Integrated cell analysis on biological cells sampled from a bioreactor

Abstract

Integrated cell analysis on biological cells sampled from a bioreactor biological cells from a bioreactor for integrated cell analysis may be provided by: a bioreactor; a sensor; a processor; a memory including instructions that when executed by the memory perform operations that comprise, automatically: extracting a sample from cells being cultured in the bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

Description

INTEGRATED CELL ANALYSIS ON BIOLOGICAL CELLS SAMPLED FROM A BIOREACTORPRIORITY

[0001] The present disclosure claims the benefit of and priority to U.S. Provisional Patent Application No.: 63 / 616,117 titled “INTEGRATED CELL ANALYSIS ON BIOLOGICAL CELLS SAMPLED PROM A BIOREACTOR” and filed on December 29, 2023, which is incorporated herein it its entirety.BACKGROUND

[0002] There has been ever growing investment and interest in developing cell and gene therapy for treating difficult diseases. Lor cell therapy, live biological cells, typically taken from patients or healthy donors, are often processed and genetically modified, cultured, or produced in a bioreactor in order to generate therapeutic materials for infusion into a patient. However, there are many unique challenges posed to the industry for the production and quality control of such cell therapies. Lor at least these reasons, there are ever increasing needs for monitoring, measuring and characterizing the cells during the course of cell production in a bioreactor (e.g., where the cells are cultured and grown).

[0003] Thus, there exists a desire and need to develop new methods and systems that can automate and integrate the processes of cell production, cell sampling, sample monitoring, and cell analysis, such that feedback from these processes can effectively guide and improve cell production in a time-efficient manner to enhance the quality and speed up the production processes for the therapeutic cells.SUMMARY

[0004] The present disclosure provides for managing the production of (e.g., culturing, sampling, analyzing, and monitoring) biological cells from a bioreactor for integrated cell analysis.

[0005] In one embodiment, a system is provided, comprising: a bioreactor; a sensor; a processor; a memory including instructions that when executed by the processor perform operations that comprise, automatically: extracting a sample from cells being cultured in the bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample toidentify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

[0006] In some such embodiments, the sample is taken at a first time, and the operations further comprise: extracting an additional sample from the cells being cultured in the bioreactor at a second time after the first time; preparing the additional sample to produce an additional prepared sample; analyzing the additional prepared sample to identify an additional value for the associated cell analysis parameter; and in response to determining that the additional value is within the window for the associated cell analysis parameter, maintaining the setting of the bioreactor.

[0007] In some such embodiments, a first duration between the first time and the second time is equivalent to a second duration between the second time and a third time at which a subsequent sample from the cells being cultured in the bioreactor is extracted to determine a subsequent value for the associated cell analysis parameter.

[0008] In some such embodiments, a duration between the first time and the second time is one of: at least thirty minutes; at least one hour; at least two hours; at least six hours; at least twelve hours; at least twenty-four hours; at least thirty-six hours; at least forty-eight hours; at least seventy-two hours; at least eighty-four hours; at least ninety-six hours; at least one hundred twenty hours; at least one hundred forty-four hours; or at least one-hundred sixty-eight hours.

[0009] In some such embodiments, the operations further comprise: extracting a second sample from the cells being cultured in the bioreactor at a first time; preparing the second sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for a second associated cell analysis parameter; in response to determining that the second value is within a second window for the second associated cell analysis parameter, maintaining the setting of the bioreactor until a second time; extracting a third sample from the cells being cultured in the bioreactor at the second time; preparing the third sample to produce a third prepared sample; analyzing the third prepared sample to identify a third value for the second associated cell analysis parameter; in response to determining that the third value is within the second window for the second associated cell analysis parameter, identifying a trend for the second associated cell analysis parameter between the first time and the second time; and in response to determining that a projected value of the trend for a third time at which a fourth sample is scheduled to be extractedis outside of the window, adjusting a second setting of the bioreactor based on a difference between the trend and the window between the second time and the third time.

[0010] In some such embodiments, preparing the sample to produce the prepared sample comprises one, two, three, four, or all of: identifying or registering the sample; incubating the sample; staining the sample; adding a buffer or medium to the sample; or adding a therapeutic agent to the sample.

[0011] In some such embodiments, the associated cell analysis parameter comprises one, two, three, four, five, six, seven, or all of: a pH; a temperature; a level or concentration of a constituent or agent within the bioreactor or the sample; a cell identity; a cell number; a cell purity; a cell size; or a run time.

[0012] In some such embodiments, the operations further comprise: automatically discarding the prepared sample after identifying the value for the associated cell analysis parameter.

[0013] In some such embodiments, the cells grown in the biorcactor arc extracted from a first biological subject prior to growth of the cells in the bioreactor, and are retrieved from the bioreactor for treatment or prophylaxis of a disorder in a second biological subject as a modified cell culture, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

[0014] In some such embodiments, the bioreactor remains self-contained and operates automatically based on instructions received exclusively before extracting the sample.

[0015] In one embodiment, a method is provided, comprising: extracting a cell culture from a first biological subject; inserting the cell culture into a bioreactor; growing, in the bioreactor, the cell culture from a first time to a second time; modifying, in the bioreactor from the first time to the second time, the cell culture to produce a modified cell culture; retrieving the modified cell culture from the bioreactor; and supplying a therapeutically effective amount of the modified cell culture to a second biological subject that is experiencing or at risk of a disorder that is treatable, preventable, or manageable via application of the modified cell culture.

[0016] In some such embodiments, growing and modifying the cell culture further comprise automatically culturing the cell culture according to operations that comprise: extracting a cell sample from the bioreactor at an intermediate time between the first time and the second time; preparing the cell sample to produce a prepared sample; analyzing the prepared sample to identifya value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting, before the second time, a setting of the bioreactor based on a difference between the value and the window.

[0017] In some such embodiments, the prepared sample is discarded after identifying the value for the associated cell analysis parameter.

[0018] In some such embodiments, growing and modifying the cell culture further comprise automatically culturing the cell culture according to operations that comprise: extracting a first cell sample from the bioreactor at a first intermediate time between the first time and the second time; preparing the first cell sample to produce a first prepared sample; analyzing the first prepared sample to identify a first value for an associated cell analysis parameter; in response to determining that the first value is within a window for the associated cell analysis parameter, maintaining a setting of the bioreactor until a second intermediate time between the first intermediate time and the second time; extracting a second cell sample from the biorcactor at the second intermediate time; preparing the second cell sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for the associated cell analysis parameter; in response to determining that the second value is within the window for the associated cell analysis parameter, identifying a trend for the associated cell analysis parameter between the first intermediate time and the second intermediate time; and in response to determining that a projected value of the trend for a third intermediate time between the second intermediate time and the second time at which a third sample is scheduled to be extracted is outside of the window, adjusting the setting of the bioreactor based on a difference between the trend and the window between the second intermediate time and the third intermediate time.

[0019] In some such embodiments, a prepared sample is extracted from the bioreactor via a needle and is inserted into an analysis vessel that is moved to an analysis module comprising a sensor that is separate from the bioreactor and in which a value for a cell growth parameter is determined.

[0020] In some such embodiments, between the first time and the second time, the bioreactor remains self-contained and operates automatically based on instructions received exclusively before the first time.

[0021] In some such embodiments, the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

[0022] In one embodiment, a method is provided, comprising: extracting a sample from cells being cultured in a bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

[0023] In some such embodiments, the sample is extracted at a first time, and the method further comprises: extracting an additional sample from the cells being cultured in the bioreactor at a second time after the first time; preparing the additional sample to produce an additional prepared sample; analyzing the additional prepared sample to identify an additional value for the associated cell analysis parameter; and in response to determining that the additional value is within the window for the associated cell analysis parameter, maintaining the setting of the bioreactor.

[0024] In some such embodiments, the method further comprising: extracting a second sample from the cells being cultured in the bioreactor at a first time; preparing the second sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for a second associated cell analysis parameter; in response to determining that the second value is within a second window for the second associated cell analysis parameter, maintaining the setting of the bioreactor until a second time; extracting a third sample from the cells being cultured in the bioreactor at the second time; preparing the third sample to produce a third prepared sample; analyzing the third prepared sample to identify a third value for the second associated cell analysis parameter; in response to determining that the third value is within the second window for the second associated cell analysis parameter, identifying a trend for the second associated cell analysis parameter between the first time and the second time; and in response to determining that a projected value of the trend for a third time at which a fourth sample is scheduled to be extracted is outside of the window, adjusting a second setting of the bioreactor based on a difference between the trend and the window between the second time and the third time.

[0025] In some such embodiments, the method further comprising automatically discarding the prepared sample after identifying the value for the associated cell analysis parameter.

[0026] In some such embodiments, the cells grown in the bioreactor are extracted from a first biological subject prior to growth in the bioreactor, and are retrieved from the bioreactor for treatment or prophylaxis of a disorder in a second biological subject as a modified cell culture once a predefined growth value is reached, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

[0027] Additional features and advantages of the disclosed method and apparatus are described in, and will be apparent from, the following Detailed Description and the Figures. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and not to limit the scope of the inventive subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 illustrates a system for automatically sampling biological cells from a bioreactor for integrated cell analysis and production, according to embodiments of the present disclosure.

[0029] Figure 2 is a flowchart of an example method for automatically sampling biological cells from a bioreactor for integrated cell analysis and production, according to a non-limiting embodiment of the present disclosure.

[0030] Figure 3 is a flowchart of an example method for automatically managing the growth and culturing of a cell sample for use in research, treatment, or prophylaxis, according to embodiments of the present disclosure.

[0031] Figure 4 illustrates an example process setup, according to embodiments of the present disclosure.

[0032] Figure 5 illustrates a computing device, according to embodiments of the present disclosure.

[0033] Figures 6A-6L show cell analysis parameters for CAR-T cells produced in G-REX® 24-well bioreactor devices, (available from Wilson Wolf Corp.), according to embodiments of the present disclosure.

[0034] Figures 7A-7F show the cell analysis parameters for T cells as cultured in presence of different cytokines (IL2, or IL7 or IL15) in G-REX 24 well devices, according to embodiments of the present disclosure.

[0035] Figure 8 illustrates an overview of an example T-cell production process with a feedback loop, according to embodiments of the present disclosure. The T-cell production process begins with thawing and overnight resting of PBMCs on the day before production (D-l). On day zero ( DO), the rested cells are seeded and activated in a bioreactor well plate at a seeding density of IxlO6cells per well. Activation is terminated on day three (D3) by replacing the activation medium with the culture medium. The feedback loop is initiated on day four (D4 ), which involves cell analysis to assess selected quality parameters. Based on these results, culture conditions arc adjusted for conditions that fail to meet quality control (QC) standards. Additional cell analyses are performed on day six (D6) and the final harvest occurs on day ten (D10).

[0036] Figure 9 is a schematic diagram illustrating the tested groups and the process for initiating an feedback loop for a sample groups, according to embodiments of the present disclosure.

[0037] Figure 10 illustrates kinetics of T-cell growth during an example production process for a control sample group, according to embodiments of the present disclosure. In the illustrated results, the overnight- rested PBMCs were seeded in a bioreactor well plate at a density of IxlO6cells per well on day zero and cultured in activation medium (e.g., RPMI 1640 with 10 % FBS, supplemented with soluble CD3 / CD28 activator and 200 U / mL IL2) from day zero to day three. From day three onward, the cells were maintained in an expansion medium containing 200 U / mL IL-2. Cell counts were performed on days three, four, fix, and ten using a flow cytometer.

[0038] Figures 11A-11C illustrate the assessment results of cell quality parameters on day four using a flow cytometer for the control and two identical experimental groups, according to embodiments of the present disclosure. Figure 11A illustrates the total cell number and the x-fold change thereof relative to the control group in Sample 1. Figure 1 IB illustrates the percentage of stem cell memory T-cells (Tscm) and the x-fold change thereof relative to the control group inSample 1. Figure 11C illustrates the cell counts of Tscm and the x-fold change thereof relative to the control group in Sample 1.

[0039] Figures 12A-12C illustrate the assessment results of cell quality parameters that were evaluated throughout the production process using a flow cytometer for the control and two identical experimental groups, according to embodiments of the present disclosure. Following the initial cell analysis on day four, Sample 3 was transferred to and maintained under modified culture conditions to apply feedback to the production process for the remainder of the production process. Figure 12A illustrates the kinetics of cell proliferation over time and the x-fold changes in total cell number relative to the control group in Sample 1. measured on days four, six, and ten. Figure 12B illustrates the kinetics of the percentage change of Tscm throughout the manufacturing process and the x-fold changes in Tscm percentage relative to the control group in Sample 1, determined on days four, six, and ten. Figure 12C illustrates the kinetics of the change in Tscm cell numbers throughout the manufacturing process and the x-fold changes in Tscm cell numbers relative to the control group in Sample 1, assessed on days four, six, and ten.DETAILED DESCRIPTION

[0040] The present disclosure provides for sampling biological cells from a bioreactor for integrated cell analysis, that allow practitioners to reduce, minimize, or eliminate the variations in the cell analysis process and results for the cells taken out of a cell culture bioreactor through automated, or semi- automated, integrated cell analysis and measurement, including the cell sampling, cell processing, cell-measurement and cell-analysis result reporting for optional cell culture bioreactor process change or maintenance and / or for optional follow-up administration of cells cultured to a biological subject (e.g., a patient). The systems, apparatuses, and methods described herein can be used for sampling biological cells from a bioreactor for integrated cell analysis and achieve important benefits and advantages over conventional approaches.

[0041] Cell therapy is a broad concept that describes, for example, the injection, grafting or implanting of viable cells into a patient in order to treat difficult diseases. The production of cells for cell therapy may involve taking live biological cells from patients or healthy donors. The cells may be processed, genetically modified, cultured, or produced in a bioreactor (e.g., for use in therapeutic materials for infusion into a patient).

[0042] One of the most important requirements for cell therapy is consistency in the product quantity and quality. For example, attributes for determining product quantity and quality in cell therapy may include, but are not limited to: a quantity of cells (cell number), cell identity, cell purity, cell potency, cell viability and other cell quality parameters. Although maintaining consistency and reliability of the quantity and quality of the cell samples is important for effective cell therapies, achieving such consistency and reliability is difficult for a number of reasons. For example, the starting materials for cell therapy, which typically include live biological cells, can be significantly different for each production lot. Furthermore, since the biological cells are live and growing during the production process, any changes in cell growth media, cell growth conditions, or any other growth parameters (known or unknown), may cause dramatic differences in the final product.

[0043] Typically, manual procedures are employed for taking the cell samples out from biorcactors or for processing the ‘taken-out cells’ for assays and analysis. These manual steps for cell sampling (e.g., withdrawing certain volumes of the cells in culture) often lead to inconsistencies in sampling volume or to suboptimal homogeneity of cell suspension (e.g. depending on the operator conducting the cell sampling, the production- site being used, or the time at which the cell sampling is done). Manual sampling may also restrict the time when such sampling may be conducted (e.g., during the weekend or late evenings) and critical time points the sample needs to be analyzed may be missed. Thus, manual sampling is an inefficient process and is subject to human errors, leading to variations in cell production, adversely affecting the results of analytical measurements, and eventually adversely affecting the cell therapy provided to patients.

[0044] Manual processing of the sampled cells for analysis may involve the addition of cell analysis reagents to the samples, incubation of the cells with the reagents in some apparatus or setups, washing and re-suspending the cells, aliquoting the cell samples to different containers (e.g., tubes, or wells for a microtiter plate), placing the cell-container to a cell analyzer for running the experiment. Depending on the cell analyzer apparatus being used, additional manual steps may be involved in operating the samples, operating the instruments, performing the experiments for data acquisition as well as data analysis. These manual steps in cell processing and in cell-assay experiments would introduce additional variables or uncertainties to the cell analysis process, leading to increased variations in the assay results. Again, the manual steps would also limit thetime windows when the cell assay experiments are performed and conducted. Furthermore, in a manual process, the cell assay results are not used for guiding or controlling possible cell production process in an automatic or semi-automatic manner. That is to say, data from different cell analyzer apparatus or systems are not synchronized in a timely manner to be effectively used to guide the culture process of cell therapy products in bioreactors.[00451 Accordingly, the present disclosure describes systems, apparatuses, and methods for automatically sampling, processing and analyzing biological cells from a bioreactor for integrated cell analysis and production that address and overcome one or more of known shortcomings of conventional approaches for manually cell sampling and analysis in cell therapy. Without wishing to be bound by theory, the systems, apparatuses, and methods described herein can achieve one or more of the following: (a) reducing variation in cell sampling from a bioreactor (e.g., for cell samples intended to be same); (b) reducing variation in cell preparation or cell processing for cell measurement steps; (c) reducing variation in cell measurement and cell analysis; (d) reducing labor-intensive, and time-limiting steps for cell sampling, cell processing and cell analysis, data analysis, and reporting; (e) allowing for real-time feedback provided from the cell analysis to guide the cell culture (e.g., long-term cell culture) to achieve optimal cultured or produced cells in the bioreactor; (f) allowing for systematic process monitoring and control during cell culture (e.g., long-term cell culture) and analytical measurements to meet regulatory requirements, combinations thereof, and other benefits that will be apparent to the practitioner of ordinary skill in the relevant art on reading the present disclosure.

[0046] In some embodiments, the systems, apparatuses, and methods described herein are designed for the entire cell analysis procedures, including, for example, the cell sampling, cell processing, cell measurement, and cell-analysis result reporting. In some embodiments, the systems, apparatuses, and methods achieve one or more of the following benefits and advantages, including, but not limited to, (a) automated, or semi- automated, integrated cell analysis and measurement; (b) consistent cell sampling; (c) potentially standardizing the sampling of cells from a bioreactor as a general approach; (d) consistent and reproducible cell preparation and cell processing; (e) less variation in cell measurement and cell monitoring; (f) integrated sample-to- result for entire cell analysis process with cell samples taken from a bioreactor; (g) easier integration for multiple types of cell measurement and cell analysis; (h) better compatibility of cell analytics process with the automated, closed cell culture bioreactors; (i) better measurementresults; (j) using cell sample analytics to guide the culture conditions and method to improve and optimize cell quality, and to reduce culture time and increase efficiency, for example, to implement a closed-loop feedback mechanism, taking the information from the cell analytics to guide and control the cell culture process (e.g., adjustment of culture media ingredients, pH, etc.) in real time; (k) meeting the regulatory compliance requirements with closed, fully automated system including therapeutic cell product culture bioreactors, cell sampling apparatus, sample preparation system, cell analytical instruments, and related software and informatics, for example, to maintain and guarantee the data integrity and process compliance for cellular therapy production and QC, and with the automated workflow described here, every step is traceable with data stored in a centralized and secure storage, which provides significant benefits and advantages of the automated process described herein versus the manual sampling and measurements which may have inherent limitations in meeting the compliance requirements such as process and data traceability, combinations thereof, and other benefits that will be apparent to the practitioner of ordinary skill in the relevant art on reading the present disclosure.

[0047] In various embodiments of the present disclosure include one or more of the following: (i) a bioreactor where cells are cultured and grown with the possibility of an automated media exchange, an automated cell culture nutrient supply, etc.; (ii) a mechanism or an apparatus for moving a tube, a cartridge, a micro-titer plate, or any vessel or container from a stack of such vessels or containers to a pre-defined location relative to the bioreactor; (iii) a mechanism or an apparatus for moving a desired volume of cell samples at pre-determined schedules from the bioreactor to the vessel or container or the tube, the cartridge, or the micro-titer plate (optionally a mechanism for mixing or homogenizing the cell samples in the bioreactor before moving the cell sample to the vessel); (iv) a mechanism or apparatus (e.g., as previously described in (ii)) for moving the vessel or container (with the cell samples being loaded) to cell preparation or processing station; (v) a mechanism for preparing or processing the cell samples for cell measurement, the mechanism including adding appropriate cell-measurement reagents to cell samples or adding cell samples to appropriate cell-measurement reagents, incubating the cell samples with reagents at appropriate conditions for desired length of time, or processing the cell samples ready for cell measurement; (vi) a mechanism or apparatus for moving the vessel or container to cell measurement apparatus (e.g., after the cell samples are processed or prepared); (vii) a mechanism for initiating cell measurements for data acquisitions according to appropriatemeasurement schedules or steps; (viii) optionally, a mechanism for initiating data analysis, creating an analysis report, or relaying the analysis report to another computing system (e.g., for use by another software); (ix) a mechanism for possible change of cell culture conditions or parameters in the bioreactor to optimize the cell culture (e.g., long-term cell culture) based on the analysis results, and combinations thereof.[00481 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0049] As used herein, the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0050] About” or “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error arc within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0051] “Automatic,” “Automated”, and variations thereof as used herein shall generally mean a process performed without direct human interaction or commands. Actions that are performed automatically may be performed according to parameters or setting that are defined by a human user, but are performed without user commands. For example, an action of opening a sample tray may be performed or directed by a human user (e.g., by pushing a button) or may be performed automatically by a computerized system in response to a triggering condition occurring. Actions that are described as semi-automated may refer to a process that is partly performed by a human and partly performed automatically. For example, a computer may request confirmation from a human before automatically performing an action, thereby rendering the sequence to be semiautomated. In another example, a computer may pause execution of an automatically performed operation to give a human operator an opportunity to cancel or modify the operation, thereby rendering the sequence to be semi-automated.

[0052] ‘Culture” or “cell culture” as used herein may refer to the process of growing cells over a prolonged period of time under controlled conditions. In some embodiments, culture occurs, and the controlled conditions are facilitated by a bioreactor. Such conditions, also referred to herein as “parameters,” may include but are not limited to a pH, a temperature, a level or concentration of a constituent or agent (e.g., O2, CO2, an amino acid, a protein, an amino acid, a protein, a nutrient, adrug, a hormone, or a medium), a process parameter (e.g., the frequency of medium change, or the flow rate used to introduce new media to bioreactor during media change) or a run time (e.g. the duration at which the cells are grown). In some embodiments, the culture involves the cells growing in an aqueous solution of nutrients referred to as a cell culture medium. The cell culture media may include a source of energy (e.g., carbohydrates) and can be used in various cell culture processes. “Culture” or “cell culture” may also refer to the composition of cells grown through the above described process. The present disclosure describes examples of a cell culture used for manufacturing or producing cells for cell-based therapies, and therefore may use the terms “cell culture”, “cell production”, or “cell manufacturing” interchangeably to refer to the process of the cells being cultured, grown, or produced in a bioreactor.

[0053] Module” as the term used herein may refer to a subsystem. The module may include a program that, when executed, effects an individual functionality or aspect of the overall automatic or semi-automatic process of integrated cell analysis and production including automatically sampling biological cells from a bioreactor. In some embodiments, the module is a computer subsystem. In some embodiments, the module may be used to also refer to an underlying device being used to execute the individual functionality or aspect. For example, as described herein, modules may include, but are not limited to, a “bioreactor module,” a “sample extraction module,” a “sample preparation module,” a “sample analysis module,” or a “controller module” (also referred to herein as “controller”).

[0054] “Or” is used herein to mean the inclusive sense of “or” (c . the exclusive sense of “or”), and is used interchangeably with, the term “and / or”, unless context clearly indicates otherwise. The use of the term “and / or” in some places herein does not mean that uses of the term “or” are not interchangeable with the term “and / or” unless the context clearly indicates otherwise.

[0055] “Sample” as the term used herein refers to a biological sample obtained or derived from a source of interest. For example, a “cell sample” as the term is used herein, refers to a sample that includes at least one cell. In some embodiments, the cell sample includes a plurality of cells. In some embodiments, the cell is disposed in a medium. In some embodiments, the source of interest includes an organism, such as an animal or human. The source of the sample can be blood or a blood constituent; a bodily fluid; a solid tissue as from a fresh, frozen or preserved organ, tissue, biopsy, resection, smear, or aspirate; or cells from any time in gestation or development of a subject. For example, a cell sample can be isolated or harvested directly or indirectly from asubject, organ, or tissue. In some embodiments, the sample is a primary sample, e.g., obtained directly from a source of interest by any appropriate means. In some embodiments, the sample is a preparation that is obtained by processing (e.g., by removing one or more components of or by adding one or more agents to) the primary sample. Processing the primary samples may include biological manipulation of the primary cells such as transfecting the genes into the primary cells, or knocking out certain genes from the primary cells, or culturing and passaging the primary cells in appropriate culture media. Accordingly, a sample may include transfected cells from a biological subject, cultured cells, or cell lines.

[0056] As used herein, various chemical compounds may be referred to by associated element abbreviations set by the International Union of Pure and Applied Chemistry (IUPAC), which one of ordinary skill in the relevant ait will be familiar with. Similarly, various units of measure may be used herein, which are referred to by associated short forms as set by the International System of Units (SI), and various applied prefixes thereto, which one of ordinary skill in the relevant art will be familiar with.

[0057] Certain terms are used throughout the description and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not function.

[0058] Figure 1 illustrates a system 100 for integrated cell analysis and production including automatically sampling biological cells from a bioreactor, according to embodiments of the present disclosure. As shown in Figure 1, the system 100 includes a plurality of modules for devices that directly produce, sample, or analyze biological cell cultures (the modules referred to collectively as “integrated cell therapy modules” 102) and a controller 170 for processing information received from the integrated cell therapy modules 102 and transmitting commands based on the information. In some embodiments, the system 100 further includes an external storage medium 190. One or more components of system 100 may communicate over a communication network 160.

[0059] In various embodiments, the controller 170 includes various hardware (e.g., a printed circuit-board populated with electronic circuit components) that is configured to process information (e.g., analog or digital signals) received from one or more of the integrated cell therapy modules 102, and execute and transmit commands based on computer-executable instructions. Insome embodiments, the controller 170 is or includes a microcontroller embedded within any of the modules of the integrated cell therapy modules 102 (e.g., as an embedded system) controlling the processes performed for automatically sampling biological cells from a bioreactor for integrated cell analysis and production.

[0060] As shown in Figure 1, the controller 170 includes one or more processors 172 and a memory 174. The one or more processors 172 may be or include any one or more types of digital circuits configured to perform operations on a data stream, including functions described in the present disclosure. In some embodiments, the one or more processors 172 include a first processor (e.g., microprocessor) and a second processor (e.g., second microprocessor) for performing separate functionalities or a functionality in parallel. The memory 174 be or include any type of long term, short term, volatile, nonvolatile, or other memory device, and is not to be limited to any particular type of memory or number of memories, or type of media upon which memory is stored. The memory 174 may store instructions that, when executed by the processor 172, can cause the controller 170 to perform one or more methods discussed herein. As used herein, the memory 174 is an example of a device that includes computer-readable storage media, and is not to be interpreted as transmission media or signals per se.

[0061] In some embodiments, the controller 170 includes one or more of an integration application 184, a network interface 176, a user interface 178, and a samples database 180. The integration application 184 be or include a software, program, or code used to allow the scheduling or initiation of different stages, steps, or actuations involved in the process for automatically sampling biological cells for integrated cell analysis and production. In some embodiments, the integration application 184 causes the controller 170 to communicate with the integrated cell therapy module 102 (optionally via communication network 160) and cause one or more of the integrated cell therapy modules 102 to perform an action based on a scheduling of the action. For example, the integration application 184 may receive an indication that a sample has been extracted (e.g., from the bioreactor module 110) and may then cause the sample extraction module 120 to transfer the sample to a sample preparation module 130 (e.g., by sending a command signal to a robotic control 134).

[0062] The network interface 176 allows the controller 170 to communicate with other systems or subsystems (e.g., any one of the integrated cell therapy modules 102) over the communication network 160. For example, the network interface 176 may include a wired interface (e.g., electrical,RF (via coax), optical (via fiber)), a wireless interface, a modem, etc. In some embodiments, one or more of the integrated cell therapy modules 102 also have respective network interfaces to allow communication with other systems or subsystems.

[0063] In various embodiments, a user interface 178 may be or include a portal allowing a user or operator of the controller 170 to set one or more aspects of the process of automatically sampling biological cells for integrated cell analysis and production. For example, a user or operator may use the user interface 178 to set parameters for growing cells in the bioreactor (bioreactor parameters or bioreactor module parameters), or set a sample size for extraction and analysis. After setting these functionalities, the controller 170 may perform the various operations without further human intervention.

[0064] In various embodiments, the samples database 180 may be or include a repository, list, or record of cell cultures and samples of cell cultures being managed by the controller 170. The samples or cell cultures may be identifiable in the samples database 180 through one or more of a sample identifier, a cell culture identifier, or a bioreactor device identifier. In some aspects, sensitive identifying information about the sample or culture (e.g., patient data) may be encrypted or stored as metadata. In some aspects, the samples database 480 may also record attributes, characteristics, or conditions (referred to collectively as “parameters”) (e.g., parameter records 182) set for the sample or the culture of the sample. The parameters may include: bioreactor parameters for the conditions set for the culture or growth of cells in the bioreactor; and cell analysis parameters for the attributes or characteristics of cells that are samples and analyzed. In some embodiments, the samples database 180 may be stored externally (e.g., at an external storage medium 190) to conserve bandwidth, processing, and other computing resources of the controller 170. Additionally or alternatively, the controller 170 may store an abridged or temporary record of information pertaining to the samples, while the external storage medium 190 may store a more complete version of the information (e.g., as shown by samples database 192 and parameter records 194 stored in the external storage medium 190).

[0065] The integrated cell therapy modules 102 may include, but are not limited to: a bioreactor module 110, a sample extraction module 120, a sample preparation module 130, and a sample analysis module 150. The bioreactor module 110 may be or include a subsystem, a program, or logic that causes an associated device (e.g., bioreactor) to support a biologically active environment for a cell culture process based on a configuration of a set of parameters. In someembodiments, the bioreactor module 110 may also include the associated device that is altered or maintained based on the program or logic. In some embodiments, the bioreactor module 110 includes a microprocessor 111 (e.g., an embedded microprocessor). In some embodiments, execution of the program (e.g., by the microprocessor 111) may cause an activation of controls (e.g., parameter controls 112) within the bioreactor device that cause one or more parameters for the cell culture (referred to herein as bioreactor parameters) to be maintained (e.g., a pH; a temperature; a level or concentration of a constituent or agent, e.g., O2, CO2, an amino acid, a protein, a nutrient, a drug, a hormone, or a medium; a process parameter such as the frequency of medium change or the flow rate used to introduce new media to a bioreactor during media change, or a run time).

[0066] For example, the bioreactor module 110 may adjust the pH by allowing the inflow or outflow of acids, bases, or buffer media into a container of the device that contains cell culture media. Furthermore, the biorcactor module 110 may adjust the temperature by adjusting (e.g., electronically) the heat provided to the container. In another example, the bioreactor module 110 may adjust the level or concentration of a constituent or agent, by adjusting the inflow or outflow of the constituent or agent into the container. Furthermore, the runtime may be adjusted via a timer that sets the time by which the cell culture process terminates, a sample is extracted, / or a user is notified that the cell culture process is completed. The bioreactor module 110 may further include a network interface 114 which allows a controller 170 or an operator of the controller 170 (e.g., via user interface 178) to be informed about and control various parameters of the bioreactor module 110. The network interface 114 may share one or more subcomponents or functionalities as network interface 176.

[0067] The sample extraction module 120 may be or include a subsystem, a program, or logic that cause an associated device to extract and hold a sample of a cell culture from the bioreactor, in order to place the sample in a position for delivery to another module (e.g., sample preparation module 130). In some embodiments, the sample extraction module 120 may also include the associated device that is controlled or activated (e.g., via associated actuators or ports) based on the program or logic. In some embodiments, the sample extraction module 120 may include a microprocessor 121 (e.g., an embedded microprocessor). In some aspects, execution of the program (e.g., by microprocessor 121) may cause the activation and movement of one or more actuators 128 (e.g., motors, levers, pumps, etc.) facilitating the mixing of the cells and media withinthe bioreactor (e.g., so that the cells are uniformly distributed with the volumes of the media in the bioreactor), or facilitating the withdrawal or extraction of the sample from the bioreactor module 110 and delivery of the sample or an aliquot of the sample into another module (e.g., the sample preparation module 130). The one or more actuators 128 may be positioned to perform both the withdrawal and the delivery (e.g., by having one actuator face the bioreactor module 110 and another actuator face the sample preparation module 130).

[0068] Furthermore, the withdrawal or delivery may occur via a port for receiving the sample from the bioreactor module 110 (receiving port 122), and a port for delivering the sample or the aliquot of the sample to the second module (delivery port 124), respectively. The receiving port 122 may include, or may be connected to a pump (e.g., a syringe pump) (receiving pump 123). The receiving pump 123 may be configured to withdraw the sample (e.g., a predetermined volume of sample) at a pre-defined schedule from the bioreactor module 110.

[0069] For example, the receiver pump 123 may be equipped with a sensor to accurately detect when a specified volume of sample has been extracted from the bioreactor. Also or alternatively, the receiving pump 123 may be designed to only hold the specified volume such that the receiver pump 123 automatically stops extracting upon reaching the specified volume. In some embodiments, the receiver pump 123 may be placed in (automatically (e.g., via a robot or mechanism of the bioreactor) or manually) or located within the bioreactor module 110 to facilitate the withdrawal of the sample. However, in other embodiments, the receiver pump 123 may be outside the bioreactor module 110 (e.g., within the sample extraction module 120 as shown).

[0070] In some embodiments, the delivery port 124 may include or may be connected to, a sample dispensing needle 125. The sample dispensing needle 125 may be configured to deliver the sample or the aliquot of sample to the second module (e.g. sample preparation module 130, or sample analysis module 150). For example, one or more actuators 128 (e.g., a robotic arm, pulley, motor, lever, etc.) may situate the sample dispensing needle 125 to be filled with the sample at a predefined amount and may move the sample dispensing needle 125 towards the second module. Additionally or alternatively, the actuators 128 may move the sample dispensing needle 125 to the exterior of the sample extraction module 120 to allow a user or the automated system to easily move the sample dispensing needle 125 to the second module (e.g. sample preparation module 130, or sample analysis module 150).

[0071] Furthermore, the sample extraction module 120 may include a network interface 126 to allow communication with the controller 170 and other modules over the communication network 160. The network interface 126 may share one or more subcomponents or functionalities as network interface 176. The network interface 126 may allow the sample extraction module 120 to receive signals from other modules (e.g., the controller 170) optionally via communication network 160. The received signals may cause the sample extraction module 120 (e.g., by relaying electrical signals to the actuators 128) to withdraw the sample from the bioreactor module 110 or deliver the sample or the aliquot of sample to the second module.

[0072] The sample preparation module 130 may be or include a subsystem, a program, or logic that cause an associated device to: obtain a sample or an aliquot of a sample (e.g., from the sample extraction module 120); add one or more reagents (a first reagent and a second reagent (e.g., cell suspension buffer)) to the sample or the aliquot of sample; mix the reagent with the sample or the aliquot of sample; incubate the sample or the aliquot of sample with the reagent; wash the sample or the aliquot of the sample (e.g., cell washing); deliver a prepared sample to another module (e.g., the sample analysis module 150).

[0073] In some embodiments, the sample extraction module 120 may also include the associated device that is controlled or activated (e.g., via associated actuators or ports) based on the program or logic. In some embodiments, the sample preparation module 130 includes a microprocessor 131 (e.g., an embedded microprocessor). In some embodiments, execution of the program (e.g., by microprocessor 131) causes the activation and movement of one or more robotic controls 134 (e.g., robotic arms, motors, levers, pumps, etc.) for performing one or more of the functions described herein. For example, the sample preparation module 130 may use a motor to cause a stirrer to mix reagents with the sample or the aliquot with the sample, or may use a robotic arm (e.g., activated via an electronic pulley and belt system) to obtain or deliver the sample or aliquot of the sample.

[0074] The sample preparation module 130 may include a port for receiving the sample or the aliquot of the sample from a second module (e.g., the sample extraction module 120) (receiving port 136); and a port for delivering the sample or the aliquot of the sample to a second module (e.g., the sample analysis module 150) (delivery port 138), respectively. In some embodiments, the receiving port 136 may include, or may be connected to, a test tube. The test tube may be configured to receive the sample or the aliquot of the sample (e.g., a predetermined volume ofsample, at a pre-defined schedule) from the sample extraction module 120 or the bioreactor module 110. For example, the test tube may be held or extended outward from the sample preparation module 130 (e.g., by an element or arm of the sample preparation module 130) for the sample dispensing needle 125 of the sample extraction module 120 to place the sample or the aliquot of the sample into the test tube.[00751 Additionally or alternatively, the receiving port may include, or may be connected to, a microfluidics cartridge (e.g., microfluidics cartridge for flow cytometry assay). The microfluidics cartridge may be configured to receive the sample or the aliquot of sample from the second module (e.g., the sample extraction module 120). For example, the microfluidics cartridge (e.g., microfluidics cartridge for flow cytometry assay) may be connected to a microfluidics cartridge stack (e.g., a microfluidics cartridge stack for flow cytometry assay). The microfluidics cartridge stack may be powered via a motor or an electronic pulley to move the microfluidics cartridge to an exposed position exterior to the sample preparation module 130 where the sample or the aliquot of the sample can be received from another module such as sample extraction module or bioreactor module. Additionally or alternatively, the microfluidics cartridge stack may dispatch the microfluidics cartridge (e.g., microfluidics cartridge for flow cytometry assay) to a dispensing position, or to may facilitate barcode reading.

[0076] Additionally or alternatively, the receiving port 136 may include, or may be connected to, a sampling manifold. The sampling manifold may be configured to receive the sample or the aliquot of sample from the second module (e.g., the sample extraction module 120). For example, the sampling manifold may include a plurality of chambers loaded with different samples or different aliquots of samples from a bioreactor. For example, the sampling manifold may include a plate with a plurality of wells. The sampling manifold may be on a platform of the sampling preparation module 130 that may be exposed to the exterior for the sample or the aliquot of the sample to be received (e.g., via the sample dispensing needle 125 in sample extraction module). In some embodiments, a robotic control 134 may move the platform holding the sampling manifold in order to prepare the samples or aliquots of the sample contained therein.

[0077] In some embodiments, the test tube, the microfluidics cartridge, or the sampling manifold may be preloaded with a reagent for analyzing the cells (e.g., a lyophilized antibody for a flow cytometry assay). In some embodiments, the sample preparation module 130 may include a slot for a user or operator to load reagents in a desired amount for mixing with the sample or thealiquot sample in the test tube, microfluidics cartridge, or sampling manifold. In other embodiments, the reagent may not be preloaded, but is loaded by the system or a human user before or after the cells for analysis or other sample is added to the receiving port 136 or sampleholding device.

[0078] In some embodiments, the sample preparation module 130 may include a staining and incubation station 140. In some aspects, the staining and incubation station 140 may include a submodule with a stored program or logic that may cause one or more robotic controls 134 to perform one or more functionalities related to staining or incubating the sample or aliquot of the sample.

[0079] For example, the staining and incubation station 140 may be configured to: dispense a staining buffer (e.g., a predetermined volume of staining buffer) into the test tube, microfluidics cartridge, or sampling manifold; cause the sample and reagents (e.g. staining buffer) in the test tube, microfluidics cartridge, or sampling manifoldcr to mix via convection or fluid motion by different means (e.g. agitate the test tube, microfluidics cartridge, or sampling manifold to facilitate mixing or incubation), or incubate the sample or the aliquot sample (e.g., for a predetermined incubation time). In some embodiments, the sample preparation module 130 may include a compartment for storing staining buffer, from which the predetermined amount may be dispensed to the test tube, microfluidics cartridge, or sampling manifold, any of which may be held in place by the staining and incubation station 140. The dispensing may be responsive to a received signal indicating that the sample or aliquot of the sample is ready for staining. In one embodiment, the mixing of the sample and reagents in the test tube, microfluidics cartridge, or sampling manifold may be conducted via actuators underneath a platform, causing the fluid motion such as fluid convection. In some embodiments, agitation may be conducted via actuators underneath a platform, causing the platform or the test tube, microfluidics cartridge, or sampling manifold to perform a rocking, linear, or orbital motion so that the sample and reagents in the test tube, microfluidics cartridge, or sampling manifold would mix. In some embodiments, the incubation may be performed by controlling one or more parameters of the environment in which the sample or aliquot of the sample is stored in. After the incubation, the sample or aliquot of the sample may be referred to as a prepared sample.

[0080] In some embodiments, the delivery port 138 may include, or may be connected to, a test tube movement arm. The test tube movement arm may be configured to deliver the preparedsample or the aliquot of the prepared sample (e.g., a predetermined volume of the prepared sample or at a pre-defined schedule) to another module (e.g., the sample analysis module 150). For example, an end of the test tube movement arm may hold the test tube (e.g., via a gripping surface or via a test tube holder) while one or more joints of the test tube movement arm may be rotated using the robotic controls 134 (e.g., motors), responsive to an electrical signal, so as to move the end of the test tube movement arm towards the sample analysis module 150.

[0081] Additionally or alternatively, the delivery port 138 may include, or may be connected to, a conveyor. The conveyor may be configured to deliver the prepared sample to another module (e.g., the sample analysis module 150). For example, the conveyor may include an electronic pulley that is powered by motors of the robotic controls 134, responsive to a signal (e.g., received from the controller 170). Using the motors, the conveyor can move any compartments or holders of the prepared sample (e.g., the test tube or the microfluidics cartridge) to the sample analysis module 150. For example, the conveyor may be configured to move the microfluidics cartridge from a dispensing position to feed a cell analyzer 158 (e.g., flow cytometer) of the sample analysis module 150.

[0082] Additionally or alternatively, the delivery port 138 may include, or may be connected to, a plate handler (e.g., a robotic plate handler). The plate handler may be configured to deliver the prepared sample to another module (e.g., the sample analysis module 150). For example, the plate handler may include a plurality of wells on a plate, whereby the plate may be picked up and moved to a location on the sample analysis module 150, by a robotic arm of the robotic controls 135. The robotic arm may include, at one end, a gripping element or a locking element to pick up the plate by gripping or locking, respectively, Furthermore, the robotic arm may include one or more joints that may be powered and rotated via motors, responsive to a signal (e.g., received from the controller 170). Relying on the motors, the robotic arm may move the plate handler, including, which includes a plurality of wells holding the prepared sample, to the sample analysis module 150. In some embodiments, the plate handler may refer to the combination of the plate and the robotic arm. In some embodiments, the sampling manifold may include the plate.

[0083] In some embodiments, the test tube, microfluidics cartridge, or sampling manifold containing the sample, the aliquot of the sample, or the prepared sample may be barcoded. In such embodiments, the test tube movement arm, conveyor, or plate handler may move the test tube, microfluidics cartridge, or sampling manifold so that the barcode can be read (e.g., via a barcodereader 142). Additionally or alternatively, the test tube movement arm, conveyor, or plate handler may move the test tube, microfluidics cartridge, or sampling manifold towards the staining and incubation station 140 of the sample preparation module 130 (e.g., for the staining and incubation processes described above).

[0084] Furthermore, the sample preparation module 130 may include a network interface 144 to allow communication with the controller 170 and other modules over the communication network 160. The network interface 144 may share one or more subcomponents or functionalities as network interface 176. The network interface 144 may allow the sample preparation module 130 to receive signals from other modules (e.g., the controller 170) that cause the robotic controls 134 to perform one or more of the functions described herein. For example, in response to receiving a signal from the controller 170, the sample preparation module 130 may prompt the robotic controls 134 to deliver the prepared sample to the sample analysis module 150. Thus, the controller 170 may function as a single software control system that provides instructions to the robotic controls 134 (e.g., robotic arm), the staining and incubation station 140, and the barcode reader 142.

[0085] The sample analysis module 150 may be or include a subsystem, a program, or logic that cause an associated device to receive a sample, aliquot of the sample, or a prepared sample from another module (e.g., the sample preparation module 130 or the sample extraction module 120), detect one or more parameters of the sample or prepared sample (referred to herein as cell analysis parameters), and generate a value for each of the one or more cell analysis parameters. In some embodiments, the sample analysis module 150 may also include the associated device that is controlled or activated (e.g., via its sensors, ports, or actuators) based on the program or logic. In some embodiments, the sample analysis module 150 may include a microprocessor 151 (e.g., an embedded microprocessor). In some embodiments, execution of the program (e.g., by microprocessor 151) may cause a port (receiving port 152) to receive the sample, and may cause sensors to measure or detect one or more parameters of the sample. In some embodiments, the sample analysis module 150 includes a receiving port 152, a delivery port 154, one or more sensors 156, and a cell analyzer 158 (e.g., a flow cytometer). The receiving port 152 may receive the sample or prepared sample from a second module (e.g., the sample extraction module 120 or sample preparation module 130, respectively) and the delivery port 154 may be used for deliveringthe sample or prepared sample after the sample has been analyzed (referred to herein as analyzed sample) to another module or container (e.g., waste).

[0086] The value for each cell analysis parameter may be generated in response to the sample or prepared sample being received by the sample analysis module 150 (e.g., via receiving port 152). For example, the receiving port 152 may have a latch or other mechanical element that locks as the sample or prepared sample as the sample enters, such that the locking triggers the sensors 156 to begin measuring or detecting the one or more cell analysis parameters. In some embodiments, the value may be transmitted as a signal to the controller 170 via a network interface 159. For example the strength of the signal (e.g., an analog signal or electromagnetic wave) may be proportional to the value for the corresponding cell analysis parameters. The cell analysis parameters may relate to the cells being cultured or the cells in the sample or prepared sample. The cell analysis parameters may include but are not limited to: a cell identity or a cell type; a cell number; a cell purity; a cell size; a cell potency; a cell viability, a possession by a cell of a marker; a metabolic state of the cell; or an ability of the cell to uptake or generate a substance. In some embodiments, for example, where the parameter concerns an identification (e.g., a cell identity, or a cell type), a digital encoding of cell identification or cell type may be transmitted to the controller 170. In some embodiments, for example, where the parameters concern information about cell heterogenous populations (e.g. different cell types within the cell population, percentage of each cell type), digital communication may take place so that the comprehensive information about cell types in the cell population may be transmitted to the controller 170.

[0087] The sample analysis module 150 may use one or more sensors 156 or a cell analyzer 158 (e.g., a flow cytometer) to detect, and generate values for, the one or more cell analysis parameters. For example, the sensors 156 may include a cell density sensor that may take images of the cells to measure the density of cells, and thereby determine a number of cells, in a sample or prepared sample with a predefined volume. In some embodiments, the cell analyzer 158 may be or include a flow cytometer that is configured to dispense a buffer to the test tube, the microfluidics cartridge or the manifold containing the sample or prepared sample. For example, the flow cytometer may include a container holding the buffer, for which a predefined amount is released into the test tube, microfluidics cartridge, or manifold positioned so as to receive the predefined amount of buffer. The release may occur in response to the flow cytometer being activated or commanded to perform measurements. The cell analyzer 158 may thus allow thebuffer to mix with the sample, and then have the sample incubate (e.g., for a predetermined period of time), before a flow cytometry analysis is performed. In some embodiments, the cell analyzer 158 may be portable (e.g., portable flow cytometer).

[0088] Furthermore, the sample analysis module 150 may include a network interface 159 to allow communication with the controller 170 and other modules over the communication network 160. The network interface 159 may share one or more subcomponents or functionalities as network interface 176. The network interface 159 may allow the sample analysis module 150 to transmit signals to other modules (e.g., the controller 170). For example, in response to the sample analysis module 150 generating values for one or more cell analysis parameters for a sample or prepared sample, the sample analysis module 150 may transmit a signal to the controller 170 reporting the values for the cell analysis parameters.

[0089] The communication network 160 may include wired and wireless networks. Examples of the wired networks may include a personal area network (PAN), wide area network (WAN) or a local area network (LAN), a client-server network, and so forth. Examples of the wireless networks include Wi-Fi and a general packet radio service (GPRS) network, an enhanced data GSM environment (EDGE) network, 802.5 communication networks, code division multiple access (CDMA) networks, Bluetooth networks or long term evolution (LTE) network, LTE- advanced (LTE- A) network or 5th generation (5G) network.

[0090] Figure 2 is a flowchart of an example method 200 for automatically sampling biological cells from a bioreactor for integrated cell analysis and production, according to a non-limiting embodiment of the present disclosure. Specifically, Figure 2 illustrates how the controller 170 and the integrated cell therapy modules 102 (e.g., the bioreactor module 110, the sample extraction module 120, the sample preparation module 130, or the sample analysis module 150) culture, sample, extract, prepare, analyze, and use feedback to further produce biological cells automatically in real-time or near real-time, which helps to reduce time and inconsistencies, and results in more effective cell therapy.

[0091] Method 200, shown in Figure 2, may be performed by the microprocessors of one or more of the integrated cell therapy modules (e.g., one or more microprocessors 111, 121, 131, or 151) and by one or more processors 172 of the controller 170 based on information received from the integrated cell therapy modules 102. Furthermore, the processors or microprocessors may perform the method 200 based on machine-readable or computer-executable instructions stored inmemory (e.g., memory 174). Although blocks are shown as being performed by one of the integrated cell therapy modules, the present disclosure contemplates that the performers may be interchanged or may perform one or more steps in parallel or in a different order than is shown, and that method 200 is a continuous process that may have several operations described in the various blocks performed multiple times over the course of operations. As used herein, the term “automatically” refers to a process performed without human intervention. Accordingly, method 200 may be initiated by or performed with respect to various parameters or input products that are initially set or provided by a human, and may conclude with a human user receiving an output product, but is performed atomically by the identified systems to generate that output product from the input product.

[0092] Method 200 may begin with block 210, where the bioreactor module 110 causes the culture of cells in a bioreactor. For example, the bioreactor module 110 may cause parameter controls 112 to activate or control one or more biorcactor parameters (e.g., a pH; a temperature; a level or concentration of a constituent or agent; or a run time) so that cells contained within a container of the bioreactor module 110 may grow. The activation or control of the one or more bioreactor parameters may be responsive to a signal received from the controller 170 or by user input.

[0093] In some embodiments, the cells may be obtained or derived from a subject. The subject may be a human, or a non-human animal (e.g., a mouse). In some embodiments, the subject has, or is at risk of having, a disorder, (e.g., a cancer). In some embodiments, the cells may be genetically altered to express one or more heterologous genes. Furthermore, in some embodiments, the cells are cultured for a therapy (e.g., a cell therapy) for the treatment or prophylaxis of a disorder. Non-limiting examples of such therapy may include but are not limited to an autologous cell therapy, an allogeneic cell therapy, a xenogeneic cell therapy, a stem cell therapy, an immunotherapy, or a combination thereof. For example, the cells may be cultured for an immunotherapy such as a chimeric antigen receptor (CAR) therapy, a tumor-infiltrating lymphocyte (TIL) therapy, or the like. In some embodiments (e.g., where the cells are cultured for immunotherapy), the cells may include immune cells (e.g., immune effector cells), T cells or natural killer (NK) cells. In some embodiments, the cells may be cultured for a stem cell therapy such as an embryonic stem cell therapy, neural stem cell therapy, mesenchymal stem cell therapy,or hematopoietic stem cell transplantation. Furthermore, the therapy may be differentiated or may include a mature cell transplantation.

[0094] At block 215, the sample extraction module 120 extracts a cell sample from the bioreactor module 110. For example, the microprocessor 121 of the sample extraction module 120 may cause an actuator 128 of the sample extraction module 120 to extend a receiver pump 123 to cause or facilitate the intake of a sample of the cells being cultured in the bioreactor module 110. In some embodiments, the sample extraction module 120 may additionally facilitate the transfer of the cell sample or an aliquot of the cell sample to another module (e.g., the sample preparation module 130 or the sample analysis module 150). For example, a sample dispensing needle 125 may be used to obtain a predefined amount of the extracted cell sample and transfer the amount to one or more of a test tube, a microfluidics cartridge, or a sample manifold or a microtiter plate of the sample preparation module 130.

[0095] At block 220, the sample preparation module 130 prepares the extracted cell sample to produce a prepared sample. For example, preparing the extracted cell sample may include identifying or registering the cell sample (e.g., via reading a barcode on the test tube, microfluidics cartridge, or sampling manifold or a microtiter plate via a barcode reader 142), moving the extracted cell sample to the staining and incubation station 140 of the sample preparation module 130, and staining and incubating the cell sample. The movement may be caused by robotic controls 134 of the sample preparation module 130, which may be activated, triggered, or commanded based on a signal received from the controller 170, a user input, or a detection of the extracted cell sample being received at the receiving port 136.

[0096] In some embodiments, the sample preparation module 130 may additionally facilitate the transfer of the prepared sample to the sample analysis module 150. For example, after the preparation of the cell sample (e.g., based on a timer detecting the end of incubation (e.g., based on a predetermined duration having occurred)), one or more of a tube movement arm, conveyor, or plate handler may move the prepared sample to the sample analysis module 150 or to the exterior of the sample preparation module 130 to facilitate the transfer to the sample analysis module 150.

[0097] At block 225, the sample analysis module 150 analyzes a parameter related to the cells being cultured or the cells in the sample (e.g., a cell analysis parameter). In some embodiments, the value for each cell analysis parameter is generated after the sample or prepared sample being received by the sample analysis module (e.g., receiving port 252).

[0098] For example, after the microprocessor 151 receives an indication of the receiving of the sample or prepared sample via receiving port 152, one or more sensors 156 or the cell analyzer 158 may be activated to detect cell analysis parameters of the sample or the prepared sample. The cell analysis parameters may relate to the cells being cultured or the cells in the sample or prepared sample. The cell analysis parameters may include but are not limited to: a cell identity or a cell type; a cell number; a cell purity; a cell size; a cell potency; a cell viability, a possession by a cell of a marker; a metabolic state of the cell; or an ability of the cell to uptake or generate a substance. In some embodiments, for example, where the parameter concerns an identification (e.g., a cell identity or a cell type), a digital encoding of cell identification or cell type is transmitted to the controller. In some embodiments, for example, where the parameters concerns information about cell heterogenous populations (e.g. different cell types within the cell population, percentage of each cell type), digital communication may take place so that the comprehensive information about cell types in the cell population may be transmitted to the controller. The sample analysis module 150 may thus use on one or more sensors 156 or a cell analyzer 158 (e.g., a flow cytometer) to detect, and generate values for, the one or more cell analysis parameters.

[0099] In some embodiments, a buffer is dispensed to the sample or prepared sample to facilitate the detection. The cell analyzer 158 may allow the buffer to mix with the sample, and then have the sample incubate (e.g., for a predetermined period of time), before a flow cytometry analysis is performed. The flow cytometry analysis may result in values for a variety of cell analysis parameters (e.g., cell identity or cell type, cell number, cell purity, cell size, cell viability etc.).

[0100] At block 230, the sample analysis module 150 transmits a signal for the analyzed parameter to the controller 170. In some embodiments, the signal includes the value of a cell analysis parameter, or may include a composite or set of values for a set of cell analysis parameters. In some embodiments, the signal may include digital signals containing cell analysis parameters that may be transmitted via the communication network 160 to the controller 170. In some embodiments, the signal may include an analog signal (e.g., an electromagnetic wave) sent wirelessly to the controller 170. For example the strength of the signal (e.g., an amplitude of an analog signal or electromagnetic wave) may be proportional to the value for the corresponding cell analysis parameters. The signal may be transmitted via the communication network 160.

[0101] In various embodiments, the sample analyses module 150, in response to transmitting the signal for the analyzed parameter, disposes of the prepared sample at block 260. The analyzed sample may be discarded (e.g., in a waste disposal receptacle) or returned to the bioreactor module, depending on the steps taken to prepare the extracted cell sample, contamination procedures specified for the bioreactor module 110, ease of returning the extracted cell sample from the plate or other analysis receptacle, and combinations thereof.

[0102] At block 235, once a signal is received from the sample analysis module 150, the controller 170 processes the signal to determine a response for the cell culture. For example, the response may pertain to the end goal of the cell culture occurring in the bioreactor module 110. In some embodiments, the end goal may be selected at the controller 170, which may result in a set of desired or ideal bioreactor parameter values. However, the received values from the cell analysis parameters may necessitate an adjustment of one or more of the preset bioreactor parameter values. The determined response may thus be modulated into a signal (e.g., a second signal) to be transmitted back to one or more integrated cell therapy modules (e.g., the bioreactor module 110).

[0103] In various embodiments, the controller 170 determines to alter the parameter in response to a value for an associated cell analysis parameter being outside of a window of acceptable values therefor, and identifies one or more actions for the bioreactor module 110 to take to bring the value back within the window. Additionally or alternatively, the controller 170 may determine to alter the parameter in response to an identified trend in the value for that parameter that is projected to place the value outside of the window (or a margin thereof) before the next scheduled sample is taken. For example, a parameter that is within the window at time tl and time t2 and is projected to be outside of the window at time t3 may result in the controller 170 generating a signal at time t2 (or before time t3) to alter the parameter before the value goes outside of the window at time t3.

[0104] Various scheduling and monitoring or triggering schemes may be used by the controller 170 and the integrated cell therapy modules 102 to determine whether any signal is received or a signal is ready for transmission.

[0105] At block 240, the controller 170 transmits a second signal of a control signal to the integrated cell therapy module (e.g., the bioreactor module 110) over the communication network 160.

[0106] At block 245, the bioreactor module 110 receives the second signal and determines whether the second signal indicates to alter various parameters for the culture of cells. The second signal may be processed to determine whether the response involves altering or not altering (and therefore maintaining) any one or more bioreactor parameters. The bioreactor parameters may include but are not limited to a pH; a temperature; a level or concentration of a constituent or agent (e.g., O2, CO2, an amino acid, a protein, a nutrient, a drug, a hormone, or a medium); or a run time for the cell culture process. When the bioreactor module 110 is signaled to modify a parameter, the bioreactor module 110 may draw media or other consumable material for encouraging or discouraging cell growth from various reservoirs, hoppers, or dispensers; activate or deactivate temperature control devices (e.g., heating elements, cooling tubes, fans); or the like.

[0107] If the response is to maintain a bioreactor parameter, or if no altering is found necessary, method 200 proceeds to block 250 where the bioreactor module 1 10 maintains the biorcactor parameter accordingly. In various embodiments, when the biorcactor parameter includes an ongoing supply, flow, or mechanical setting (e.g., fan or agitator), maintaining the bioreactor parameter includes maintaining operation of the device affecting the supply, flow, or mechanical setting at a current setting (e.g., continuing to supply a reagent at a current flow rate, continuing to agitate the sample at a current revolutions per minute).

[0108] If the response is to alter a bioreactor parameter, method 200 proceeds to block 255 where the bioreactor module 110 alters the bioreactor parameter accordingly (e.g., via parameter controls 112). In various embodiments, the extent of the alteration is based on a difference between the measured value and a window for the cell growth analysis parameter, such that larger differences result in more drastic alterations than smaller differences to bring the parameter back within the window. Alternations may include the addition of acids, bases, buffer media, therapeutic agents, water, O2, CO2, and various growth media, which may be introduced into the bioreactor module 110 from various reservoirs maintained by the system. Additionally or alternatively, the bioreactor module 110 may activate or deactivate various fans, cooling elements, heating elements, agitators, or the like to change the temperature, atmosphere, or exposed surfaces of the cells being cultured to alter the bioreactor parameter, or terminate the modification thereof once a previously out-of-window value is brought back in-window.

[0109] After block 250 or block 255, the bioreactor module 110 continues culturing of the cells at block 265, and one or more steps of method 200 may be repeated. Accordingly, the method200 may perform several iterations in which one of block 250 or block 255 is performed to adjust cell culture without human intervention, but in which the system automatically adjusts or maintains parameters over a period of time per desired growth parameters. In various embodiments, the duration for which method 200 is preformed may last for one day, two days, three days, four days, five days, six days, seven days, etc. with various samples being extracted and examined at various sampling rates, which may include having a sample taken for analysis at scheduled intervals (e.g., at least once every 30 minutes, one hours, two hours, three hours, six hours, twelve hours, twenty- four hours, thirty-six hours, forty-eight hours, seventy-two hours, eighty-four hours, ninety-six hours, one hundred twenty hours, one hundred forty-four hours, or one-hundred sixty-eight hours) or in response to triggering conditions (e.g., a value for a different cell analysis parameter or environmental condition). The duration between successive samples may therefore be equivalent or different over the duration of cell culturing, and different cell analysis parameters may be analyzed at the same or different rates.

[0110] Figure 3 is a flowchart of an example method 300 for automatically managing the growth and culturing of a cell sample for use in research, treatment, or prophylaxis, according to embodiments of the present disclosure.

[0111] Method 300 begins at block 310 where an operator extracts a cell sample from a biological subject. In various embodiments, the cell sample may be extracted as a tissue, blood, or fluid sample from the biological subject, which may be intended for return to the same or a different biological subject from which the cell sample was extracted after growth and culturing. In various embodiments, the operator may filter or select a subset of the extracted cells from the biological subject to constitute the cell sample (e.g., to exclude unwanted cells or media).

[0112] At block 320, an operator inserts the cell sample into a bioreactor (e.g., the bioreactor module 110). In various embodiments, the operator may deposit the cells into various containers or holding vessels for insertion into the bioreactor, or may provide the cell sample to a port or an actuator-platform assembly that accepts the cell sample from the operator and automatically transfers the cell sample into a culturing chamber of the bioreactor. In various embodiments, the cell sample may be placed into various sample containment vessels that the sample may remain in for the duration of growth / culture, including but not limited to: test tubes, wells of microtiter plates, microfluidics cartridges, fluidic manifolds, or the like.

[0113] At block 330, the bioreactor automatically grows and cultures the cells from the cell sample. This process is automated, and requires no further input from an operator after accepting the cell sample until delivering the cell sample as cultured back to an operator. Accordingly, the system may operate based on instructions received before a first time and grow / culture a cell sample until a second time without receiving further instructions from an operator between the first and second times. Various conveyors, robotic arms, pumps, syringes, and the like may be used to automatically move some or all of the cell sample between various modules of the system, which are programmatically controlled to adjust the growth conditions by changing one or more parameters (e.g., adding growth media / buffers / therapeutic agents, changing temperatures / humidifies / atmospheric conditions, agitating the cell sample, etc.). These instructions may include time- scheduled or trigger-based operations so that various operations are performed at prescribed times or in response to a sensor identifying a particular parameter in the cell sample or growth environment.

[0114] The operations performed automatically by the bioreactor allow for the system to be closed - thereby reducing the possibility of introducing human error, improving environmental control for cell growth / culture, reducing the escape of controlled atmosphere, reducing the risk of outside contamination, reducing cleaning requirements during or after operation, and extending the operational period beyond when human operators are available to monitor the system, among other benefits.

[0115] At block 335, which may be understood with reference to method 200 discussed in relation to Figure 2, the bioreactor automatically controls the culture of the cell sample for the duration of the growth and culture operator of block 330. The system monitors the growth of the cells in the bioreactor to set or maintain growth conditions for a desired output from the cell sample. In various embodiments the system maintains various cell parameters within predefined windows for those values over the course of cell growth / culturing, and may adjust various parameters by changing environmental conditions within the bioreactor, adding various reagents to the cell samples, agitating the cell samples, etc., to maintain or bring various parameters back within the windows. In various embodiments, the windows may remain static for the duration of culturing, or may change over the course of culturing according to predefined patterns for cell growth / culture.

[0116] Block 335 may be performed continuously between a first time and a second time for a duration of cell growth and modification, and various readings of cell growth parameters are taken at intermediate times thereto to determine how to adjust or maintain cell growth within the desired windows. The bioreactor may remain self-contained during this time period, and operate automatically based on instructions received exclusively before the first time. Accordingly, an operator may leave the system to operate per these instructions with no further action on the operator’s part until the cultured / modified cells are ready for retrieval.

[0117] At block 340, an operator retrieves the cultured / modified cells from the bioreactor. Depending on the intended use for the cultured / modified cells, method 300 may conclude at block 340, and the operator may discard the cultured / modified cells or perform further experiments or assays on the sample outside of the bioreactor, or return some or all of the samples to the bioreactor for continued or alternative automated culturing or modification.

[0118] In various embodiments, the growth and culture of the cell sample may be part of an immunophenotyping assay, cell activation and proliferation assay, immune-cell induced cytolysis assay, cell metabolism measurement assay, cytokine measurement, or the like. In some embodiments, the growth and culture of the cell sample is used as a precursor for the treatment or prophylaxis of a disorder in a biological subject, which uses a cell culture grown from and / or modified from the cell sample as a therapeutically effective dose. These grown / modified cells may include: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; and cells that are cultured for a stem cell therapy. When the sample is to be used in a therapeutic treatment, method 300 may proceed to block 350.

[0119] At block 350, an operator supplies the cultured / modified cells to a biological subject for the treatment or prophylaxis of a disorder that is treatable, preventable, or manageable via application of the cell culture (or is experimentally supplied therefore). In various embodiments, the operator may mix the cells with various buffers, delivery media, contrast agents, additional therapeutic agents or the like before delivery to the biological subject. The biological subject may be the same biological subject from whom the cell sample was originally extracted (per block 310) or a different subject. The present disclosure contemplates that the person of ordinary skill in the art will be able to determine what dose constitutes a therapeutically effective dose, which may be affected by the age, weight, gender, genetics, metabolism, comorbidities, tolerances, additionaltherapeutic agents in the system of, and combinations thereof of the biological subject. Method 300 may then conclude.

[0120] Figure 4 illustrates an example process set-up for automatically sampling biological cells from a bioreactor for integrated cell analysis and production is described, where a test tube is used as a cell sample container. The test tube 420 is moved through various integrated cell therapy modules 202, and is used to conduct a flow cytometry assay (e.g. an immunophenotyping assay). While flow cytometry may be used as an illustrative example of an assay, other cell-based assays may also be performed (e.g. cell activation and proliferation assay, immune-cell induced cytolysis assay, cell metabolism measurement assay, cytokine measurement). Similarly, although a test tube base approach may be used, microfluidic cartridges, of manifolds may be used in offline integrated cell therapy systems, at-line integrated flow cytometers, at-line microfluidics chip-based flow cytometers and combinations thereof. For some of the assays, the sample cells in the test tube may be transferred into the wells of a microtitcr plate. For such assays, additional sample handling and / processing may be required for conducting the appropriate cell-based assays.

[0121] As shown in Figure 4, a pump 410 may be used to extract a sample of cells from a bioreactor module 110. In some embodiments, the bioreactor module 110 pre-mixes the sample before the specified volume of the sample is pumped out. The sample of cells may fill a sample dispensing needle 125, which is used to place the sample of cells into test tubes 420. In some embodiments, the sample pump 410 or the sample dispensing needle 125 are designed for accurately and effectively dispensing the specified volume of sample from the bioreactor module 110. In some embodiment, the sample dispensing needle 125 allows for motion in Cartesian (x, y, and z) directions, and may be used to pierce through the test tubes 420. In some embodiments, the test tubes 420 are pre-loaded with reagents (e.g., lyophilized antibody cocktails) to facilitate flow cytometry assays.

[0122] At specified time, a robotic arm 430 (e.g., controlled via robotic controls 134 of the sample preparation module 130) may move a test tube 420 from a pre-defined (e.g., dedicated) tube position (with information of antibody defined), cause a barcode reader 440 to read a barcode from the test tube 420, and transfer the test tube 420 to a sample dispensing position (e.g., a dedicated position to match the sample dispensing probe position of a bioreactor module 110).

[0123] Additionally, the robotic arm 430 moves the test tube 420 to the sample preparation module 450 (e.g., a staining and incubation station of the bioreactor module 1 10). At the samplepreparation module 450, a specific volume of staining buffer is dispensed into the test tube 420, allowing the sample to incubate. In some embodiments, a gentle agitation may be used to facilitate incubation.

[0124] After a specified incubation time, the robotic arm 430 moves the test tube 420 into a flow cytometer 460 to allow for the flow cytometry measurement. The type of flow cytometer used in this example is specifically selected so that the cytometer has a smaller foot-print or has a more compact form, relative to other cell analyzers. In some embodiments, flow cytometry software performs an automatic analysis on the acquired flow cytometry measurement data. Optionally the analyzed results can be automatically transmitted, as feedback, to a bioreactor software (e.g., integration application 184 of the controller 170), which can provide real time control, change, or adjustment of cell culture conditions (e.g., bioreactor parameters). In some embodiments, the robotic arm 430 for test tube movement, sample staining station, and barcode reader is integrated into a single software control- system (e.g., the controller 170).

[0125] The integrated software (e.g., integration application 184) is used for controlling the workflow, editing the working protocol, receiving and relaying feedback from the analyzed flow cytometry data (e.g., CD3 % & count, CART cell % & count, etc.) to the bioreactor module 110. The bioreactor module 110 may modify various bioreactor parameters (e.g., cell culture controls) to adjust the culture condition according to the results measured from the flow cytometer 460.

[0126] Advantages of this example system and process include, but are not limited to, there being a closed system and a streamlined workflow for integrated cell therapy, data traceability with optional bar code-reading of the bar codes on the test tubes 420, a single sampling via a single test tube 420, and the lack of need for rinsing or cleaning of fluidics path, resulting in a cleaner architecture.

[0127] Although Figure 4 illustrates one process set-up, the present disclosure contemplates that the methodologies described herein may be practiced with various hardware to analyze cells for various characteristics.

[0128] Figure 5 illustrates a computing device 500, as may be used as the controller 170 or a localize control device for a module, according to embodiments of the present disclosure. The computing device 500 may include at least one processor 510, a memory 520, and a communication interface 530.

[0129] The processor 510 may be any processing unit capable of performing the operations and procedures described in the present disclosure. In various embodiments, the processor 510 can represent a single processor, multiple processors, a processor with multiple cores, and combinations thereof.

[0130] The memory 520 is an apparatus that may be either volatile or non-volatile memory and may include RAM, flash, cache, disk drives, and other computer readable memory storage devices. Although shown as a single entity, the memory 520 may be divided into different memory storage elements such as RAM and one or more hard disk drives. As used herein, the memory 520 is an example of a device that includes computer-readable storage media, and is not to be interpreted as transmission media or signals per se.

[0131] As shown, the memory 520 includes various instructions that are executable by the processor 510 to provide an operating system 522 to manage various features of the computing device 500 and one or more programs 524 to provide various functionalities to users of the computing device 500, which include one or more of the features and functionalities described in the present disclosure. One of ordinary skill in the relevant art will recognize that different approaches can be taken in selecting or designing a program 524 to perform the operations described herein, including choice of programming language, the operating system 522 used by the computing device 500, and the architecture of the processor 510 and memory 520. Accordingly, the person of ordinary skill in the relevant art will be able to select or design an appropriate program 524 based on the details provided in the present disclosure.

[0132] The communication interface 530 facilitates communications between the computing device 500 and other devices, which may also be computing devices as described in relation to Figure 5. In various embodiments, the communication interface 530 includes antennas for wireless communications and various wired communication ports. The computing device 500 may also include or be in communication, via the communication interface 530, one or more input devices (e.g., a keyboard, mouse, pen, touch input device, etc.) and one or more output devices (e.g., a display, speakers, a printer, etc.).

[0133] Although not explicitly shown in Figure 5, it should be recognized that the computing device 500 may be connected to one or more public or private networks via appropriate network connections via the communication interface 530. It will also be recognized that softwareinstructions may also be loaded into the memory 520 from an appropriate storage medium or via wired or wireless means.

[0134] Accordingly, the computing device 500 is an example of a system that includes a processor 510 and a memory 520 that includes instructions that (when executed by the processor 510) perform various embodiments of the present disclosure. Similarly, the memory 520 is an apparatus that includes instructions that when executed by a processor 510 perform various embodiments of the present disclosure.

[0135] Figures 6A-6L show cell analysis parameters for CAR-T cells produced in G-REX® 24-well bioreactor devices (available from Wilson Wolf Corp.), according to embodiments of the present disclosure. The data demonstrate that the characteristics of CAR-T cells such as cell number, cell potency, cell type compositions, cell ATP production rate and / or cell spare respiratory capacity change as CAR-T are cultured and manufactured in the bioreactor, illustrating the need for preforming integrated cell analysis for cells sampled from a biorcactor to guide an cell culture time for high cell quality.

[0136] Figure 6A shows the cell number folder change on day six, eight, and ten relative to cell number in day zero when cells are introduced into G-REX 24-well devices. Cells were cultured at a volume density of 0.33xl06cells / ml (i.e., 1.36xl06 / cm2) in a XV1VO 15 medium with 5% human serum and 200 U / ml IL2 (after an overnight rest in the medium without IL2). UT refers the un-transduced CART sample, CAR-T refers to EMCAM CAR transduced samples for which the CAR transduction occurred on day one. The cell expanded well in the Grex 24-well bioreactor devices, by day ten, up to 19.2-fold for the un-transduced T-cells (UT), and 17.1-fold for the EpCAM CAR transduced T-cells (CAR-T).

[0137] Figure 6B shows the change of percentage of CD3+ and CAR+ cells during the course of cell culture in G-REX 24-well bioreactor devices. EpCAM CAR-positive and CD3-positive cells changed during the course of cell culture, reached 15% on day six and gradually decrease to 9.5% on day ten.

[0138] Figure 6C shows the change of the percentage of CD3+ cells within total cell population, and percentage of CD4+ and CD8+ cells within CD3+ population during the course of cell culture. CD4 / CD8 ratio changed over time. As the cell production progressed, percentage of CD8+ cells increased and correspondingly percentage of CD4+ cells decreased.

[0139] Figure 6D shows the change of the percentage of different T cell sub-types within total cell population. Tn: naive T cells, Tcm: central memory T cells, Tscm: stem-cell like central memory T cells, Tern: effector memory T cells and Temra: terminally differentiated effector memory T cells. As the cell cultured progressed, more fully differentiated effector cells (Temra) and effector memory T cells (Tern) appeared. Simultaneously, the percentage of central memory cells (Tcm) decreased over time.

[0140] Figure 6E shows the time course of percentage of cytolysis of T47D cells as killed by CAR-T cells harvested on day six of cell culture on G-REX 24-well devices. The E:T ratio (i.e. Effector to Target cell ratio) here was calculated based on the total effector cells, which includes CAR T-cells and un-transduced cells. UT refers to un-transduced control sample and CAR refers to EpCAM CAR transduced cell samples.

[0141] Figure 6F shows the time course of percentage of cytolysis of T47D cells as killed by CAR-T cells harvested on day eight of cell culture on G-REX 24-well devices. The E:T ratio here was calculated based on the total effector cells, which includes CAR T-cells and un-transduced cells.

[0142] Figure 6G shows the time course of percentage of cytolysis of T47D cells as killed by CAR-T cells harvested on day ten of cell culture on G-REX 24-well device. The E:T ratio here was calculated based on the total effector cells, which includes CAR T-cells and un-transduced cells.

[0143] Figure 6H shows the cell potency parameter of KT50 - the time for reaching 50% cytolysis based on time course curve for % cytolysis, for EpCAM Cai’ T cells harvested on day six, eight, and ten. The E:T ratio here was calculated based on the total effector cells, which includes CAR T-cells and un-transduced cells. The killing potency of the EpCAM CAR T-cell gradually decreased over time from day six to day tend, as demonstrated by an increase in KT50 for E:T ratio 4:1 samples.

[0144] Figure 61 shows the cell potency parameter of Slope - the slope of time course curve for % cytolysis, for EpCAM Cai’ T cells harvested on day six, eight, and ten. The E:T ratio here was calculated based on the total effector cells, which includes CAR T-cells and un-transduced cells. The killing potency of the EpCAM CAR T-cell gradually decreased over time from day six to day ten, as demonstrated by a decrease in slope for E:T ratio 4:1 samples.

[0145] Figure 6J shows the cell potency parameter of AUC of % cytolysis - the Area Under the Curve for time course curve of % cytolysis (0-48 hrs), for EpCAM Car T cells harvested on day six, eight, and ten. The E:T ratio here was calculated based on the total effector cells, which includes CAR T-cells and un-transduced cells. The killing potency of the EpCAM CAR T-cell gradually decreased over time from day six to day ten, as demonstrated by a decrease in 48-hr AUC for % cytolysis curve for E:T ratio 4:1 samples.

[0146] Figure 6K shows the ATP production rate for CAR T cells on day six, eight, and ten as they were cultured in G-REX 24-well devices. ATP production rate of CAR T cells was measured in the XF assay, and gradually decreased over time. In addition, CART cells produced in G-REX devices are more mitochondrial bioenergetic than glycolytic

[0147] Figure 6L shows the spare respiratory capacity of CAR T cells on day six, eight, and ten as they were cultured in G-Rex 24-well devices. Spare respiratory capacity of CAR T cells was measured in the XF assay and gradually decreased over time.

[0148] Figures 7A-7F show the cell analysis parameters for T cells as cultured in presence of different cytokines (IL2, or IL7 or IL15) in G-REX 24 well devices, according to embodiments of the present disclosure. The data demonstrate T cells have different characteristics such as cell potency, cell ATP production rate, and cell spare respiratory capacity which depend on which cytokines are present in cell culture media, illustrating that the choice or change of cell culture media condition may allow the control or improvement of cell quality based on measured cell analysis parameters.

[0149] Figure 7A shows the time course of percentage of cytolysis of T-47D cells as killed by T cells, via BITE molecule-mediated engagement mechanism, harvested on day seven of cell culture in presence of different cytokines IL-2, IL-7 or IL 15, in G-REX 24-well devices. Seeding density of T47D target cells was 8,000 cells / well, and the effector cells were added at 24-40 hours after target cell seeding at an effector to target cell ratio of 6: 1 for the potency assay. CD3xEpCAM BiTE concentration: 500 ng / ml. The T cells expanded in presence of IL- 15 showed higher % cytolysis (higher killing potency) of target T47D (an EpCAM positive cancer cell line) as compared with the T cells expanded in presence of IL-2 and IL-7.

[0150] Figure 7B shows the time course of percentage of cytolysis of T-47D cells as killed by T cells, via BITE molecule-mediated engagement mechanism, harvested on day ten of cell culture, in presence of different cytokines IL-2, IL-7 or ILL5, in G-REX 24-well devices. Seeding densityof T47D target cells was 8,000 cells / well, and the effector cells were added at 24-40 hours after target cell seeding at an effector to target cell ratio of 6:1 for the potency assay. CD3xEpCAM BiTE concentration: 500 ng / ml. The T cells expanded in presence of IL- 15 showed higher % cytolysis (higher killing potency) (higher killing potency) of target T47D (an EpCAM positive cancer cell line) as compared with the T cells expanded in presence of IL-2 and IL-7.[01511 Figure 7C shows the ATP production rate for T cells on day seven as they were cultured in presence of IL2, IL7 or IL15 in G-REX 24-well devices. The T cells expanded in presence of IL- 15 showed higher ATP production rate, as compared with the T cells expanded in presence of IL-2 and IL-7.

[0152] Figure 7D shows the spare respiratory capacity of T cells on day seven as they were cultured in presence of IL2, IL7 or IL15 in G-REX 24-well devices. The T cells expanded in presence of IL- 15 showed higher Spare Respiratory Capacity, as compared with the T cells expanded in presence of IL-2 and IL-7.

[0153] Figure 7E shows the ATP production rate for T cells on day ten as they were cultured in presence of IL2, IL7 or IL15 in G-REX 24-well devices. The T cells expanded in presence of IL- 15 showed higher ATP production rate, as compared with the T cells expanded in presence of IL-2 and IL-7.

[0154] Figure 7F shows the spare respiratory capacity of T cells on day ten as they were cultured in presence of IL2, IL7 or IL15 in G-REX 24-well devices. The T cells expanded in presence of IL-7 showed higher Spare Respiratory Capacity, as compared with the T cells expanded in presence of IL-2 and IL- 15.

[0155] The cells whose parameters shown in Figures 6A-6L and Figures 7A-7F were prepared, cultured and analyzed according to the methods described herein. As will be appreciated, the experimental processes described herein involve the use of live cells.

[0156] In the preparation of the stalling materials, a first operation is performed the day before cell production or expansion, by thawing CD3 enriched (i.e. CD4+ and CD8+ positive selected) PBMC samples (i.e., Peripheral Blood Mononucleated Cells). This may be done by removing the cell vials from the -150°C freezer and thawing the cell vials in a water bath (e.g., for 2 minutes) until only a small ice pellet remains.

[0157] In a second operation, an operator or robot, using an aseptic technique and a pipette, gently transfers cell suspensions from the cryovial to a 50 mL centrifuge tube. The operator or robot then may add the thawing medium to the cell suspension at 1:20 dilution.

[0158] In a third operation, an operator or robot, dropwise adds 5 mL of warmed thaw media to the expansion medium, but without the growth factor, such as IL2, at a rate of about 1 drop every 10 seconds, swirling occasionally. The operator or robot then brings the volume of each cell suspension at a final dilution of 1:20 with the remaining warmed thawing media dropwise.

[0159] In a fourth operation, the cells are centrifuged at 250G (250 times the force of gravity) for ten minutes at room temperature (RT).

[0160] In a fifth operation, the operator or robot, resuspends the cells in the thawing medium at 2-5 xlO6cells / ml overnight in the incubator at 5% CO2 and 37°C.

[0161] In the CAR-T cell production (or T cell expansion), a first operation is performed on day zero, the immune cells prepared in the Preparation operations above arc centrifuged at 400G (400 times the force of gravity) for eight minutes and resuspended in the activation media (i.e., the expansion medium supplemented with activation solution), such as CD3 and CD28, at 0.5-1 xlO6cells / ml.

[0162] In a second operation, on day one for CAR-T cell production (e.g., for data in Figures 6A-6L): the activated immune cells are transduced by lentiviruses carrying a CAR construct for one day. In a second operation, on day one for T cell expansion (e.g., for data in Figures 7A-7F): the activation media is replaced with expansion media (100% medium change).

[0163] In a third operation, on day two, 100% medium change is performed by replacing the inoculation medium with the pre-warmed expansion medium.

[0164] In a fourth operation, the medium is refreshed every 2-3 days depending on the cell culture vessel used in the expansion.

[0165] For cell sample collection during the process, the produced CART cells or expanded T cells were sampled from G-REX 24-well devices on the desired days during the expansion / production for the in-process analysis.

[0166] To perform a cell count, in a first operation, the cells are well mixed by pipetting the cell suspension in the G-REX 24 well devices up and down three times.

[0167] In a second operation, the desired amount of cell suspension is collected from the G- REX 24 well devices using a pipette or serological pipette.

[0168] In a third operation, a 10 pL cell sample is mixed with 90 pL cell staining buffer containing 1 pL 7AAD (cell viable dye) and incubated for five minutes, followed by cell count using a flow cytometer system (e.g., the NOVOCYTE® QUANTEON®, offered by Agilent Technologies, Inc.), and the total cell number and total viable cell number are recorded.

[0169] To split cells for the follow-up analytical assays, in a first operation, the desired volume of cultured CART cells or expanded T-cells for each assay are aliquoted, and the volume of cell suspension is calculated based on the concentration of cell suspension (total cells or viable cells), seeding density (number of cells / well) for each assay, the number of replicates (N) using the following equation:Volume of cells (ml) = seeding density X N / concentration of cell suspension (number of cells / ml).

[0170] For, the XCELLIGENCE functional potency assay, use the concentration of viable cells. For XF bioenergetics assays, use the concentration of total cells. For immunophenotyping using a flow cytometer, use the concentration of viable cells.

[0171] In a second operation, the cell suspension is centrifuged at 250G for five minutes.

[0172] In a third operation, the cells are resuspended in the proper buffer used in each followup assay.

[0173] For the XCELLIGENCE functional potency assay, shown in the figures, an Agilent XCELLIGENCE RTCA MP or ESIGHT instrument is used for the functional potency / killing assay, but other laboratory assay instruments may be used in various embodiments.

[0174] In the functional potency assay, target cells are prepared the day before the potency assay. In a first operation of target cell preparation, pre-warmed 50 pL of potency assay medium, e.g., RPMI 1640 with 10% FBS, is added to the wells of an E-Plate, followed by the plate background measurement on the RTCA system. In a second operation, adherent target cancer cells expressing CAR-antigen for an antigen recognized by a bispecific T-cell engager (BiTE) are disassociated from a cell tissue culture vessel. In this example, the EpCAM antigen expressing T- 47D was used. In a third operation, 50 pL of cell suspension is added to each well of E-Plate that contains 50 pL assay medium at an optimal seeding density, e.g. T-47D cells at a seeding density of 8,000 cells / well. In a fourth operation, the cell plate is left in the lamina flow hood at roomtemperature for thirty minutes for an even cell distribution in the well. In a fifth operation, the cell plate is engaged in the RTCA station, and data recording is started at 15-minute intervals for the entire duration of the experiment. In a sixth operation, data acquisition is paused when adding the immune cells to the target cell plate.

[0175] In the preparation of the effector cells on the assay day, in a first operation, immune cells are collected and counted as described previously.

[0176] hr a second operation, the number of immune cells is added according to the different effector-to-target (E:T) ratios (e.g. 8:1, 4:1, 2:1, and 1:1, E:T ratio was based on the total cell, which includes both CAR-T cells and un-transduced cells, data shown in Figures 6A-6L) in a volume of 100 pL. Multiple E:T ratios are recommended, including high intermediate, and low E:T ratios. The E:T ratios here are calculated based on 100% CAR-T cells. For BiTE killing potency assay (data shown in Figures 7A-7F), a certain concentration of BiTE (e.g., 500 ng / ml CD3XEpCAM BiTE) is added to the immune cells before adding effectors to the target cell plate.

[0177] In a third operation, the effector cell-only controls, target plus mock effector controls or target plus un-transduced T-cells, target cell-only controls, and a full lysis control (treat target cells with a final concentration of 0.25% Triton X-100) according to the plate layout map. Set up at least five replicates for each condition. The user can modify the plate layout according to their experiment design. However, including the right controls and adequate replicates for each sample and control in the same plate is critical for proper data interpretation and obtaining reliable potency data.

[0178] In a fourth operation, after effector cell addition, the E-Plate is placed back into the RTCA station and real-time recording is immediately resumed at 15-minute intervals for up to two days. The duration of the potency assay could vary depending on the purpose of the potency test. For cell product QC, 6-24 hours is recommended. For the process development, 48-72 hours is recommended.

[0179] The real-time measurement of impedance is initiated right after the seeding of target cells in the E-Plate (e.g., within 30 seconds). However, the changes in impedance are reported as Cell Index (Cl). The Cl is further normalized to the time point right before CAR T-cell addition, named Normalized Cell Index (NCI).

[0180] In various embodiments, various key parameters are used which may include, but are not limited to: 1 ) Percentage of cytolysis (% cytolysis): to determine the level of immune-cell-mediated killing. The percentage of cytolysis utilizes the Normalized Cell Index from the samples (NCIs) and the average Normalized Cell Index from the target alone control (NCIt) according to the following equation, % Cytolysis = [1- NCIs / NCIt] *100; 2) Area Under the Curve (AUC): to convert real-time kinetic data to an endpoint readout. AUC of the percentage of cytolysis is to calculate the area under the time course of % cytolysis, starting from the Normalized Time point that corresponds to the time when effectors are added to the target cells to the selected time point on the curve. The larger the AUC of % cytolysis more potent the effectors would be; 3) KT: to determine the speed of the immune cell-mediated cell killing. KT is measured as the time of killing and is calculated starting from the Normalization Time Point that corresponds to the time when effectors and other relevant conditions are added to samples. The RTCA Pro software provides the options for KT of 20, 40, 50, 60, and 80% cytolysis; and 4) The Slope: to describe the steepness, incline, gradient, or changing rate of a curve within a given time window. For each selected well, the Software calculates the Slope of the Cell Index (or Normalized Cell Index) curve over a chosen Time frame. Data points within this time frame are fit to a straight line.

[0181] In the XF Assay, the sample(s) collected from the expansion system (at least 1-2 x 106cells per sample) are, in a first operation, centrifuged (10 min x 1000 g) and cells were resuspended in an appropriate volume of assay media (e.g., XF RPM1 Assay Media pH 7.4 containing 10 mM Glucose, 1 mM Pyruvate and 2 mM Glutamine) to reach the recommended cell density for the cell type of analysis (for example, for pre-activated T cells, a cell density of 2 mill total cells / mL is recommended). A sample (typically 50 pL) of the cell suspension was added to the same volume of buffer containing the viability dye 7- A AD (2X) and the total and live cell numbers in the sample were counted using the NOVOCYTE flow cytometer. If cell density was higher or less than 60% of the recommended density, cell suspension volume was adjusted to reach the desired cell density range, and the cell sample was counted again to confirm the final cell density. A sample (typically 50 uL) of the cell suspension are seeded in XF96 multiwell plates pre-coated with PDL and prewarmed overnight at 37C (in general 3 or more replicate wells per cell sample). Multiwell plates are centrifuged, (e.g., for one minute at 100G), assay media is added to complete the recommended volume for the particular plate type (e.g., 200 pL) and incubated at 37°C in a non-CO2 incubator for forty-five minutes.

[0182] In a second operation of the XF assay, the appropriate XF SEAHORSE Analyzer is programmed with command instructions to conduct, (e.g., three measurements, inject sequentiallythe solution from the ports in a cartridge disposed above the cell sample in a well and conduct three measurements after each injection).

[0183] In a third operation of the XF assay, the following metabolic modulators working solutions were prepared: Oligomycin A stock solution is prepared to a working concentration of 13.5 pM in assay media. BAM15 stock solutions is prepared at an optimized concentration (generally 25 pM for human T cells) and rotenone plus antimycin A mix stock solution is prepared to a working concentration of 5.5 pM each.

[0184] In a fourth operation of the XF assay, a sufficient volume of each modulator working solution is added to an assay cartridge such that upon injection the working solution is diluted into the assay medium to the final desired concentration. For example, in human T cells, the final desired concentration is 1.5 pM of oligomycin A, 2.5 pM of BAM15 and 0.5 pM of Rotenone plus Antimycin A mixture. These concentrations were determined by titration for optimal effectiveness.

[0185] In a fifth operation of the XF assay, the hydrated assay cartridge containing the indicated reagent is loaded into the instrument.

[0186] Figures 8, 9, 10, 11A-11C, and 12A-12C are provided with respect to an experimental validation of the concepts presented in the present disclosure, and demonstrate the surprising efficacy of the techniques and methodologies discussed herein. The present disclosure contemplates that the general results discussed in the example herein may be substantially replicated with altered experimental set ups, within the spirit and scope of the concepts discussed herein, to demonstrate that low-quality cell samples can be used to manufacture high-quality therapeutic manufactured cells.

[0187] Chimeric antigen receptor (CAR) T-cell immunotherapies show astonishing promise as a treatment for cancer and other diseases; however, many challenges remain, including unaffordable manufacturing processes, short-term persistence of cells, lengthy manufacturing processes, and poor cell expansion. These concerns are present in both allogeneic (also referred to as heterologous) and autologous therapies (in which the basis cells are respectively provided from a different or the same biological subject to whom the manufactured cells are provided), but are felt particularly keenly in autologous therapies. Although autologous therapy reduces the immune rejection risk of the manufactured cell, the quality of the starting cells for therapeutic cell production can vary depending on the health of the individual health conditions of the patient. Accordingly, clinicians may evaluate the health of a patient before extracting cells for use as thebasis of an autologous therapy, thereby delaying treatment for the health of the patient to reach certain threshold and potentially delaying treatment via an allogeneic therapy if the patient cannot exhibit threshold health criteria. Hence, the presently described CAR-T product manufacturing processes involves ex vivo expansion of T cells to obtain enough viable T-cells for infusion into patients from whom the basis cells for manufacturing were extracted, this approach should be also used for allogeneic cell therapy instead of limiting it to autologous cell therapy. Although, the present disclosure provides examples primarily with respect to an autologous approach, the present disclosure contemplates that even given the starting cells used in allogeneic cell therapy manufacturing come from healthy donors and meet the criteria for proceeding with the product, variability between donors still exists, and that the present disclosure may advantageously be used with allogeneic treatments.

[0188] Therefore, using identical cell culturing conditions to expand autologous adoptive cell therapies may not be the most effective approach for producing immune cells with high efficacy and good persistence. Additionally, apart from the number of cells, what qualifies as having “high efficiency or good persistence” has not been, to date, defined in any standards by medical bodies (such as the Food and Drug Administration (FDA) in the United States), so determining whether an experimental process can produce improved results for patients has been left for individual experimentation and definition. Nonetheless, retrospective studies have correlated better disease remission rates with the enrichment of less differentiated T-cell subsets in leukapheresis products.

[0189] T-cell differentiation is a progressive process characterized by phenotypic and functional changes. Following the theory of a linear hierarchical system, upon antigen exposure, naive T-cells undergo proliferative expansion and differentiation into memory T-cell subsets culminating into terminally differentiated effector T-cells. During this differentiation process, T- cells mature and progressively acquire effector functions while losing their self-renewal and persistence abilities.

[0190] Advances in multiparameter flow cytometry over the past decades have enabled dissection of the heterogeneity of the T-cell phenotyping with growing precision. Moreover, the combinatorial expression of markers such as CD3, CD4, CD8, CD45RA, CD45RO, CCR7 and CD95 have allowed the identification of Stem cell memory T-cells (Tscm), Central memory T- cells (Tern), effector memory T-cells (Tern) and terminal effector T-cells (Teff). Tscm subset represents the earliest and longest-lasting developmental stage of memory T-cells, displaying stemcell-like properties, and exhibiting a gene profile between naive and central memory T cells. As such, Tscm self-renewal capacity, proliferative potential, telomere length and long-term survival has sparked interest in the cancer therapy field. Indeed, Tscm cells have shown properties of high self-renewal, high proliferative and superior antitumor responses compared with other memory T- cell subsets in adoptive T-cell therapy studies in mice, and evidence suggests that adjusting certain process parameters, such as cytokines, pH, dissolved oxygen level, amino acid concentrations, temperature, and the types of bio-reactors used can successfully affect T-cell expansion and differentiation state in ex vivo cultures with an emphasis on generating higher numbers of less differentiated T-cells. Due to the relatively low frequency of Tscm in the peripheral blood, expansion techniques to increase the frequency of Tscm subsets prior to adoptive transfer is encouraged. Hence, any methods that could guide the generation of higher quantities of Tscm subset could be coupled with any genetically engineered T-cell therapy for use in cancer treatment. Hence, the development of advanced process analytical tools and methods that can automate and integrate the processes of cell production, cell sampling, sample monitoring, and cell analysis, such that feedback from these processes can efficiently guide and improve cell number and frequency of Tscm to enhance the quality of T-cells produced and speed-up the production processes.

[0191] In the present disclosure, setup and results from a proof-of-concept study are provided to demonstrate the effectiveness of incorporating a feedback loop into cell product manufacturing. T cells were stimulated and expanded in a standard condition and in conditions mimicking clinical samples not meeting certain quality attributes. Early in the production stage, T-cells from each group were examined using various analytical tools. The cell analysis results, such as cell proliferation and differentiation status, of sample 2 and sample 3 were then compared against the quality attributes (QAs) of a control / S ample 1. If the cells in the experimental samples did not meet or exceed the QAs of the control, the cell culture conditions were adjusted accordingly, and the cells were expanded with the revised culture conditions. Hence, by intermittently conducting cell analysis throughout the production, if required, the cell production process was continuously modified. Accordingly, it was found that using feedback control resulted in more T-cell products will meet the established standards by the end of manufacturing, regardless of the initial cell status at the beginning of production.

[0192] Due to individual patient differences in health conditions, such as varying expression levels of specific genes and proteins, patients with the same type of cancer may have cells with distinct characteristics, resulting in different quality attributes during autologous therapy manufacturing. The feedback loop methods described in the present disclosure can help standardize cell therapies produced from different patients or donors (autologous or allogeneic), ensuring that the patients achieve consistent quality attributes by the end of production. In addition, the feedback loop methods described in the present disclosure can also provide benefits to current / conventional solutions when a patient is deemed too sick to provide cells to be worth trying autologous treatment with. When some patients are too sick to currently supply cells for effective autologous treatment, doctors / care providers may opt to offer allogeneic therapy (with cells from a heathy person who does have health conditions that meet the threshold for sample extraction) or wait to see if a patient’s health improves, wait for some number of days after a chcmothcrapy / antibiotic course, etc., so that the patient’s health conditions improve to later meet some conditions so that patients can provide some cells for autologous therapy production under some circumstance. The present disclosure avoids the downsides and risks associated with such conventional solutions (e.g., allogeneic and waiting approaches) among other benefits. Accordingly, rather than waiting for a patient to exhibit health conditions associated with threshold values for sample extraction of cells deemed “healthy enough” or “in sufficient quantities / percentages” for manufacturing effective therapies for autologous treatments, or determining to use a different biological subject as the source for the when the patient cannot or has not exhibited health conditions associated with threshold sample extraction, as is conventional, the present disclosure demonstrates that superior autologous treatments with therapeutically effective amounts and potencies of manufactured cells are possible even when the patient cannot or has not exhibited health conditions associated with conventional threshold sample extraction. Although generally discussed in relation to autologous therapies, the present disclosure contemplates application of the teachings herein for allogeneic (e.g., heterologous) therapies.

[0193] Figure 8 illustrates an overview of an example T-cell production process 800 with a feedback loop, according to embodiments of the present disclosure.. Enriched CD4+ and CDS+ cells from peripheral blood mononuclear cells (PBMCs) from a healthy donor were provided. The cells were centrifuged at 250x g for ten minutes after thawing in a 37 degree C water bath for approximately two minutes. Cells were resuspended, in 5 mL of resting medium, supplementedwith 5 % human serum, followed by cell counting using a flow cytometer. After counting, the cells were transferred into a flask, rested, and incubated at 5-10xl06cells / mL in a 37 degree C incubator with 5% CO2 overnight. The following day, the rested PBMCs were centrifuged at 400x g for eight minutes. The cell sample was resuspended in 2 mL of basal glucose-free medium, and viable cells were counted using the flow cytometer. The cells were seeded at a density of IxlO6cells / well in 2 mL of activation medium, which included the expansion medium and 50 pL of human CD3 / CD28 T-cell activator well plate of a bioreactor on day zero. On day one, an additional 6 mL of the expansion medium was added to the wells of the well plate. A 75% medium refresh was performed on day three and day six during the production process.

[0194] To mimic variations in cell performance and status caused by donor differences, which can lead to samples potentially failing to meet the certain thresholds for quality parameters (e.g. total cell number, total Tscm numbers, percentage of Tscm), the overnight rested PBMCs from the same donor were initially stimulated and expanded in the basic RPMI-1 and supplemented with varying concentrations of IL2 (as is shown in Figure 9). The cells cultured in medium containing 200 U / mL IL2 for Sample 1 were used as the standard control, while those cells expanded in medium containing IL 250 U / mL were designated as Sample 2 and Sample 3 to represent possible patient variability in starting cells material - for low-quality initial sampling). Among Sample 2 and Sample 3, Sample 2 was continuously expanded under the same culture conditions to represent the test without applying feedback for cell growth, hr contrast, feedback for cell growth was applied to Sample 3. The first cell analysis was conducted on day four, which represents the moment the cells had recovered from activation-induced cell death, as indicated by the cell expansion profde shown in Figure 10.

[0195] Briefly, after gently removing 6 mL of the old medium from the top of the culture, the cells were resuspended in, the remaining medium. A small volume of cell suspension was taken for cell counting, and a fraction of cells were prepared for a flow cytometry assessment of T-cell differentiation state on day four. The analytic results of the technical replicates Sample 2 and Sample 3 were compared against those of the control group (Sample 1). If the cells in Sample 3 did not meet the control-determined standards for the total cell number, percentage of Tscm, or the number of Tscm cells, the manufacturing conditions were adjusted immediately in a feedback loop.

[0196] Cells were carefully resuspended homogeneously by pipetting the medium up and down. After resuspension, 50 pL of the cell suspension was transferred to a well plate and 50 pLof a solution containing the cell staining buffer was added. The mixture was incubated for five minutes at room temperature in the dark. Without wash, the samples were loaded and each sample absolute count was acquired in duplicates using precise volumetric measurements. The sample loading settings were the following: automatic mixing setting lOOOrpm / lOsec, one mix and one rinse per well, stop condition of 100 pL, sample flow rate of 66 pL / min. Following sample acquisition, proper gating was applied in the measurement software, and the absolute count of 7AAD negative cells were reported as total live cell numbers. The kinetic cell growth of the positive control group throughout the expansion is presented in Figures 11A-11C.

[0197] The flow cytometry method was also used to assess the purity and differentiation state of T-cells throughout the cell culture. First, a scientifically sound multiparametric 8-colors / 10 parameters flow cytometry panel was designed and validated. The example panel presented herein included antibodies to identify CD3, CD4, CD8, viability, CCR7, CD95, CD45RA, and CD45RO. Evidence has shown that based on differential expression of CD45RA, CD45RO, CD95 and CCR7 markers, naive T cells (Tn, CD45RA+CCR7+CD95-), stem cell memory T cells(Tscm, CD45RA+CCR7+CD95+), central memory T cells (Tern, CD45RO+CD45RA-CCR7-), effector memory T cells (Tern, CD45RO+CD45RA-CCR7-) and terminal effector T cells (Teff, CD45RA+CD45RO+CCR7-) can be identified. The panel design included antibody titration of each antibody to determine the optimal antibody concentrations. Flow cytometry assays were performed on fresh cells on day zero, day four, day six, and day ten. Between 0.3xl06to IxlO6cells were collected, washed at 350g / 5min and resuspended in 100 pL of a cell staining buffer. The Fc receptors of cells were blocked using a blocking buffer for eight minutes at room temperature (RT). Without washing, the cells were directly stained with a mix of all the surface antibodies of interest in the presence of a brilliant stain buffer to block fluorescent dye interactions. The labeled cells were incubated or twenty minutes at room temperature in the dark, and washed once a 350g / 5min. Per FDA guidelines regarding CART-cells manufacture recommendations, a viability dye should always be added in flow cytometry panel to remove dead cells. As such, after washing, the cells were resuspended 500 pL of cell staining buffer in the presence of a viability dye 7AAD (l / 100e). The samples were then incubated for five minutes at room temperature with no wash. The samples were split, loaded into a sampler, and acquired as technical replicates. An average number of 0.83xl0ncells per sample were analyzed. Single stained compensation controls, fluorescence minus one (FMO) controls and unstained controls were performed todetermine the fluorescence spread and the gating boundaries during data analysis. Moreover, evidence-backed gating strategies were designed and applied to phenotypic ally characterize T-cell subsets (e.g., %CD3+T, %CD4+ T-cell helper, and %CD8+ cytotoxic T-cell populations) as well as T-cell differentiation subsets (%Tn, %Tscm, %Tcm, %Tem, %Teff). Finally, daily instrument quality control and maintenance were performed according to manufacturer recommendations to ensure the accuracy of the results.

[0198] Absolute cell counting was acquired using precise volumetric counting. Figure 10 shows the kinetic cell growth under the reference control condition for Sample 1. Based on these data, day four appears as a suitable starting point for the feedback because the cell numbers exhibit cells starting to recover from activation-induced cell death, which is a known phenomenon following T-cell activation. Based on these data, the first cell analysis was conducted on day four. This cell analysis included cell counting and T-cell differentiation phenotyping. Indeed, the FDA guidance document has recognized that beside CAR expression, other factors such as viability and total cells number should be considered in determining the cell dose administered to the patient receiving cell therapies. Consequently, the final cell number of T-cells product can considerably influence the therapeutic dose of immunotherapies. Other studies demonstrated at Tscm subsets are good candidates for adoptive cell therapy due to their longevity and aptitude for self-renewal. Indeed, clinical studies have demonstrated that the infusion of Tscm CAR T-cells results in favorable outcomes. Therefore, we selected the total cell number, the percentage, and the number of Tscm subsets as the cell quality parameters to assess during T-cell expansion. The data from the Sample 1 were used as benchmarks. Clinical starting material samples have shown great variability from donors. As such, we chose a 20% deviation threshold for parameters from these standard values to initiate cell cult recondition adjustments. Figures 11 A-l 1C present the total cell number, percentage of Tscm, and expansion fold change observed on day four. The cell number in Sample 2 and Sample 3 was 0.82x106 cells; representing 28% fewer than in Sample 1. Similarly, the Tscm percentage in Sample 2 and Sample 3 was 35% compared to 49% in Sample 1. Likewise, further analysis revealed that Sample 2 and sample 3's Tscm fold change to control represented approximately 71% of Sample 1. Equally, when considering the cell number of Tscm cells, data revealed that the number of Tscm cells in Sample 2 and Sample 3 group was approximately 54% of that in Sample 1.

[0199] Taken together, these cell analysis results indicated that the selected quality attributes in Sample 2 and Sample 3 were below the benchmark threshold on day four, necessitating an adjustment of the culture conditions to hopefully improve the production of high-quality T-cell products by the end of the process. In the example verification, the interleukin-2 (IL-2) and interleukin- 15(IL- 15) cytokine concentration were adjusted in Sample 3 during feedback.

[0200] IL-2 is a common supplement in ex vivo T-cell culture, recognized as booster of T-cell expansion. Alternatively, IL- 15 has been reported to promote the proliferation and preserve the stem cell memory phenotype of CART-cells, thereby enhancing their antitumor activity. Additionally, both IL-2 and IL-15 are cytokines that have been known to be involved in different stages of T-cell differentiation in vivo, and they are supplements in T-cell culture media that play a role in the ex vivo activation, proliferation and survival of T cells. To improve the quality of the final products in Sample 3 via feedback, the IL-2 concentration was increased to match that in Sample at the same time, and 10 ng / mL of IL- 15 was added to the medium. Proceeding from day four, Sample 1, Sample 2, and Sample 3 were expanded for the remainder of the ten-day culture.

[0201] Two more cell analyses were conducted on day six and day ten to assess changes in cell quality attributes. Figures 12A-12C demonstrate that Sample 2 and Sample 3 each presented approximately 28% fewer cells than Sample 1 on day four. However, adjusting the culture conditions in Sample 3 (with feedback) resulted in a faster growth rate compared to Sample 2 (maintained in the same culturing conditions). Indeed, Sample 2 reached only 37% of the Sample 1 cell count on day six, whereas the adjusted Sample 3 reached 58% of the Sample 1 cell count on day six. Additionally, cell analysis on day ten revealed that Sample 3 reached 80% of the Sample 1 total cell count (not fully matching the control), whereas Sample 2 showed minimal proliferation after day six, with its total cell count at day ten similar to that on day six, and representing only 20% compared to that of Sample 1 at the same time (Figure 12A).

[0202] Interestingly, the percentage of Tscm subsets fluctuated throughout the manufacturing process (Figure 12B). starting at 19% on day zero, the Tscm percentage peaked on day four at 49% in the Sample 1 and at 35% in the Sample 2 and Sample 3 groups. The %Tscm later declined to 20% across all groups on day six, indicating that the culture condition adjustment initiated on day 4 in Sample 3 had not yet significantly influenced T-cell differentiation. Surprisingly, data analysis of T-cell differentiation toward the end of production (day ten) displayed significant changes. Indeed, day ten results showed significantly higher %Tscm in Sample 3 than compared eitherSample 1 or Sample 2, in which Sample 3 reached 63% more %Tscm when compared the Sample 1 (Figure 12C), while Sample 2 remained the lowest level among all the sample groups. Accordingly, data also revealed significantly higher Tscm cell number in Sample 3 compared to Sample 1, while Sample 2 displayed the lowest Tscm cell number (Figure 12C).

[0203] Interestingly, the changes in total cell number together with changes in percentage of Tscm could explain, at least partially, the changes in Tscm number as presented in Figure 12C. Taken together, the findings revealed that the Tscm cell count and percentage in Sample 2 gradually decreased, and remained low after day four, whereas Tscm cell number in the feedback- adjusted Sample 3 and control Sample 1 progressively increased over time. Surprisingly, the Tscm cell count in Sample 3 showed fewer Tscm cell numbers compared to that in Sample 1 at day six (Figure 12C), despite media adjustment day four. This suggest that the treatment duration of the feedback was potentially not long enough at day six to observe a significant recovery (from day four) of the Tscm cell number as it has been suggested in other studies that not only the cytokine concentration, but also the cytokine treatment duration impact the T-cell differentiation and cell expansion. Nevertheless, a significant increase in Tscm cell count was observed by the end of the production (day ten) in Sample 3, which was even 30% higher than in Sample 1; indicating the effectiveness of the modified culture conditions using feedback as described herein, and the delayed impacts thereof of cell quality.

[0204] These findings highlight the surprising improvements offered by the integrated cell analysis methods described herein to monitor T-cells quality attributes during T-cell product manufacturing. Indeed, developing methods that can provide a rapid and reliable T-cell characterization, cell count, and T-cell differentiation state can provide clinicians the ability to guide the adjustment of the cell culture conditions to further enrich the clinical dose and the frequency of less-differentiated T-cell populations in the final product. As such, real-time monitoring and optimizing the cell number and T-cell differentiation in ex vivo culture could hold an immense potential for the development of sufficient dose-efficacious CAR T-cell products, particularly in autologous treatment settings where the provision of such efficacious concentrations has been a long felt need. The feedback mechanisms described herein can ease the manufacturing workflow, thereby enabling widespread adoption of these therapies and possibly addressing the issue of scaling and commercialization of both autologous and allogenic T-cell therapies.

[0205] The present disclosure may also be understood with reference to the following numbered clauses.

[0206] Clause 1: A system, comprising: a bioreactor; a sensor; a processor; a memory including instructions that when executed by the processor perform operations that comprise, automatically: extracting a sample from cells being cultured in the bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

[0207] Clause 2: The system of any one or more of clauses 1 and 3-10, wherein the sample is taken at a first time, and the operations further comprise: extracting an additional sample from the cells being cultured in the bioreactor at a second time after the first time; preparing the additional sample to produce an additional prepared sample; analyzing the additional prepared sample to identify an additional value for the associated cell analysis parameter; and in response to determining that the additional value is within the window for the associated cell analysis parameter, maintaining the setting of the bioreactor.

[0208] Clause 3: The system of clause 2 and (optionally) any one or more of clauses 4-10, wherein a first duration between the first time and the second time is equivalent to a second duration between the second time and a third time at which a subsequent sample from the cells being cultured in the bioreactor is extracted to determine a subsequent value for the associated cell analysis parameter.

[0209] Clause 4: The system of clause 2 and (optionally) any one or more of clauses 3 and 5- 10, wherein a duration between the first time and the second time is one of: at least thirty minutes; at least one hour; at least two hours; at least six hours; at least twelve hours; at least twenty-four hours; at least thirty-six hours; at least forty-eight hours; at least seventy-two hours; at least eighty- four hours; at least ninety- six hours; at least one hundred twenty hours; at least one hundred forty- four hours; or at least one-hundred sixty-eight hours.

[0210] Clause 5: The system of any one or more of clauses 1-4 and 6-10, wherein the operations further comprise: extracting a second sample from the cells being cultured in the bioreactor at a first time; preparing the second sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for a second associated cell analysis parameter; inresponse to determining that the second value is within a second window for the second associated cell analysis parameter, maintaining the setting of the bioreactor until a second time; extracting a third sample from the cells being cultured in the bioreactor at the second time; preparing the third sample to produce a third prepared sample; analyzing the third prepared sample to identify a third value for the second associated cell analysis parameter; in response to determining that the third value is within the second window for the second associated cell analysis parameter, identifying a trend for the second associated cell analysis parameter between the first time and the second time; and in response to determining that a projected value of the trend for a third time at which a fourth sample is scheduled to be extracted is outside of the window, adjusting a second setting of the bioreactor based on a difference between the trend and the window between the second time and the third time.

[0211] Clause 6: The system of any one or more of clauses 1-5 and 7-10, wherein preparing the sample to produce the prepared sample comprises one, two, three, four, or all of: identifying or registering the sample; incubating the sample; staining the sample; adding a buffer or medium to the sample; or adding a therapeutic agent to the sample.

[0212] Clause 7: The system of any one or more of clauses 1-6 and 8-10, wherein the associated cell analysis parameter comprises one, two, three, four, five, six, seven, or all of: a pH; a temperature; a level or concentration of a constituent or agent within the bioreactor or the sample; a cell identity; a cell number; a cell purity; a cell size; or a run time.

[0213] Clause 8: The system of any one or more of clauses 1-7 and 9-10, wherein the operations further comprise: automatically discarding the prepared sample after identifying the value for the associated cell analysis parameter.

[0214] Clause 9: The system of any one or more of clauses 1-8 and 10, wherein the cells grown in the bioreactor are extracted from a first biological subject prior to growth of the cells in the bioreactor, and are retrieved from the bioreactor for treatment or prophylaxis of a disorder in a second biological subject as a modified cell culture, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

[0215] Clause 10: The system of any one or more of clauses 1-9, wherein the bioreactor remains self-contained and operates automatically based on instructions received exclusively before extracting the sample.

[0216] Clause 11: A method, comprising: extracting a cell culture from a first biological subject; inserting the cell culture into a bioreactor; growing, in the bioreactor, the cell culture from a first time to a second time; modifying, in the bioreactor from the first time to the second time, the cell culture to produce a modified cell culture; retrieving the modified cell culture from the bioreactor; and supplying a therapeutically effective amount of the modified cell culture to a second biological subject that is experiencing or at risk of a disorder that is treatable, preventable, or manageable via application of the modified cell culture.

[0217] Clause 12: The method of any one or more of clauses 11 and 13-17, wherein growing and modifying the cell culture further comprise automatically culturing the cell culture according to operations that comprise: extracting a cell sample from the bioreactor at an intermediate time between the first time and the second time; preparing the cell sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting, before the second time, a setting of the bioreactor based on a difference between the value and the window.

[0218] Clause 13: The method of clause 12 and any (optionally) one or more of clauses 14-17, wherein the prepared sample is discarded after identifying the value for the associated cell analysis parameter.

[0219] Clause 14: The method of any one or more of clauses 11-13 and 15-17, wherein growing and modifying the cell culture further comprise automatically culturing the cell culture according to operations that comprise: extracting a first cell sample from the bioreactor at a first intermediate time between the first time and the second time; preparing the first cell sample to produce a first prepared sample; analyzing the first prepared sample to identify a first value for an associated cell analysis parameter; in response to determining that the first value is within a window for the associated cell analysis parameter, maintaining a setting of the bioreactor until a second intermediate time between the first intermediate time and the second time; extracting a second cell sample from the bioreactor at the second intermediate time; preparing the second cell sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for the associated cell analysis parameter; in response to determining that the second value is within the window for the associated cell analysis parameter, identifying a trend for the associated cell analysis parameter between the first intermediate time and the second intermediatetime; and in response to determining that a projected value of the trend for a third intermediate time between the second intermediate time and the second time at which a third sample is scheduled to be extracted is outside of the window, adjusting the setting of the bioreactor based on a difference between the trend and the window between the second intermediate time and the third intermediate time.[02201 Clause 15: The method of any one or more of clauses 11-14 and 16-17, wherein a prepared sample is extracted from the bioreactor via a needle and is inserted into an analysis vessel that is moved to an analysis module comprising a sensor that is separate from the bioreactor and in which a value for a cell growth parameter is determined.

[0221] Clause 16: The method of any one or more of clauses 11-15 and 17, wherein, between the first time and the second time, the bioreactor remains self-contained and operates automatically based on instructions received exclusively before the first time.

[0222] Clause 17: The method of any one or more of clauses 11-16, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

[0223] Clause 18: A method, comprising: extracting a sample from cells being cultured in a bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

[0224] Clause 19: The method of any one or more of clauses 18 and 19-22, wherein the sample is extracted at a first time, and the method further comprises: extracting an additional sample from the cells being cultured in the bioreactor at a second time after the first time; preparing the additional sample to produce an additional prepared sample; analyzing the additional prepared sample to identify an additional value for the associated cell analysis parameter; and in response to determining that the additional value is within the window for the associated cell analysis parameter, maintaining the setting of the bioreactor.

[0225] Clause 20: The method of any one or more of clauses 18-19 and 21-22, further comprising: extracting a second sample from the cells being cultured in the bioreactor at a first time; preparing the second sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for a second associated cell analysis parameter; inresponse to determining that the second value is within a second window for the second associated cell analysis parameter, maintaining the setting of the bioreactor until a second time; extracting a third sample from the cells being cultured in the bioreactor at the second time; preparing the third sample to produce a third prepared sample; analyzing the third prepared sample to identify a third value for the second associated cell analysis parameter; in response to determining that the third value is within the second window for the second associated cell analysis parameter, identifying a trend for the second associated cell analysis parameter between the first time and the second time; and in response to determining that a projected value of the trend for a third time at which a fourth sample is scheduled to be extracted is outside of the window, adjusting a second setting of the bioreactor based on a difference between the trend and the window between the second time and the third time.

[0226] Clause 21 : The method of any one or more of clauses 18-20 and 22, further comprising automatically discarding the prepared sample after identifying the value for the associated cell analysis parameter.

[0227] Clause 22: The method of any one or more of clauses 18-21, wherein the cells grown in the bioreactor are extracted from a first biological subject prior to growth in the bioreactor, and are retrieved from the bioreactor for treatment or prophylaxis of a disorder in a second biological subject as a modified cell culture once a predefined growth value is reached, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

[0228] Certain terms are used throughout the description and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not function.

[0229] As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of the referenced number, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number.

[0230] Furthermore, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in thatrange. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0231] As used in the present disclosure, a phrase referring to “at least one of’ a list of items refers to any set of those items, including sets with a single member, and every potential combination thereof. For example, when referencing “at least one of A, B, or C” or “at least one of A, B, and C”, the phrase is intended to cover the sets of: A, B, C, A-B, B-C, and A-B-C, where the sets may include one or multiple instances of a given member (e.g., A-A, A-A-A, A- A-B, A- A-B-B-C-C-C, etc.) and any ordering thereof. For avoidance of doubt, the phrase “at least one of A, B, and C” shall not be interpreted to mean “at least one of A, at least one of B, and at least one of C”.

[0232] As used in the present disclosure, the term “determining” encompasses a variety of actions that may include calculating, computing, processing, deriving, investigating, looking up (e.g., via a table, database, or other data structure), ascertaining, receiving (e.g., receiving information), accessing (e.g., accessing data in a memory), retrieving, resolving, selecting, choosing, establishing, and the like.

[0233] Without further elaboration, it is believed that one skilled in the art can use the preceding description to use the claimed inventions to their fullest extent. The examples and aspects disclosed herein are to be construed as merely illustrative and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having skill in the art that changes may be made to the details of the above-described examples without departing from the underlying principles discussed. In other words, various modifications and improvements of the examples specifically disclosed in the description above are within the scope of the appended claims. For instance, any suitable combination of features of the various examples described is contemplated.

[0234] Within the claims, reference to an element in the singular is not intended to mean “one and only one” unless specifically stated as such, but rather as “one or more” or “at least one”. Unless specifically stated otherwise, the term “some” refers to one or more. No claim element is to be construed under the provision of 35 U.S.C. § 112(1) unless the element is expressly recited using the phrase “means for” or “step for”. All structural and functional equivalents to the elements of the various embodiments described in the present disclosure that are known or come later to be known to those of ordinary skill in the relevant art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed in the presentdisclosure is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims

Claims

CLAIMSThe invention is claimed as follows:

1. A system, comprising: a bioreactor; a sensor; a processor; a memory including instructions that when executed by the processor perform operations that comprise, automatically: extracting a sample from cells being cultured in the bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

2. The system of claim 1, wherein the sample is taken at a first time, and the operations further comprise: extracting an additional sample from the cells being cultured in the bioreactor at a second time after the first time; preparing the additional sample to produce an additional prepared sample; analyzing the additional prepared sample to identify an additional value for the associated cell analysis parameter; and in response to determining that the additional value is within the window for the associated cell analysis parameter, maintaining the setting of the bioreactor.

3. The system of claim 2, wherein a first duration between the first time and the second time is equivalent to a second duration between the second time and a third time at which asubsequent sample from the cells being cultured in the bioreactor is extracted to determine a subsequent value for the associated cell analysis parameter.

4. The system of claim 2, wherein a duration between the first time and the second time is one of: at least thirty minutes; at least one hour; at least two hours; at least six hours; at least twelve hours; at least twenty-four hours; at least thirty-six hours; at least forty-eight hours; at least seventy-two hours; at least eighty-four hours; at least ninety-six hours; at least one hundred twenty hours; at least one hundred forty-four hours; or at least one-hundred sixty-eight hours.

5. The system of claim 1, wherein the operations further comprise: extracting a second sample from the cells being cultured in the bioreactor at a first time; preparing the second sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for a second associated cell analysis parameter; in response to determining that the second value is within a second window for the second associated cell analysis parameter, maintaining the setting of the bioreactor until a second time; extracting a third sample from the cells being cultured in the bioreactor at the second time; preparing the third sample to produce a third prepared sample;analyzing the third prepared sample to identify a third value for the second associated cell analysis parameter; in response to determining that the third value is within the second window for the second associated cell analysis parameter, identifying a trend for the second associated cell analysis parameter between the first time and the second time; and in response to determining that a projected value of the trend for a third time at which a fourth sample is scheduled to be extracted is outside of the window, adjusting a second setting of the bioreactor based on a difference between the trend and the window between the second time and the third time.

6. The system of claim 1, wherein preparing the sample to produce the prepared sample comprises one, two, three, four, or all of: identifying or registering the sample; incubating the sample; staining the sample; adding a buffer or medium to the sample; or adding a therapeutic agent to the sample.

7. The system of claim 1, wherein the associated cell analysis parameter comprises one, two, three, four, five, six, seven, or all of: a pH; a temperature; a level or concentration of a constituent or agent within the bioreactor or the sample; a cell identity; a cell number; a cell purity; a cell size; or a run time.

8. The system of claim 1, wherein the operations further comprise:automatically discarding the prepared sample after identifying the value for the associated cell analysis parameter.

9. The system of claim 1, wherein the cells grown in the bioreactor are extracted from a first biological subject prior to growth of the cells in the bioreactor, and are retrieved from the bioreactor for treatment or prophylaxis of a disorder in a second biological subject as a modified cell culture, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

10. The system of claim 1 , wherein the bioreactor remains self-contained and operates automatically based on instructions received exclusively before extracting the sample.

11. A method, comprising: extracting a cell culture from a first biological subject; inserting the cell culture into a bioreactor; growing, in the bioreactor, the cell culture from a first time to a second time; modifying, in the bioreactor from the first time to the second time, the cell culture to produce a modified cell culture; retrieving the modified cell culture from the bioreactor; and supplying a therapeutically effective amount of the modified cell culture to a second biological subject that is experiencing or at risk of a disorder that is treatable, preventable, or manageable via application of the modified cell culture.

12. The method of claim 11, wherein growing and modifying the cell culture further comprise automatically culturing the cell culture according to operations that comprise: extracting a cell sample from the bioreactor at an intermediate time between the first time and the second time; preparing the cell sample to produce a prepared sample;analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting, before the second time, a setting of the bioreactor based on a difference between the value and the window.

13. The method of claim 12, wherein the prepared sample is discarded after identifying the value for the associated cell analysis parameter.

14. The method of claim 11, wherein growing and modifying the cell culture further comprise automatically culturing the cell culture according to operations that comprise: extracting a first cell sample from the bioreactor at a first intermediate time between the first time and the second time; preparing the first cell sample to produce a first prepared sample; analyzing the first prepared sample to identify a first value for an associated cell analysis parameter; in response to determining that the first value is within a window for the associated cell analysis parameter, maintaining a setting of the bioreactor until a second intermediate time between the first intermediate time and the second time; extracting a second cell sample from the bioreactor at the second intermediate time; preparing the second cell sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for the associated cell analysis parameter; in response to determining that the second value is within the window for the associated cell analysis parameter, identifying a trend for the associated cell analysis parameter between the first intermediate time and the second intermediate time; and in response to determining that a projected value of the trend for a third intermediate time between the second intermediate time and the second time at which a third sample is scheduled to be extracted is outside of the window, adjusting the setting of the bioreactor based on a difference between the trend and the window between the second intermediate time and the third intermediate time.

15. The method of claim 11, wherein a prepared sample is extracted from the bioreactor via a needle and is inserted into an analysis vessel that is moved to an analysis module comprising a sensor that is separate from the bioreactor and in which a value for a cell growth parameter is determined.

16. The method of claim 11, wherein, between the first time and the second time, the bioreactor remains self-contained and operates automatically based on instructions received exclusively before the first time.

17. The method of claim 11, wherein the modified cell culture comprises: cells that are genetically altered to express one or more heterologous genes; cells that arc cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

18. A method, comprising: extracting a sample from cells being cultured in a bioreactor; preparing the sample to produce a prepared sample; analyzing the prepared sample to identify a value for an associated cell analysis parameter; and in response to determining that the value is outside a window for the associated cell analysis parameter, adjusting a setting of the bioreactor based on a difference between the value and the window.

19. The method of claim 18, wherein the sample is extracted at a first time, and the method further comprises: extracting an additional sample from the cells being cultured in the bioreactor at a second time after the first time; preparing the additional sample to produce an additional prepared sample; analyzing the additional prepared sample to identify an additional value for the associated cell analysis parameter; andin response to determining that the additional value is within the window for the associated cell analysis parameter, maintaining the setting of the bioreactor.

20. The method of claim 18, further comprising: extracting a second sample from the cells being cultured in the bioreactor at a first time; preparing the second sample to produce a second prepared sample; analyzing the second prepared sample to identify a second value for a second associated cell analysis parameter; in response to determining that the second value is within a second window for the second associated cell analysis parameter, maintaining the setting of the bioreactor until a second time; extracting a third sample from the cells being cultured in the bioreactor at the second time; preparing the third sample to produce a third prepared sample; analyzing the third prepared sample to identify a third value for the second associated cell analysis parameter; in response to determining that the third value is within the second window for the second associated cell analysis parameter, identifying a trend for the second associated cell analysis parameter between the first time and the second time; and in response to determining that a projected value of the trend for a third time at which a fourth sample is scheduled to be extracted is outside of the window, adjusting a second setting of the bioreactor based on a difference between the trend and the window between the second time and the third time.

21. The method of claim 18, further comprising automatically discarding the prepared sample after identifying the value for the associated cell analysis parameter.

22. The method of claim 18, wherein the cells grown in the bioreactor are extracted from a first biological subject prior to growth in the bioreactor, and are retrieved from the bioreactor for treatment or prophylaxis of a disorder in a second biological subject as a modified cell culture once a predefined growth value is reached, wherein the modified cell culture comprises:cells that are genetically altered to express one or more heterologous genes; cells that are cultured for an immunotherapy; or cells that are cultured for a stem cell therapy.

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