in the first bioreactor 526. If it is determined that the parameters have reached a threshold, method 502 may further include automatically transferring the cells from the first bioreactor to a second bioreactor 528 in order to grow additional cells. The second bioreactor may be located within the first cartridge. In some modifications, the second bioreactor may be located within a second cartridge. If it is determined that the parameters have not reached a threshold, the cells may be retained within the first bioreactor.

[0106] Transferring cell samples from the first bioreactor to the analytical instruments of the sampling system may be carried out by using a sterile liquid transfer device and a robot. The sampling system may determine parameters at predetermined time intervals. Culturing cells in the first bioreactor may include one or more of the following: cell concentration, cell washing, cell incubation, cell selection, cell activation, cell transduction, and cell transfection. The method may further include retaining the cells in the first bioreactor if it is determined that the parameters have not reached a threshold.

[0107] As shown in Figure 5C, Method 503 may target optimizing cell growth by maintaining or adjusting conditions within a bioreactor. As illustrated, Method 503 may include providing a first cartridge containing a first bioreactor for containing and culturing cells 530. The cells may include allogeneic cells. The cells may be transferred to the first bioreactor before the first cartridge is supplied to the work cells. In some modifications, an empty cartridge may be supplied to the work cells, and then the cells are transferred to the cartridge, for example, via a robot. Method 503 may further include culturing cells in the first bioreactor 532. Culturing cells may include one or more of the following: cell concentration, cell washing, cell incubation, cell selection, cell activation, cell transfection, and cell transfection. Culturing cells may be carried out according to a pre-programmed workflow, which may be based on user input via a controller. Method 503 may then include transferring a sample of cells from the first bioreactor to an analytical instrument for measuring parameters 534. For example, the cartridge may be transferred to a sterile liquid transfer device within the work cell, from which a sample of cells may be removed by the sterile liquid transfer device and transferred to the sterile liquid transfer device. The sterile liquid transfer device may be transferred to an analytical instrument, for example, via a robot. The analytical instrument may be inside or outside the work cell. Based on the measured parameters, method 503 may include one or more optional steps. As illustrated, method 503 may include maintaining existing conditions in the first bioreactor 536. For example, conditions may be maintained if it is determined that the measured parameters do not meet predefined conditions, such as cell concentration values. Alternatively, method 503 may include performing a perfusion with a medium exchange of 25% or more. For example, perfusion may be performed if it is determined that the measured parameters meet or exceed predefined conditions. The amount of medium perfused may be proportional to the difference between the measured parameters and the predefined conditions. In yet another alternative example, method 503 may include transferring cells from the first bioreactor to the second bioreactor.For example, the measured parameters may indicate that the cells can be used to seed additional cellular processes.

[0108] As shown in Figure 5D, Method 504 may include providing transfection reagents and transduction reagents to cells. As illustrated, Method 504 may include providing a first cartridge containing a first bioreactor for containing and culturing cells, as described with reference to Figure 5C 550. Method 504 may further include providing transfection reagents to cells via a first sterile liquid transfer device 552 for transfecting the cells in the first bioreactor of the first cartridge. The transfection reagents may be provided at first predetermined time intervals. For example, the cartridge may be transferred to a sterile liquid transfer device. The first sterile liquid transfer device may be removed from the reagent storage unit by a robot and transferred to the sterile liquid transfer device so that the contents of the first sterile liquid transfer device may be transferred to the cartridge. Once the first sterile liquid transfer device can be emptied, the first sterile liquid transfer device may be transferred back to the reagent storage unit, or in some modifications, it may be transferred to a work cell waste unit for subsequent removal. Additionally, method 504 may include providing a transduction reagent to cells via a second sterile fluid transfer device for transduction of cells in a first bioreactor of a first cartridge 554. The transduction reagent may be provided at a second predetermined time interval. The second sterile fluid transfer device may be transferred in a manner similar to that of the first sterile fluid transfer device.

[0109] Method 504 may also include culturing cells in a first bioreactor 556. Culturing cells may include one or more of cell enrichment, cell washing, cell incubation, cell selection, and cell activation. Method 504 may then include transferring a sample of cells from the first bioreactor to an automated sampling system in a work cell 558. Transferring a sample of cells may be done at a third predetermined time interval. For example, the sampling system may comprise a sterile liquid transfer device and an analytical instrument. Thus, the sampling system may be used to determine parameters in the first bioreactor 560. Including its analytical instrument, the sampling system may be configured to measure one or more parameters associated with the cell solution. Once it is determined that the parameters have reached a threshold, Method 504 may include transferring cells from the first bioreactor to a second bioreactor 562 to grow additional cells. The transfer from the first bioreactor to the second bioreactor may be referred to as seeding.

[0110] As shown in Figure 5E, Method 505 may include a plurality of cell processes and comparing a plurality of measured parameters to each of a plurality of conditions according to a pre-programmed workflow. As illustrated, Method 505 may include providing a first cartridge containing a first bioreactor for containing and culturing cells 570. Method 505 may further include carrying out a first cell process and measuring a first parameter 572. The first cell process may include enrichment. The first parameter may be measured at a first time interval. For example, the first parameter may be a cell concentration value, such as a leukocyte concentration value. The leukocyte concentration value may be measured by a flow cytometer. The first parameter may then be compared to a first condition 574. The first condition may be, for example, a leukocyte concentration value of about 80%. If the first parameter satisfies or exceeds the first condition, the workflow may continue. If the first parameter does not meet or exceed the first condition, the workflow may be stopped, and / or an alert may be generated via the controller.

[0111] Method 505 may further include performing a second cell process and measuring one or more of the second, third, and fourth parameters 576. The second cell process may include selection. One or more of the second, third, and fourth parameters may be measured at a second time interval. For example, the second parameter may be a cell concentration value, such as a CD4+ and / or CD8+ concentration value. The CD4+ and / or CD8+ concentration value may be measured by a flow cytometer. The third parameter may be a cell recovery value, such as a CD4+ and / or CD8+ recovery value. The fourth parameter may be a target cell value. Then, one or more of the second, third, and fourth parameters may be compared to the second, third, and fourth conditions, respectively 578. The second condition may be a CD4+ and / or CD8+ concentration value of about 90%. The third condition may be a CD4+ and / or CD8+ recovery value of about 30%. The fourth condition may be a target cell value of approximately 800E6 cells.

[0112] If the second parameter is below the second condition and / or the third parameter is below the third condition, an alert may be generated via the controller. If both the second and third conditions are met or exceeded, and the fourth parameter meets or exceeds the fourth condition, a certain amount of cells may be removed from the cell solution. The amount of cells removed may correspond to the difference between the measured fourth parameter and the fourth condition, such that, after removal, the cell solution may contain an amount of target cells below the threshold represented by the fourth condition. If none of the second, third, and fourth conditions are met or exceeded, the workflow may continue.

[0113] Method 505 may further include performing a third cellular process and measuring one or more of the fifth and sixth parameters 580. The third cellular process may include activation. One or more of the fifth and sixth parameters may be measured at a third time interval. For example, the fifth parameter may be a cell diameter value. The sixth parameter may be a cell viability value. Then, one or more of the fifth and sixth parameters may be compared to the fifth and sixth conditions, respectively 582. The fifth condition may be a cell diameter value of about 10 micrometers (μm). The sixth condition may be a cell viability value of about 70%. If the fifth parameter is less than the fifth condition and / or the sixth parameter is less than the sixth condition, an alert may be generated via the controller. If both the second and third conditions are met or exceeded, the workflow may continue.

[0114] Method 505 may further include performing a fourth cell process and measuring one or more of the seventh and eighth parameters 584. The fourth cell process may include perfusion. One or more of the seventh and eighth parameters may be measured at a fourth time interval. For example, the seventh parameter may be a glucose value. The eighth parameter may be a lactate value. Then, one or more of the seventh and eighth parameters may be compared to the seventh and eighth conditions, respectively 586. The fifth condition may be a glucose value of about 2 g / L. The eighth condition may be a lactate value of about 2 g / L. If the seventh parameter is less than the seventh condition and / or the eighth parameter is greater than the eighth condition, the cell solution may be perfused up to a predetermined perfusion value. For example, the predetermined perfusion value may be about 50% such that about 50% of the medium can be removed and replaced while retaining substantially all of the cells in the cell solution. If the seventh parameter satisfies or exceeds the seventh condition, and the eighth parameter is less than the eighth condition, the workflow may continue.

[0115] Method 505 may further include performing a fifth cell process and measuring one or more of the ninth parameter, the tenth parameter, and the eleventh parameter 588. The fifth cell process may include proliferation and / or perfusion. One or more of the ninth, tenth, and eleventh parameters may be measured at the fifth time interval. For example, the ninth parameter may be the total cell count value. Similar to the fourth cell process described above, the tenth parameter may be the glucose value and the eleventh parameter may be the lactate value. Then, one or more of the ninth, tenth, and eleventh parameters may be compared to the ninth, tenth, and eleventh conditions, respectively 590. The ninth condition may be the total cell count value of a predetermined value. The predetermined value may correspond to a historical average based on one or more previous workflows. If the ninth parameter is less than the ninth condition, another sample may be obtained and measured. If the ninth parameter satisfies or exceeds the ninth condition, then one or more of the tenth and eleventh parameters may be compared to the ninth and tenth conditions, respectively. The fifth cell process may be similar to the fourth cell process, such that the tenth parameter may be a glucose value and the eleventh parameter may be a lactate value. Similarly, the tenth condition may be equivalent to the seventh condition and the eleventh condition may be equivalent to the eighth condition. Therefore, if the tenth parameter is less than the tenth condition and / or the eleventh parameter is greater than the eleventh condition, the cell solution may be perfused up to a predetermined perfusion value. If the tenth parameter satisfies or exceeds the tenth condition and the eleventh parameter is less than the eleventh condition, the workflow may continue.

[0116] Method 505 may further include performing a sixth cell process and measuring one or more of the twelfth and thirteenth parameters 592. The sixth cell process may include perfusion. The sixth cell process may be similar to the fourth cell process, such that the twelfth parameter may be a glucose value and the thirteenth parameter may be a lactate value. Similarly, the twelfth condition may be equivalent to the seventh condition and the thirteenth condition may be equivalent to the eighth condition. Method 505 may include comparing the twelfth parameter with the twelfth condition and the thirteenth parameter with the thirteenth condition 594. Thus, if the twelfth parameter is less than the twelfth condition and / or the thirteenth parameter is greater than the thirteenth condition, the cell solution may be perfused to a predetermined perfusion value. If parameter 12 satisfies or exceeds condition 12, and parameter 13 is less than condition 13, the workflow may continue.

[0117] Method 504 may further include performing a seventh cell process and measuring one or more of the 14th and 15th parameters 595. The seventh cell process may include proliferation. One or more of the 14th and 15th parameters may be measured at a seventh time interval. For example, the 14th parameter may be the total cell count value. The 15th parameter may be the chimeric antigen receptor expression value. Then, one or more of the 14th and 15th parameters may be compared to the 14th and 15th conditions, respectively 596. The 14th condition may be the total cell count value of a predetermined value. The predetermined value may correspond to a historical mean based on one or more previous workflows, such as about 4E9 cells. The 15th condition may be a chimeric antigen receptor expression value of about 10%. If the 14th parameter is greater than the 14th condition and / or the 15th parameter is greater than the 15th condition, the workflow may continue. If parameter 14 is less than condition 14, and parameter 15 is less than condition 15, the growth process may continue.

[0118] Method 505 may further include carrying out an eighth cell process 597. The eighth cell process may include harvesting cells from a cell solution. In some modifications, the cell solution may be transferred to a sterile liquid transfer device so that at least a portion of the cell solution can be removed from the cartridge and then transferred to an SLTD. The SLTD may be moved by a robot to a reagent storage unit for storage or to a feedthrough for removal from work cells. In this way, the contents can be stored for subsequent use in cell therapy and / or used for future workflows for growing additional cells.

[0119] While described above as including certain steps, please understand that a cell processing method may include any subset of the cell processing steps in any preferred order.

[0120] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in error in the device or method employed to determine that value, or variations present between samples being measured. Unless otherwise stated or evident from the context, “approximately” means within 10 percent of the reported value (except where such a value is greater than 100% of the possible value or less than 0%). When used in conjunction with a range or set of values, the term “approximately” applies to each of the endpoints of the range or the values ​​listed in the set of values, unless otherwise indicated. As used herein, the terms “approximately” and “about” are used synonymously.

[0121] While embodiments of the present invention have been shown and described herein, those skilled in the art will understand that such embodiments are provided only as examples. Those skilled in the art will recall numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the invention. The following claims define the scope of the present invention, and methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby.

Claims

1. A method for processing cells, Automatically executing a pre-programmed workflow that defines a set of cell processing steps, wherein at least one execution of the cell processing steps includes measuring at least one parameter based on a set of pre-programmed conditions and corresponding results for that step. Based on the aforementioned at least one measured parameter, it is determined whether the condition is met. A method comprising continuing, modifying, or stopping the workflow based on the aforementioned determination.

2. The method according to claim 1, wherein the set of cell processing steps includes one or more of concentration, selection, activation, proliferation, perfusion, sampling, and harvesting.

3. The method according to claim 1, wherein two or more cell processing steps are performed simultaneously.

4. The method according to claim 1, wherein the parameter includes one or more of the following: total cell count, target cell count, cell concentration, cell recovery, cell diameter, cell viability, glucose value, lactate value, dissolved oxygen value, pH value, chimeric antigen receptor expression value, and transgenic T cell receptor expression value.

5. The method according to claim 1, wherein the parameter is measured at predetermined time intervals.

6. The method according to claim 1, wherein the condition defines a threshold for the parameter.

7. The method according to claim 1, wherein a cell processing step is performed when it is determined that the measured parameters satisfy the conditions.

8. The method according to claim 1, wherein, when it is determined that the measured parameters satisfy the conditions, the cells are transferred from the first module to the second module.

9. The method according to claim 1, wherein a notification is generated to the user when it is determined that the measured parameter does not satisfy the conditions.

10. The method according to claim 1, wherein the workflow is stopped if it is determined that the measured parameters do not satisfy the conditions.

11. The method according to claim 1, wherein if it is determined that the measured parameters do not satisfy the conditions, at least one cell processing step is repeated.

12. A controller for the conditional execution of an automated cell processing method, A pre-programmed workflow that defines a set of cell processing steps, A set of pre-programmed conditions and corresponding results for use when modifying at least one of the cell processing steps, A controller in which at least one of the pre-programmed conditions is selectable by the user from the pre-programmed set.

13. The controller according to claim 12, wherein the controller is configured to receive input from a user in order to pre-program one or more workflows, conditions, and results.

14. The controller according to claim 12, wherein the controller is configured to generate an alert to the user if at least one of the pre-programmed conditions is not met.

15. The controller according to claim 12, wherein the controller is configured to control a robot configured to automatically execute the pre-programmed workflow.

16. The controller according to claim 12, wherein the controller communicates with a sterile liquid transfer device.

17. The controller according to claim 12, wherein the controller communicates with a cell sampling system configured to perform one or more measurements of a cell solution.

18. The controller according to claim 12, wherein the controller communicates with one or more devices configured to perform one or more cell processing steps.

19. A method for automated cell processing, Receiving a workflow, wherein the workflow defines one or more cell processing steps to be performed on cells in a cartridge within a work cell, and the cell processing steps are selected from the group consisting of cell washing, isolation, purification, concentration, dilution, and growth. The aforementioned workflow is to be executed automatically, The method involves automatically measuring at least one parameter within the cartridge, wherein the at least one parameter is selected from the group consisting of cell count, cell diameter, cell concentration, cell viability, glucose, lactate, cell recovery, dissolved oxygen, and pH. The process involves comparing the at least one measured parameter with a predefined condition, A method comprising automatically continuing, modifying, or stopping the workflow based on the comparison described above.

20. The method according to claim 19, wherein the workflow is automatically performed by a robot.

21. The method according to claim 19, wherein the workflow is pre-programmed in the controller.

22. The method according to claim 19, wherein the predefined conditions are pre-programmed in the controller.

23. The method according to claim 19, wherein the predefined conditions define a threshold for the measured parameter.

24. The method according to claim 19, wherein continuing the workflow includes completing the first cell processing step and performing the second cell processing step.

25. The method according to claim 19, wherein an alert is generated if the comparison indicates that the measured parameter does not satisfy the predefined condition.

26. The method according to claim 19, wherein the workflow is stopped if the comparison indicates that the measured parameter does not satisfy the predefined condition.

27. The method according to claim 19, wherein if the comparison indicates that the measured parameter does not satisfy the predefined conditions, at least one cell processing step is repeated.

28. The method according to claim 19, wherein the one or more cell processing steps are performed at one or more predefined time intervals.

29. The method according to claim 19, wherein measuring at least one parameter is performed by an analytical instrument.

30. The method according to claim 19, wherein the cell sample in the cartridge is transferred to an analytical instrument.

31. The method according to claim 19, wherein the cells are diluted, including partial depletion.

32. A method for automated cell processing, Perform the first cell processing step on the cells in the cartridge, Based on the first cell processing step, the first parameter is measured, To determine whether the first condition is met, the measured first parameter is compared with a first set of pre-programmed conditions, After determining that the first condition is met, a second cell processing step is performed on the cells in the cartridge. Based on the second cell processing step described above, the second parameter is measured, This includes, in order to determine whether the second condition is met, comparing the measured second parameter with a second set of pre-programmed conditions, A method wherein the first cell processing step and the second cell processing step each include concentration, selection, activation, or proliferation.

33. The method according to claim 32, wherein each of the first and second conditions includes a total cell count, a target cell count, a cell concentration, a cell recovery value, a cell diameter, a cell viability value, a glucose value, a lactate value, a dissolved oxygen value, a pH value, a chimeric antigen receptor expression value, or a transgenic T cell receptor expression value.

34. The method according to claim 32, wherein each of the first and second cell processing further comprises cell perfusion, cell sampling, or cell harvesting.

35. The method according to claim 32, wherein each of the first and second cell processing steps is performed automatically by a robot.

36. The method according to claim 32, wherein the user can use a controller to pre-program each of the first and second conditions.

37. The method according to claim 32, wherein the first cell processing step is performed by a first instrument, the second cell processing step is performed by a second instrument, and the first and second instruments are placed side by side in a work cell.

38. The method according to claim 32, wherein a user alert is generated if either of the first or second conditions is not met.

39. The method according to claim 32, wherein the first and second cell processing steps are each performed at a predefined time interval.

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