Systems and methods for processing cells

Abstract

The present disclosure provides systems and methods for processing cells. The systems of the present disclosure may include a compartment, a three-dimensional (3D) matrix, a fluid flow path, an analysis unit, a preservation unit, or any combination thereof. The methods of the present disclosure may process cells via, for example, expansion, culturing, gene delivery, activation, or any combination thereof. The systems and methods described herein may be useful for producing cell products including, for example, cell therapy products.

Description

WSGR Docket No.67566-702601 SYSTEMS AND METHODS FOR PROCESSING CELLS CROSS-REFERENCE

[0001] This application claims of benefit of U.S. Provisional Patent Application No.63 / 723,534, filed November 21, 2024, U.S. Provisional Patent Application No.63 / 558,804, filed February 28, 2024, and U.S. Provisional Patent Application No.63 / 623,024, filed January 19, 2024, each of which is entirely incorporated herein by reference. BACKGROUND

[0002] Cell processing techniques have applications in a variety of fields such as lifescience research and industrial and therapeutic production. Bioreactor technologies and bioprocessing systems are useful tools for facilitating culturing, processing, and manufacturing of cells and valuable cell byproducts. SUMMARY

[0003] The present disclosure provides systems and methods for processing cells. Cellprocessing systems may permit fully automated cell processing. Automated cell processing may include cellular expansion, gene delivery, development of cellular therapies, biologics manufacturing, or any combinations thereof. The systems and method described herein may include simpler fluidics as compared to other cell processing systems which may permit cell processing in smaller and lower cost instruments. The systems and methods described herein may permit cell processing with reduced cell handling and in an environment that replicates a cells natural environment which may maintain or produce healthier cells as compared to other cell processing systems and methods. Thus, the systems and methods described herein may provide for improved cell handling and processing such that produced cells or cell products have increased reproducibility, viability, and overall health as compared to cells produced using other systems and methods.

[0004] In an aspect, the present disclosure provides a system for manufacturing a cellproduct, comprising: a compartment configured to retain a plurality of cells; a fluid flow path in fluid communication with the compartment, wherein the fluid flow path is configured to (i) flow a fluid along a first direction having an angle with respect to a second direction of flow within the compartment, wherein the angle is greater than zero degrees, and (ii) provide material from the fluid to the compartment to thereby generate the cell product; and an analysis unit coupled to and within a same housing as the compartment and the fluid flowWSGR Docket No.67566-702601 path, wherein the analysis unit is configured to monitor the plurality of cells or the fluid as the materials are provided to the compartment during generation of the cell product.

[0005] In some embodiments, the material comprises reagents or nutrients. In someembodiments, the angle is greater than 45 degrees. In some embodiments, the angle is greater than 60 degrees. In some embodiments, the angle is greater than 80 degrees. In some embodiments, the cell product is a cell therapy product. In some embodiments, the system is configured to generate the cell therapy product at a scale of at least one human dose equivalent. In some embodiments, the compartment is configured to contain the plurality of cells. In some embodiments, the plurality of cells or a portion thereof is immobilized within the compartment. In some embodiments, the plurality of cells is encapsulated within the compartment.

[0006] In some embodiments, the analysis unit is configured to monitor the plurality ofcells in the compartment. In some embodiments, the analysis unit is configured to monitor the plurality of cells in real time. In some embodiments, the analysis unit monitors or detects one or more properties selected from the group consisting of marker expression, phenotype, viability, concentration, potency, sterility, endotoxins, and safety. In some embodiments, the system further comprises a cell processing unit in fluid communication with the compartment. In some embodiments, the cell processing unit is configured to provide one or more processes selected from the group consisting of cell enrichment, cell selection, cell isolation, washing, concentration, buffer exchange, and de-clumping.

[0007] In some embodiments, the compartment is configured to provide to the pluralityof cells one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection. In some embodiments, the selection is negative selection. In some embodiments, the housing has a volume of about 0.01 cubic meters (m3) to about 2 m3. In some embodiments, the volume is of at most 1 m3. In some embodiments, the volume is of at most 0.5 m3. In some embodiments, the system is an aseptically closed system.

[0008] In some embodiments, the analysis unit comprises one or more sensors configuredto monitor an environment comprising the plurality of cells. In some embodiments, the one or more sensors are selected from the group consisting of metabolites sensors, dissolved gas sensors, pH sensors, temperature sensors, viscosity sensors, and pressure sensors. In some embodiments, the analysis unit comprises one or more sensors configured to analyze the cell product. In some embodiments, the one or more sensors are selected from the group consisting of Raman spectroscopy, infrared (IR) absorption spectroscopy, near-infrared (NIR)WSGR Docket No.67566-702601 spectroscopy, mid-infrared spectroscopy, Fourier Transform infrared (FTIR) spectroscopy, optical coherence tomography, confocal microscopy, multiphoton microscopy, microwave impedance microscopy, ultrasound imaging, photoacoustic microscopy, elastography, computed tomography, conductance sensor, radio-frequency impedance sensor, dielectric spectroscopy, permittivity sensor, fluorescence imaging, mass spectroscopy, optical density, and magnetic resonance angiography. In some embodiments, the system further comprises one or more temperature sensors and a temperature control unit. In some embodiments, the system further comprises a plurality of compartments comprising the compartment and a plurality of fluid flow paths comprising the fluid flow path.

[0009] In another aspect, the present disclosure provides a system for cell processing,comprising: a cell processing unit configured to retain and process a plurality of cells to generate a cell product; a preservation unit coupled to the cell processing unit, wherein the preservation unit is configured to preserve the cell product; and an analysis unit coupled to the cell processing unit or the preservation unit, wherein the analysis unit is configured to detect or monitor one or more properties of the plurality of cells or the cell product.

[0010] In some embodiments, the cell product is a cell therapy product. In someembodiments, the system further comprises a receiving unit configured to receive the plurality of cells. In some embodiments, the plurality of cells is a plurality of cryopreserved cells. In some embodiments, the receiving unit is configured to subject the plurality of cryopreserved cells to controlled thawing to generate a plurality of thawed cells. In some embodiments, the receiving unit is configured to automatically transfer the plurality of thawed cells to the cell processing unit. In some embodiments, the system is an aseptically closed system. In some embodiments, the preservation unit is configured to generate a cryopreserved cell product. In some embodiments, the cell processing unit is configured to provide one or more processes selected from the group consisting of cell enrichment, cell selection, cell isolation, washing, concentration, buffer exchange, and de-clumping. In some embodiments, the cell processing unit is configured to contain the plurality of cells. In some embodiments, the plurality of cells or a portion thereof is immobilized within the cell processing unit. In some embodiments, the plurality of cells is encapsulated within the cell processing unit.

[0011] In some embodiments, the system further comprises a bioreactor coupled to thecell processing unit, the preservation unit, or the analysis unit. In some embodiments, the bioreactor is a solid-state bioreactor. In some embodiments, the bioreactor is configured toWSGR Docket No.67566-702601 provide to the plurality of cells one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection.

[0012] In some embodiments, the analysis unit is configured to provide in situ, real-timedetection or monitoring of the one or more properties of the plurality of cells or the cell product. In some embodiments, the one or more properties of the plurality of cells or the cell product are selected from the group consisting of cell viability, density, concentration, cytometry, pH, dissolved oxygen, and metabolites. In some embodiments, the analysis unit is configured to provide release testing of the cell product. In some embodiments, the release testing comprises one or more testing methods selected from the group consisting of imaging cytometry, quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR), enzyme-linked immunosorbent assay (ELISA), cell-based assays, cell count, viable cell count, and imaging bead count.

[0013] In some embodiments, the cell processing unit, the analysis unit, and thepreservation unit are integrated withing a housing. In some embodiments, the housing has a volume of about 0.01 cubic meters (m3) to about 2 m3. In some embodiments, the volume is of at most 1 m3. In some embodiments, the volume is of at most 0.5 m3. In some embodiments, the analysis unit comprises one or more sensors configured to monitor an environment comprising the plurality of cells. In some embodiments, the one or more sensors are selected from the group consisting of metabolites sensors, dissolved gas sensors, pH sensors, temperature sensors, viscosity sensors, pressure sensors. In some embodiments, the analysis unit comprises one or more sensors configured to analyze the cell product. In some embodiments, the one or more sensors are selected from the group consisting of Raman spectroscopy, infrared (IR) absorption spectroscopy, near-infrared (NIR) spectroscopy, mid- infrared spectroscopy, Fourier Transform infrared (FTIR) spectroscopy, optical coherence tomography, confocal microscopy, multiphoton microscopy, microwave impedance microscopy, ultrasound imaging, photoacoustic microscopy, elastography, computed tomography, conductance sensor, radio-frequency impedance sensor, dielectric spectroscopy, permittivity sensor, fluorescence imaging, mass spectroscopy, optical density, and magnetic resonance angiography.

[0014] In another aspect, the present disclosure provides a method for manufacturing acell product, comprising: providing a plurality of cells to a system comprising (i) a compartment that retains the plurality of cells, (ii) a fluid flow path in fluid communication with the compartment, and (iii) an analysis unit coupled to and within a same housing as the compartment and the fluid flow path; providing material from the a fluid in the fluid flowWSGR Docket No.67566-702601 path to the compartment to generate the cell product, wherein the fluid flow path flows the fluid along a first direction having an angle with respect to a second direction of flow within the compartment, wherein the angle is greater than zero degrees; and using the analysis unit to monitor the plurality of cells or the fluid as the materials are provided to the compartment during generation of the cell product.

[0015] In some embodiments, the material comprises reagents or nutrients. In someembodiments, the angle is greater than 45 degrees. In some embodiments, the angle is greater than 60 degrees. In some embodiments, the angle is greater than 80 degrees. In some embodiments, the cell product is a cell therapy product. In some embodiments, the cell therapy product is generated at a scale of at least one human dose equivalent. In some embodiments, the compartment is configured to contain the plurality of cells. In some embodiments, the plurality of cells or a portion thereof is immobilized within the compartment. In some embodiments, the plurality of cells is encapsulated within the compartment.

[0016] In some embodiments, the analysis unit monitors the plurality of cells in thecompartment. In some embodiments, the analysis unit monitors the plurality of cells in real time. In some embodiments, the analysis unit monitors or detects one or more properties selected from the group consisting of marker expression, phenotype, viability, concentration, potency, sterility, endotoxins, and safety. In some embodiments, the method further comprises using a cell processing unit to generate the cell product. In some embodiments, the cell processing unit provides one or more processes selected from the group consisting of cell enrichment, cell selection, cell isolation, washing, concentration, buffer exchange, and de-clumping. In some embodiments, the method further comprises subjecting the plurality of cells to one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection. In some embodiments, the selection is negative selection. In some embodiments, the housing has a volume of about 0.01 cubic meters (m3) to about 2 m3.

[0017] In another aspect, the present disclosure provides a method for cell processing,comprising: providing a plurality of cells to a system comprising (i) a cell processing unit that retains the plurality of cells, (ii) a preservation unit coupled to the cell processing unit, and (iii) an analysis unit coupled to the cell processing unit or the preservation unit; processing the plurality of cells in the cell processing unit to generate a cell product; preserving the cell product in the preservation unit; and detecting or monitoring one or more properties of the plurality of cells or the cell product during the processing or the preserving.WSGR Docket No.67566-702601

[0018] In some embodiments, the cell product is a cell therapy product. In someembodiments, the method further comprises providing the plurality of cells to receiving unit coupled to the cell processing unit, the preservation unit, or the preservation unit. In some embodiments, the plurality of cells provided to the receiving unit is a plurality of cryopreserved cells. In some embodiments, the receiving unit subjects the plurality of cryopreserved cells to controlled thawing to generate a plurality of thawed cells. In some embodiments, the receiving unit automatically transfers the plurality of thawed cells to the cell processing unit. In some embodiments, the preservation unit generates a cryopreserved cell product.

[0019] In some embodiments, the method further comprises subjecting the plurality ofcells to one or more processes selected from the group consisting of cell enrichment, cell selection, cell isolation, washing, concentration, buffer exchange, and de-clumping. In some embodiments, the method further comprises a bioreactor coupled to the cell processing unit, the preservation unit, or the analysis unit. In some embodiments, the bioreactor is a solid- state bioreactor. In some embodiments, the method further comprises using the bioreactor to subject the plurality of cells to one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection.

[0020] In some embodiments, the analysis unit provides in situ, real-time detection ormonitoring of the one or more properties of the plurality of cells or the cell product. In some embodiments, the one or more properties of the plurality of cells or the cell product are selected from the group consisting of cell viability, density, concentration, cytometry, pH, dissolved oxygen, and metabolites. In some embodiments, the method further comprises using the analysis unit for release testing of the cell product. In some embodiments, the release testing comprises one or more testing methods selected from the group consisting of imaging cytometry, qPCR, dPCR, ELISA, cell-based assays, cell count, viable cell count, and imaging bead count.

[0021] In another aspect, the present disclosure provides a system for cell processing,comprising: a plurality of three-dimensional (3D) matrices, wherein a 3D matrix of the plurality of 3D matrices is configured to retain a population of cells; and a plurality of fluid flow paths, wherein a fluid flow path of the plurality of fluid flow paths is independent from and adjacent to the 3D matrix, and wherein the fluid flow path is configured to deliver a fluidic material to the 3D matrix to process the population of cells into a cell product.

[0022] In some embodiments, the cell product is a cell therapy product. In someembodiments, the 3D matrix and the fluid flow path are separated by a membrane. In someWSGR Docket No.67566-702601 embodiments, the 3D matrix is configured as a slab comprising a first dimension and a second dimension that are larger than a third dimension. In some embodiments, the third dimension is less than or equal to about 10 millimeters (mm). In some embodiments, the third dimension is less than or equal to about 1 mm. In some embodiments, the third dimension is less than or equal to about 500 micrometers (µm). In some embodiments, the third dimension is less than or equal to about 200 µm. In some embodiments, the 3D matrix is configured as a fiber comprising a diameter that is smaller than the long dimension. In some embodiments, the diameter is less than or equal to about 10 mm. In some embodiments, the 3D matrix is configured as a sphere and wherein the tangential flow of the fluid flows along an outer circumference of the sphere. In some embodiments, the plurality of 3D matrices is fluidically coupled to one another. In some embodiments, the 3D matrix is configured to contain the plurality of cells. In some embodiments, the 3D matrix is configured to immobilize the plurality of cells. In some embodiments, the 3D matrix is configured to encapsulate the plurality of cells.

[0023] In some embodiments, the fluid flow path is disposed between a first 3D matrixand a second 3D matrix. In some embodiments, the fluid flow path is configured to deliver the fluidic material to the first 3D matrix and the second 3D matrix. In some embodiments, the 3D matrix comprises a pore size of less than or equal to about 400 micrometers (µm). In some embodiments, the 3D matrix comprises a pore size from about 0.5 µm to about 100 µm. In some embodiments, the 3D matrix comprises a porosity of greater than or equal to about 80%. In some embodiments, an interfacial area between the fluid flow path and the 3D matrix is greater than or equal to about 5 square centimeters (cm2). In some embodiments, the 3D matrix comprises a porous, solid matrix having interconnected pores with an average pore size sufficient to permit growth of lymphocyte cells within the 3D matrix. In some embodiments, the 3D matrix is a programmable 3D matrix. In some embodiments, the 3D matrix has a surface area to volume ratio of greater than or equal to about 0.1. In some embodiments, the 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m. In some embodiments, the 3D matrix is configured to provide activation of the plurality of cells via membrane signal transduction in the 3D matrix.

[0024] In another aspect, the present disclosure provides a method for cell processing,comprising: providing (i) a population of cells, (ii) a plurality of three-dimensional (3D) matrices, and (iii) a plurality of fluid flow paths, wherein the population of cells is retained within a 3D matrix of the plurality of 3D matrices, and wherein a fluid flow path of the plurality of fluid flow paths is independent from and adjacent to the 3D matrix; using theWSGR Docket No.67566-702601 fluid flow path to deliver fluidic material to the 3D matrix; and in a presence of the fluidic material delivered to the 3D matrix, processing the population of cells into a cell product.

[0025] In some embodiments, the cell product is a cell therapy product. In someembodiments, the cell therapy product is produced at a scale of at least one human dose equivalent. In some embodiments, the 3D matrix is configured to contain the plurality of cells. In some embodiments, the 3D matrix is configured to immobilize the plurality of cells. In some embodiments, the 3D matrix is configured to encapsulate the plurality of cells.

[0026] In some embodiments, the method further comprises functionalizing the 3Dmatrix to generate a functionalized 3D matrix. In some embodiments, the functionalizing comprises coupling one or more biomolecules to the 3D matrix. In some embodiments, the one or more biomolecules are selected from the group consisting of proteins, peptides, cytokines, antibodies, and nucleic acid molecules. In some embodiments, the one or more biomolecules are usable for membrane signal transduction. In some embodiments, the method further comprises polymerizing pre-functionalized polymer precursors to generate the 3D matrix. In some embodiments, the functionalizing occurs concurrently with providing (i) a population of cells, (ii) a plurality of three-dimensional (3D) matrices, and (iii) a plurality of fluid flow paths. In some embodiments, the functionalizing occurs subsequent to providing (i) a population of cells, (ii) a plurality of three-dimensional (3D) matrices, and (iii) a plurality of fluid flow paths.

[0027] In some embodiments, the method further comprises releasing the one or morebiomolecules from the 3D matrix or replacing the one or more biomolecules with additional or different biomolecules. In some embodiments, the 3D matrix and the fluid flow path are separated by a membrane, and wherein the fluidic material is transported through the membrane. In some embodiments, the 3D matrix has a dimension less than or equal to about 10 millimeters (mm). In some embodiments, the dimension is less than or equal to about 1 mm. In some embodiments, the dimension is less than or equal to about 500 micrometers (µm). In some embodiments, the dimension is less than or equal to about 200 µm. In some embodiments, the plurality of 3D matrices is fluidically coupled to one another. In some embodiments, the fluid flow path is disposed between a first 3D matrix and a second 3D matrix of the plurality of 3D matrices. In some embodiments, the fluid flow path delivers the fluidic material to the first 3D matrix and the second 3D matrix.

[0028] In some embodiments, the 3D matrix comprises a pore size of less than or equal toabout 400 micrometers (µm). In some embodiments, the 3D matrix comprises a pore size from about 0.5 µm to about 100 µm. In some embodiments, the 3D matrix comprises aWSGR Docket No.67566-702601 porosity of greater than or equal to about 80%. In some embodiments, an interfacial area between the fluid flow path and the 3D matrix is greater than or equal to about 5 square centimeters (cm2). In some embodiments, the 3D matrix comprises a porous, solid matrix having interconnected pores with an average pore size sufficient to permit growth of lymphocyte cells within the 3D matrix. In some embodiments, the 3D matrix has a surface area to volume ratio of greater than or equal to about 0.1. In some embodiments, the 3D matrix is a programmable 3D matrix.

[0029] In another aspect, the present disclosure provides a method for cell processing,comprising: providing a three-dimensional (3D) matrix containing a plurality of cells in a cell processing unit; in the cell processing unit, automatically: processing the plurality of cells within the 3D matrix to produce a plurality of processed cells; and separating the plurality of processed cells from the 3D matrix; and collecting the plurality of processed cells.

[0030] In some embodiments, the plurality of cells is encapsulated in the 3D matrix. Insome embodiments, the 3D matrix is configured to retain the plurality of cells. In some embodiments, the 3D matrix is configured to immobilize the plurality of cells. In some embodiments, the plurality of processed cells is a cell therapy product. In some embodiments, the cell therapy product is produced at a scale of at least one human dose equivalent. In some embodiments, the method further comprises providing a mixture comprising a matrix precursor and the plurality of cells to the cell processing system.

[0031] In some embodiments, the method further comprises, in the cell processing unit,applying a first stimulus to the mixture comprising the matrix precursor and the plurality of cells to generate the 3D matrix encapsulating the plurality of cells. In some embodiments, the first stimulus is a thermal stimulus. In some embodiments, the first stimulus is a chemical stimulus. In some embodiments, the first stimulus is an enzymatic stimulus. In some embodiments, the method further comprises functionalizing the 3D matrix with one or more biomolecules concurrently with the applying the first stimulus to generate the 3D matrix. In some embodiments, (ii) comprises applying a second stimulus to the 3D matrix to release the cell product from the 3D matrix. In some embodiments, the second stimulus is a thermal stimulus. In some embodiments, the second stimulus is a chemical stimulus. In some embodiments, the second stimulus is an enzymatic stimulus.

[0032] In some embodiments, the method further comprises, prior to providing the 3Dmatrix, generating the 3D matrix in presence of the plurality of cells such that the 3D matrix encapsulates the plurality of cells. In some embodiments, providing the 3D matrix comprises loading the 3D matrix encapsulating the plurality of cells is loaded into a compartment in theWSGR Docket No.67566-702601 cell processing unit. In some embodiments, providing the 3D matrix comprises separately providing the plurality of cells and the 3D matrix to the cell processing unit. In some embodiments, the 3D matrix is generated within a compartment in the cell processing unit. In some embodiments, the 3D matrix is generated external to the cell processing system and subsequently provided to the cell processing unit.

[0033] In some embodiments, the method further comprises functionalizing the 3Dmatrix to generate a functionalized 3D matrix. In some embodiments, the functionalizing comprises coupling one or more biomolecules to the 3D matrix. In some embodiments, the one or more biomolecules are selected from the group consisting of proteins, peptides, cytokines, antibodies, and nucleic acid molecules. In some embodiments, the one or more biomolecules are usable for membrane signal transduction. In some embodiments, the method further comprises functionalizing the 3D matrix with the one or more biomolecules prior to (i). In some embodiments, the method further comprises functionalizing the 3D matrix with the one or more biomolecules concurrently with (i). In some embodiments, the method further comprises releasing the one or more biomolecules from the 3D matrix or replacing the one or more biomolecules with additional biomolecules. In some embodiments, (i) comprises activating the plurality of cells within the 3D matrix to obtain the cell product. In some embodiments, (i) comprises transducing the plurality of cells within the 3D matrix to obtain the cell product. In some embodiments, (i) comprises transfecting the plurality of cells within the 3D matrix to obtain the cell product. In some embodiments, the 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m. In some embodiments, the method further comprises activating the plurality of cells via membrane signal transduction in the 3D matrix.

[0034] In another aspect, the present disclosure provides a cell processing system,comprising: a cell processing unit configured to retain a three-dimensional (3D) matrix, wherein the 3D matrix is configured to encapsulate a plurality of cells; and one or more computer processors operatively coupled to the cell processing unit, wherein the one or more computer processors are individually or collectively programmed to automatically (i) process the plurality of cells within the 3D matrix to produce a plurality of processed cells, (ii) direct the cell processing unit to separate the plurality of processed cells from the 3D matrix, and (iii) direct the cell processing unit to collect the plurality of processed cells.

[0035] In some embodiments, the method further comprises a receiving unit configuredto receive the plurality of cells from a user. In some embodiments, the one or more computer processors is operatively coupled to the receiving unit and individually or collectivelyWSGR Docket No.67566-702601 programmed to automatically direct the receiving unit to provide the plurality of cells to the cell processing unit. In some embodiments, the receiving unit is configured to receive the plurality of cells in a cryopreserved state, and wherein the receiving unit is configured to provide controlled thawing to the plurality of cells in the cryopreserved state. In some embodiments, the method further comprises an analysis unit configured to monitor the plurality of cells during processing of the plurality of cells. In some embodiments, the analysis unit is configured to monitor the plurality of cells in real time. In some embodiments, the analysis unit monitors or detects one or more properties selected from the group consisting of marker expression, phenotype, viability, concentration, potency, sterility, endotoxins, and safety. In some embodiments, the analysis unit comprises one or more sensors configured to monitor an environment comprising the plurality of cells. In some embodiments, the one or more sensors are selected from the group consisting of metabolites sensors, dissolved gas sensors, pH sensors, temperature sensors, viscosity sensors, pressure sensors. In some embodiments, the analysis unit comprises one or more sensors configured to analyze the cell product. In some embodiments, the one or more sensors are selected from the group consisting of Raman spectroscopy, infrared (IR) absorption spectroscopy, near-infrared (NIR) spectroscopy, mid-infrared spectroscopy, Fourier Transform infrared (FTIR) spectroscopy, optical coherence tomography, confocal microscopy, multiphoton microscopy, microwave impedance microscopy, ultrasound imaging, photoacoustic microscopy, elastography, computed tomography, conductance sensor, radio-frequency impedance sensor, dielectric spectroscopy, permittivity sensor, fluorescence imaging, mass spectroscopy, optical density, and magnetic resonance angiography. In some embodiments, the cell processing unit is further configured to provide one or more processes selected from the group consisting of cell enrichment, cell selection, cell isolation, washing, concentration, buffer exchange, and de-clumping. In some embodiments, the cell selection is negative selection. In some embodiments, the method further comprises a housing configured to retain the cell processing unit and the one or more computer processors. In some embodiments, the housing has a footprint of about 0.01 cubic meters (m3) to about 2 m3.

[0036] In some embodiments, the 3D matrix is configured to contain the plurality ofcells. In some embodiments, the 3D matrix is configured to immobilize the plurality of cells. In some embodiments, the 3D matrix is configured to encapsulate the plurality of cells. In some embodiments, the cell processing unit is configured to generate the 3D matrix in presence of the plurality of cells. In some embodiments, the cell processing unit is configured to receive a pre-formed 3D matrix. In some embodiments, the cell processing unit isWSGR Docket No.67566-702601 configured to receive the pre-formed 3D matrix comprising the plurality of cells. In some embodiments, the cell processing unit is configured to encapsulate the plurality of cells in the pre-formed 3D matrix. In some embodiments, the 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m. In some embodiments, the 3D matrix is configured to provide activation of the plurality of cells via membrane signal transduction in the 3D matrix.

[0037] In another aspect, the present disclosure provides a method for cell processing,comprising: providing a plurality of cells ex vivo to a cell processing unit; in the cellprocessing unit, contacting the plurality of cells with a selection reagent, wherein, upon the contacting, the selection reagent (i) couples with cells of a sub-population of the plurality of cells and (ii) perturbs a state of one or more cells of the sub-population to product (A) a population of perturbed cells and (B) a population of non-perturbed cells; and processing either the population of perturbed cells or the population of non-perturbed cells into a cell therapy product.

[0038] In some embodiments, the cell therapy product is produced at a scale of at leastone human dose equivalent. In some embodiments, the population of perturbed cells have an altered growth rate or viability as compared to the population of non-perturbed cells. In some embodiments, the method further comprises contacting the plurality of cells with the selection reagent and processing either the population of perturbed cells or the population of non-perturbed cells into a cell therapy product simultaneously. In some embodiments, the method further comprises processing either the population of perturbed cells or the population of non-perturbed cells subsequent to contacting the plurality of cells with a selection reagent.

[0039] In some embodiments, the cell processing unit comprises a three-dimensional(3D) matrix encapsulating the plurality of cells. In some embodiments, the method further comprises contacting the plurality of cells with the selection reagent within the 3D matrix. In some embodiments, the cell processing unit comprises a vessel comprising the plurality of cells. In some embodiments, the method further comprises contacting the plurality of cells with the selection reagent within the vessel.

[0040] In some embodiments, the selection reagent comprises a binding agent and aselection agent. In some embodiments, the selection agent is a cytotoxic agent. In some embodiments, the binding agent is coupled to the selection agent via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the binding agent is an antibody. In some embodiments, the binding agent is an aptamer. In some embodiments, theWSGR Docket No.67566-702601 population of perturbed cells comprises dead cells or cell degradation products. In some embodiments, the population of perturbed cells have an altered cell morphology as compared to the population of non-perturbed cells. In some embodiments, the processing comprises one or more processes selected from the group consisting of cell culture, transduction, transfection, transformation, expansion, in situ electroporation, and gene delivery.

[0041] In another aspect, the present disclosure provides a system for manufacturing acell therapy product, comprising: a three-dimensional (3D) matrix configured to retain a population of cells at a density of greater than about 80 million cells per cubic centimeter (cell / cm3), wherein the system is capable of processing cells of the population of cells, with aid of the 3D matrix, into the cell therapy product.

[0042] In some embodiments, the density is at least 100 million cells / cm3. In someembodiments, the 3D matrix is configured to contain the population of cells. In some embodiments, the 3D matrix is configured to immobilize the population of cells. In some embodiments, the 3D matrix is configured to encapsulate the population of cells. In some embodiments, the 3D matrix has a surface area to volume ratio greater than or equal to 0.1. In some embodiments, the 3D matrix has a porosity of at least about 80%. In some embodiments, the method further comprises a plurality of 3D matrices comprising the 3D matrix. In some embodiments, the method further comprises a fluid flow path in fluid communication with the 3D matrix, wherein the fluid flow path is adjacent to and independent from the 3D matrix. In some embodiments, the method further comprises a membrane disposed between the fluid flow path and the 3D matrix. In some embodiments, the 3D matrix is programmable.

[0043] In another aspect, the present disclosure provides a method for manufacturing acell therapy product, comprising (i) providing a population of cells at a density of greater than about 80 million cells per centimeters (cells / cm3) retained within a three-dimensional (3D) matrix and (ii) with aid of the 3D matrix, processing cells of the population of cells into the cell therapy product.

[0044] In some embodiments, the processing of the cells comprises culturing thepopulation of cells. In some embodiments, the processing of the cells comprises in situ electroporation of the cells. In some embodiments, the 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m. In some embodiments, the processing of the cells comprises activation of the cells in the 3D matrix.

[0045] In another aspect, the present disclosure provides a bioreactor for processing apopulation of cells comprising a three-dimensional (3D) matrix configured to retain theWSGR Docket No.67566-702601 population of cells, wherein the bioreactor has a volume of at most about 2 cubic meters (m3) and is configured to, with aid of the 3D matrix and at high reproducibility, processes the population of cells into a cell product.

[0046] In some embodiments, the cell product has a viability of greater than or equal toabout 75%. In some embodiments, the volume is of at most 1 m3. In some embodiments, the volume is of at most 0.5 m3.

[0047] In another aspect, the present disclosure provides a method for processing apopulation of cells comprising (i) providing a population of cells retained within three- dimensional (3D) matrix of a bioreactor, wherein the bioreactor has a volume of at most 2 cubic meters (m3) and (ii) processing, with aid of the 3D matrix and at high reproducibility, the population of cells into a cell product.

[0048] In some embodiments, the cell product has a viability of greater than or equal toabout 75%. In some embodiments, the method further comprises, prior to (i), generating the 3D matrix in presence of the population of cells. In some embodiments, the method further comprises subsequent to (ii), dissolving the 3D matrix and collecting the population of cells to generate the cell product.

[0049] In another aspect, the present disclosure provides a system for cell therapymanufacturing, comprising: a three-dimensional (3D) matrix configured to retain a population of cells at a density of at least about 5 million cells per cubic centimeters (cells / cm3), wherein the system is capable of processing cells of the population within the 3D matrix for a time period of at most about 48 hours to generate a cell product, and wherein the cell product is usable to generate a cell therapy.

[0050] In some embodiments, the density is at least 10 million cells / cm3. In someembodiments, the density is at least 20 million cells / cm3. In some embodiments, the 3D matrix has a surface area to volume ratio greater than or equal to 0.1. In some embodiments, the 3D matrix has a porosity of at least about 80%. In some embodiments, the 3D matrix has a dimension of less than about 200 micrometers (µm). In some embodiments, the dimension is less than about 100 µm. In some embodiments, the dimension is less than about 10 µm. In some embodiments, the dimension is from about 0.5 µm to about 100 µm. In some embodiments, the 3D matrix comprises a porous, solid matrix having interconnected pores with an average pore size sufficient to permit growth of lymphocyte cells within the 3D matrix. In some embodiments, the system further comprises a plurality of 3D matrices comprising the 3D matrix. In some embodiments, the system further comprises a fluid flow path in fluid communication with the 3D matrix, wherein the fluid flow path is adjacent toWSGR Docket No.67566-702601 and independent from the 3D matrix. In some embodiments, the system further comprises a membrane disposed between the fluid flow path and the 3D matrix.

[0051] In another aspect, the present disclosure provides a method for manufacturing acell therapy, comprising (i) providing a population of cells at a density of at least about 5 million cells per centimeters (cells / cm3) retained within a three-dimensional (3D) matrix; (ii) processing the cells of the population of cells within the 3D matrix for a time period of at most about 48 hours to generate a cell product; and (iii) using the cell product to generate the cell therapy.

[0052] In some embodiments, the processing of the cells comprises culturing thepopulation of cells. In some embodiments, the processing of the cells comprises in situ electroporation of the population of cells. In some embodiments, the 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m. In some embodiments, the processing of the cells comprises activation via membrane signal transduction in the 3D matrix.

[0053] Additional aspects and advantages of the present disclosure will become readilyapparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0054] All publications, patents, and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The novel features of the invention are set forth with particularity in the appendedclaims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrativeWSGR Docket No.67566-702601 embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which:

[0056] FIG. 1 shows example cell products that may be generated by the systems andmethods described herein;

[0057] FIG. 2 shows example chimeric antigen receptor (CAR)-T cell therapymanufacturing;

[0058] FIG. 3 schematically illustrates an example integrated cell processing system andconsumables;

[0059] FIG. 4 schematically illustrates an example cell processing unit and solid-statebioreactor;

[0060] FIGs. 5A and 5B show example processes provided by an example integrated cellprocessing system; FIG.5A schematically illustrates example processes provided by an example integrated cell processing system; and FIG.5B schematically illustrates an example cell processing workflow;

[0061] FIG. 6 schematically illustrates glucose levels as a function of time for examplebioreactor configurations;

[0062] FIG. 7 schematically illustrates an example workflow for producing a cell productfrom an example cell processing system;

[0063] FIG. 8 schematically illustrates example units integrated into an example cellprocessing system;

[0064] FIG. 9A schematically illustrates fluid, nutrient, or reagent transfer in examplebulk and microfluidic bioreactors; FIG.9B schematically illustrates fluid, nutrient, or reagent transfer in example tangential or perfusion flow type bioreactors;

[0065] FIGs. 10A and 10B schematically illustrates example 3D matrix configurationsand fluid interfaces;

[0066] FIGs. 11A and 11B schematically illustrate example 3D matrix functionalphysical properties;

[0067] FIGs. 12A and 12B schematically illustrate example 3D matrix functionalization;

[0068] FIG. 13 schematically illustrates example interfaces between 3D matrices andfluid flow paths and 3D matrices and cellular substrates;

[0069] FIG. 14 schematically illustrates example cell loading methods;

[0070] FIG. 15 schematically illustrates example 3D matrix loading into a cellcompartment;WSGR Docket No.67566-702601

[0071] FIGs. 16A and 16B schematically illustrate example reagent delivery and removalfrom 3D matrix;

[0072] FIG. 17 shows example delivery reagent sizes and corresponding size cutoffs;

[0073] FIG. 18 show example cell processing unit comprising a 3D matrix and flowpathway;

[0074] FIG. 19 schematically illustrates single and dual fluid flow path configurations;

[0075] FIG. 20 schematically illustrates an example cell processing unit configured fortangential flow;

[0076] FIGs. 21A-21C schematically illustrate example plate-like 3D matrixconfigurations and fluid flow pathways;

[0077] FIGs. 22A and 22B schematically illustrates example fiber-like 3D matrixconfigurations and fluid flow pathways;

[0078] FIG. 23 schematically illustrates example cell processing units including 3Dmatrix and fluid flow pathway;

[0079] FIG. 24 shows an image of an example cell processing unit comprising a 3Dmatrix and fluid flow pathway;

[0080] FIG. 25 shows an exploded view of an example cell processing unit;

[0081] FIG. 26 shows an example cartridge comprising a plurality of cell processingunits;

[0082] FIG. 27 schematically illustrates an example cell processing consumable;

[0083] FIG. 28 schematically illustrates an alternate vessel configuration;

[0084] FIG. 29 schematically illustrates example 3D matrix configurations andbioreactor vessel types;

[0085] FIG. 30 schematically illustrates example upstream modules for cell processing;

[0086] FIG. 31 schematically illustrates additional example upstream modules for cellprocessing;

[0087] FIG. 32 schematically illustrates example downstream modules for cellprocessing;

[0088] FIG. 33 schematically illustrates additional example upstream and downstreammodules for cell processing;

[0089] FIG. 34 schematically illustrates an example aseptic fluidic interface;

[0090] FIG. 35 schematically illustrates workflow for generating aseptic fluidic coupling;

[0091] FIG. 36 schematically illustrates a comparison between a non-aseptic connectorand aseptic fluidic connector;WSGR Docket No.67566-702601

[0092] FIG. 37 schematically illustrates another workflow for generating aseptic fluidiccoupling using in situ sterilized connectors;

[0093] FIG. 38 schematically illustrates example hybrid interconnects;

[0094] FIG. 39 schematically illustrates aseptic interconnection automation;

[0095] FIG. 40 schematically illustrates negative selection and magnetic cell selectionworkflows;

[0096] FIG. 41 shows example in situ stimulation of cells within a 3D matrix and insuspension;

[0097] FIG. 42 shows example negative cell selection of CD19 cells from peripheralblood mononuclear cells (PBMCs);

[0098] FIG. 43 shows example dose response curve for negative cell selection using anexample antibody-drug conjugate;

[0099] FIG. 44 shows example in situ processes for generating cell products;

[0100] FIG. 45 shows example cell transduction as a function of cell viability for celltransduced in a suspension and 3D matrix;

[0101] FIG. 46 shows example lentiviral transduction in suspended cells and cellsencapsulated in a 3D matrix;

[0102] FIG. 47 shows example electroporation within a 3D matrix;

[0103] FIG. 48 shows example optical density measurements in a 3D matrix andsuspension culture;

[0104] FIG. 49 shows example optical density measurements taken in a 3D matrix andliquid media;

[0105] FIG. 50 shows example monitoring of cell expansion within a 3D matrix and in asuspension culture;

[0106] FIG. 51 shows example monitoring of cell viability within a 3D matrix andsuspension;

[0107] FIG. 52 shows example flow cytometry measurements for cultures expanded in a3D matrix and suspension culture;

[0108] FIG. 53 shows example release testing;

[0109] FIG. 54 shows an example workflow for cell and gene therapy manufacturing;

[0110] FIG. 55 schematically illustrates example processes performed during workflowprocesses;

[0111] FIG. 56 shows an example workflow for CAR-T manufacturing;WSGR Docket No.67566-702601

[0112] FIG. 57 schematically illustrates another example CAR-T cell manufacturingworkflow;

[0113] FIG. 58 shows a computer system that is programmed or otherwise configured toimplement methods provided herein;

[0114] FIGs. 59A and 59B schematically illustrate example configurations for cellprocessing scale up;

[0115] FIGs. 60A-60C schematically illustrate example electroporation configurations;

[0116] FIGs. 61A-61B schematically illustrate other example electroporationconfigurations;

[0117] FIG. 62A and 62B schematically illustrates example optical densitymeasurements;

[0118] FIG. 63 schematically illustrates another example optical density measurementconfiguration;

[0119] FIG. 64 schematically illustrates an example consumable;

[0120] FIG. 65 schematically illustrates an internal portion of an example consumable;

[0121] FIG. 66 schematically illustrates an example electronics and component diagramfor an example cell processing system;

[0122] FIG. 67 schematically illustrates an example perfusion module;

[0123] FIG. 68 schematically illustrates another example perfusion module;

[0124] FIG. 69 schematically illustrates another example perfusion module;

[0125] FIG. 70 schematically illustrates an example perfusion module workflow;

[0126] FIG. 71 schematically illustrates an example filtration module;

[0127] FIG. 72 schematically illustrates another example filtration module;

[0128] FIG. 73 schematically illustrates another example filtration module;

[0129] FIG. 74 schematically illustrates another example filtration module;

[0130] FIG. 75 shows example cell expansion for donor cells in 3D cell culture andsuspension;

[0131] FIG. 76 shows example cell expansion and viability after transduction;

[0132] FIG. 77 shows an example image of a 3D matrix prior to cell harvest;

[0133] FIG. 78 shows an example 3D bioreactor during cell expansion;

[0134] FIG. 79 shows example sensors usable for 3D cell culture;

[0135] FIG. 80 shows example phenotype quality of cells subjected to 3D culture versussuspension culture;WSGR Docket No.67566-702601

[0136] FIG. 81 shows example T cell activation and expansion using functionalizedmatrices;

[0137] FIG. 82 shows example antibody conjugation to a functionalized 3D matrix;

[0138] FIG. 83 shows example cell isolation using size selection;

[0139] FIG. 84 shows an example filtration module;

[0140] FIG. 85 shows another example filtration module;

[0141] FIG. 86 shows example cell filtration; and

[0142] FIG. 87 shows another example of cell filtration.DETAILED DESCRIPTION

[0143] While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0144] Whenever the term “at least,” “greater than,” or “greater than or equal to”precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0145] Whenever the term “no more than,” “less than,” or “less than or equal to”precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0146] The term “automatic” or “automatically,” as used herein, generally refers to adevice, system, or process without or substantially without external direction or interference. For example, a system or device may be configured to run or may run a process from start to finish without or substantially without interference from a human operator. Alternatively, or in addition to, a system or device may be configured to run or may run portions of a process without or substantially without interference from a human operator.

[0147] The terms “cell processing system” or “bioprocessing system,” which may beused interchangeably, generally refer to a multicomponent system configured for processing cells. A cell processing system may comprise a bioreactor, cell processing unit, analysisWSGR Docket No.67566-702601 system, control system, preservation system, other components useful for processing cells, or any combinations thereof. In an example, a cell processing system comprises a bioreactor comprising a cell processing unit and additional cell processing unit(s) separate from the bioreactor. In another example, a cell processing system comprises a cell processing unit and does not comprise a bioreactor. In another example, a cell processing system may be a bioreactor comprising cell processing unit(s) integrated with one or more other components, such as analysis system, control system, or other system.

[0148] The term “cell processing unit” as used herein, generally refers to a device,module, or unit configured to process a cell. A cell processing unit may comprise a compartment configured to retain or which retains cells (e.g., cell compartment). A cell compartment, as described elsewhere herein, may include or comprise a three-dimensional substrate that provides cell retention. A cell processing unit may comprise one or more other components that facilitate or aid in processing cells. The one or more other components may include one or more of fluid flow path, interfaces, sensors, or other components useful for processing cells. A cell processing unit may be configured to process cells without expansion or culturing of the cells. Alternatively, or in addition to, a cell processing unit may be configured to expand or culture cells. A cell processing system or a bioreactor may comprise one or a plurality of cell processing units.

[0149] The term “bioreactor,” as used herein, generally refers to a device or system thatsupports a biologically active environment for growth or expansion of a cell population. A bioreactor may include a vessel, tank, compartment, or other environment in which whole cells are grown or cultured or in which cells are processed to generate a cell product or an expression product of a cell. A bioreactor may include a single vessel, tank, compartment, or other environment. Alternatively, or in addition to, a bioreactor may include a plurality of vessels, tanks, compartments, or other environments. A bioreactor may provide a controlled environment for biological reactions, chemical reactions, or both chemical and biological reactions. A bioreactor may include one or more cell processing units, as described elsewhere herein, that provide an environment for cellular expansion or culturing. A bioreactor may comprise one or a plurality of cell processing units.

[0150] The term “cell therapy product,” as used herein, generally refers to a therapyproduct including cells or tissues for implantation, transplantation, infusion, or transfer to a subject, such as a patient. A cell therapy product may be a cell product that is sufficient for treatment of a subject or patient. A cell therapy product may be a general product or may beWSGR Docket No.67566-702601 a personalized cell therapy product. A personalized cell therapy product may be manufactured from a subject’s own cells or from a cell line or other cell source.

[0151] The term “automated system,” as used herein, generally refers to a systemconfigured to process cells with little or no operator interventions. For example, an automated system may not have or permit the transfer of matter in or out of the system by an operator during operation. For example, an automated system may permit transfer of sample, cells, or consumables into the system by an operator when the system is not operating (e.g., before operation), but may not permit intervention during cell processing. In another example, the automated system may permit samples, cells, consumables, reagents, etc. to be input into the system before, during, or after operation, but may not permit intervention in the processing of the cells. The automated system may be coupled to one or more other modules or devices (e.g., a sequencer) such that matter (e.g., samples or cells) may be transferred to the coupled units, modules, or devices without or substantially without human intervention.

[0152] The term “closed system,” as used herein generally refers to a system configuredto processes cells with minimal or without environmental contamination. A closed system may be an aseptically closed system. An aseptically closed system may be a system that prevents microorganisms from entering or leaving the system to protect a cell product, workers, or facilities from contamination.

[0153] The term “integrated system,” as used herein, generally refers to a systemcomprising a plurality of components, devices, apparatus, or combinations thereof that are coupled together such that the components, devices, apparatus, or combinations thereof function as a single system. Individual components of the system may be disposed in a single housing. Alternatively, or in addition to, individual components of the system may be disposed in separate houses and be electronically, fluidically, or pneumatically coupled to function as a single system. In an example, an integrated system may comprise robotic or mechanical transfer of objects (e.g., consumables, reagents, samples, cells, etc.) between separate houses. In an example, some components, devices, or apparatus may be disposed in a single housing while other components, devices, or apparatus may be disposed in another housing or in an individual housing. The various components, devices, or apparatus may comprise individual controllers. For example, an individual component, device, or apparatus may comprise a controller associated with that component, device, or apparatus. The system may further include a master controller that permits the individual components, devices, or apparatus to be controlled as one functional system. The components, devices, or apparatus may be coupled together via electronic, fluidic, pneumatic, or other connections. Fluidic orWSGR Docket No.67566-702601 pneumatic connections may provide aseptic coupling between the various components of the integrated system.

[0154] The terms “three-dimensional (3D) matrix” or “3D substrate,” as usedinterchangeably herein, generally refer to a matrix or substrate configured for 3D cell processing. The 3D matrix or substrate may be configured to permit cell proliferation, migration, differentiation, or any combination thereof. The 3D matrix, as described elsewhere herein, may comprise a polymer matrix, such as a hydrogel matrix, 3D scaffold, or combination thereof.

[0155] The term “microfluidic,” as used herein, generally refers to fluid handlingcomponents on the scale of micrometers (µm). For example, a microfluidic fluid flow path (e.g., channel), chamber, or other fluid handling component may have one or more dimensions (e.g., diameter, depth, etc.) on the order of 1 µm, 10 µm, 100 µm, or larger. Microfluidic devices may be configured to or otherwise used to process volumes of fluid on the order of milliliters (mL) to picoliters (pL). For example, a microfluidic device may process fluid volumes on the order of 1 pL, 10 pL, 100 pL, 1 µL, 10 µL, 100 µL, 1 mL, 10 mL, 100 mL, or more.

[0156] The term “real time,” as used herein, generally refers to a response time of lessthan about 1 second, a tenth of a second, a hundredth of a second, a millisecond, or less. In an example, real time may refer to simultaneous or substantially simultaneous processing, detection, monitoring, analysis, or any combination thereof.

[0157] The term “subject,” as used here in, generally refers to a human or other mammal,avian, or other organism (e.g., plant). For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, or an individual that is a candidate for a therapy (e.g., cell therapy). A subject may be a patient.

[0158] The term “sample,” as used herein, generally refers to a biological sample of asubject. A biological sample may comprise a population of cells, for example, a heterogenous or homogenous population of cells. The biological sample may provide a cell line or cell sample for cell processing. The sample may be a cell line or cell culture sample. The sample can include one or more cells. The sample can include one or more microbes. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sampleWSGR Docket No.67566-702601 may be a fluid sample, such as a blood sample, urine sample, saliva sample, apheresis sample, or leukapheresis sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. Cell processing applications

[0159] Cells may be an important material resource for life science research anddevelopment across basic and applied scientific practices. Primary and secondary cells, including cell lines, are frequently propagated, expanded, and banked for use in research. Cells are also frequently modified, for example, via genome editing, for research use. Additionally, cell therapies, such as adoptive cell therapy and ex vivo cell-based gene therapies may represent an emerging therapeutic modality with the potential to treat many types of human diseases, for example, cancer, autoimmune diseases, neurological diseases, and age-related diseases.

[0160] The methods and systems described herein may be useful for production of celltherapies. Cell therapies including adoptive cell therapy (ACT) and ex vivo cell-based gene therapies may represent an emerging therapeutic modality with the potential to treat many types of human disease, including, for example, oncology, autoimmune, neurological, and aging-related diseases. In autologous therapies, cells may be collected from the patient, modified ex vivo, and reintroduced to the patient. In allogeneic therapies, a different cell source than the patient may be used, including primary donor sources (matched or unrelated), cells derived from primary cells, and secondary cell sources including stem cells and induced pluripotent stem cells (iPSC). Many emerging cell therapies may utilize genome editing technologies to engineer cells with therapeutic functions and mechanisms of action, which adds significant complexity to cell manufacturing (also referred to herein as cell processing, cell bioprocessing, or bioprocessing). Advanced cell therapies may be comprised of immune cells or cells of an immune lineage, such as T cells and hematopoietic stem and precursor cells (HSPCs). Some advanced cell therapies delivering other cell types may be approved or in various stages of development, including those which use various somatic cell types including fibroblasts, chondrocytes, keratinocytes, hepatocytes, pancreatic islet cells, cardiomyocytes, neurons, and other types of immune cells, such as dendritic cells (DCs), natural killer (NK) cells, and macrophages. Other types of cell products may include stem cells (e.g., both naturally occurring, such as in tissues, bone marrow, and cord blood, and induced, e.g., iPSC), and cell products derived from stem cells through directed differentiation or genetic programing to achieve a targeted therapeutic cell state. Despite the significant number of approved products and ongoing clinical trials, many challenges remainWSGR Docket No.67566-702601 in reproducible, cost-effective manufacturing procedures for cell and ex vivo cell-based gene therapy products and other expression products of a cell with therapeutic and industrial value.

[0161] The systems and methods described herein may be useful for development of avariety of cell products. For example, and as shown in FIG.1, the systems and methods described herein may be useful for culturing or otherwise producing cell products from adherent cell types, stem and stem derived cells, rare cell types, or any combination thereof. The systems and methods described herein may be useful for personalized medicine, point of care treatment, or both.

[0162] Cell manufacturing may be a bottleneck that limits patient access, increases cost,and reduces commercial viability of cell therapy products. For example, cell therapy products may utilize 106-109therapeutic cells per clinical dose. Increasing the efficiency and rate of cell expansion while maintaining critical attributes may be challenging. Executing complex bioprocessing procedures at dose scale may also be challenging and may use multiple instrumentation platforms to perform various processes. The various processes that comprise complex or multi-process cell processing workflows may be performed in series, increasing manufacturing time. Manufacturing challenges may be reflected in the high cost of researching and developing novel cell therapies, as well as the high cost of approved cell therapy products. Reproducible manufacturing may also be a challenge due to functional heterogeneity of cells and variations that result in different quality attributes and clinical outcomes. Sources of product variability may include, but are not limited to: Patient-to- patient variability and intra-population heterogeneity in source materials, or variability introduced during manufacturing processes, including variations in cell isolation, expansion processes, or genome engineering processes.

[0163] Approximately ten advanced cell therapies are approved for human use in the US,and despite hundreds of thousands of patients being potentially eligible for cell therapy treatments, to date tens of thousands of patients have received cell therapy treatment. Currently hundreds of cell therapies are in development or being tested in clinical trials. Over the next decade, many tens of cell therapies are expected to be approved for human use. New advances in clinical cell manufacturing may be useful to reduce the research and development burden of novel cell therapies, as well as to deliver these potent therapies to more patients. In addition, cell therapies may be manufactured in centralized plants, adding complexity in the transport of fresh or cryopreserved cell products and manufacturing logistics. Advances in clinical cell therapy manufacturing that enable alternative manufacturing paradigms, such as distributed and point-of-care manufacturing may reduceWSGR Docket No.67566-702601 cost and otherwise improve patient access. Advances in cell manufacturing that reduce cost, labor, logistical complexity, biological variation, and turnaround time of cell therapy production may increase patient access to a powerful emerging therapeutic modality.

[0164] The systems and methods described herein may be useful for gene therapy,valuable cell byproduct, and biologic therapy production. Cellular processes, both natural and modified through genetic engineering, enable the production of various chemicals and biologic compounds that are essential in the pharmaceutical, chemical, food, energy, and agricultural industries. Bioproduction of clinically and commercially important biological products and chemicals from living cells may be complex and time-consuming, requiring complex infrastructure, manufacturing equipment, and workflows. Bioreactors are crucial tools for bioproduction, enabling the controlled and scalable production of valuable cellular byproducts. These specialized systems are designed to provide an optimal environment for the growth and metabolic activity of cells, allowing for the efficient production of biologics, such as enzymes, therapeutic proteins, vaccines, viral particles, and other biological agents. By precisely controlling parameters such as temperature, pH, oxygen levels, and nutrient supply, bioreactors ensure that cells are maintained in conditions that maximize their productivity.

[0165] One application of bioreactors is for production of biopharmaceuticals through theuse of recombinant DNA technology. Cells (e.g., natural or genetically engineered cells) may be cultured in bioreactors to produce therapeutic proteins such as insulin, monoclonal antibodies, and hormones. These proteins may be useful for treating various diseases, including diabetes, cancer, and autoimmune disorders. For example, monoclonal antibodies produced in bioreactors may be used in targeted cancer therapies that may bind specifically to cancer cells and mediate their destruction without harming normal cells. Bioreactors may be useful for producing viral vectors that are capable of delivering therapeutic genes to patients in the form of in vivo gene therapies. For example, onasemnogene abeparvovec-xioi (Zolgensma) uses an adeno-associated virus vector (AAV9) to deliver a fully functional copy of the human survival motor neuron (SMN) gene directly into a patient’s cells. The SMN gene is critical for the survival of motor neurons, and mutations in this gene may cause spinal muscular atrophy (SMA). By providing a new, working SMN gene, Zolgensma may address the genetic root cause of the disease, allowing motor neuron cells to produce proteins for improving muscle movement and function. This gene therapy may be useful to treat pediatric patients under the age of two with SMA, a rare and often fatal genetic disease that causes muscle weakness and progressive loss of movement. Zolgensma may be administered as aWSGR Docket No.67566-702601 one-time intravenous infusion, highlighting the practical advantages of in vivo gene therapies. However, the production of viral vectors such as gene therapies may be tightly controlled to ensure their safety and efficacy. Bioreactors may provide a controlled environment that can support the growth of virus-producing cells to high densities, facilitating the production of sufficient quantities of vectors for clinical applications. The use of bioreactors in bioproduction may offer reliable, efficient, and scalable methods for generating high-value biological products. As biotechnology continues to advance, the capabilities of bioreactors may expand, leading to greater innovations and applications in medicine and beyond.

[0166] Cells may be a material resource for life sciences research and development acrossbasic and applied scientific practices. As such, cell culture and cell engineering workflows are integral to the practice of modern life sciences. Moreover, many research and development applications use significant quantities of cells or reproducible sources of cells over time. Examples of some research and development workflows and experimental paradigms which utilize cells include, but are not limited to, high-throughput screening, cell- based assays, and stem cell research. Primary and secondary cells including cell lines may be frequently propagated, expanded, and banked for use in research. Cells may also be frequently modified for research use, including by genome editing, through various bioprocessing workflows of considerable complexity. Across all these bioprocessing workflows, manual and semi-automated approaches require significant time, labor, and expense, which ultimately increases the cost and decreases the flexibility of cell-based research. New advances in cell manufacturing that reduce the turnaround time, labor, or cost of cell bioprocessing workflows may therefore accelerate cell-based life sciences research.

[0167] The systems and processes described herein may be useable for producing avariety of cell products. Cell products may include products comprising whole cells or products generated by whole cells. In an example, the systems and methods described herein may be usable to produce chimeric antigen receptor (CAR)-T cell therapy. As shown in FIG. 2, CAR-T cell therapy manufacturing may include rapid and non-rapid manufacturing methods. Manufacturing methods may include point of care, distributed, or centralized manufacturing. The systems and methods described herein may provide cell therapy manufacturing using smaller production volumes and higher cell densities than other cell therapy manufacturing systems and methods.

[0168] The systems described herein may be cell processing systems, integrated cellprocessing systems, automated cell processing systems, or any combination thereof. A cellWSGR Docket No.67566-702601 processing system may be an integrated cell processing system, as shown in FIG.3. The integrated cell processing system 301 may be configured to permit flexible cell inputs 302. The integrated cell processing system may include a graphical user interface (e.g., touch screen interface) that provides users with real time information related to cell processing. Cell inputs 302 may include whole blood, Leukopak, cryopreserved cells, or any combination thereof. The integrated cell processing system 301 may be configured to permit the use of flexible sample carriers 303. The system may include radio frequency identification (RFID) readers for carrier identification and cloud-based electronic documentation systems. Sample carriers may include, but are not limited to cryo bags, tubes, vessels, cartridges, or any combination thereof. The integrated cell processing system may further include an integrated preservation unit 304. The preservation unit 304 may be a cryopreservation unit. The preservation unit 304 may be configured to freeze or thaw cells, cell samples, or other biological products. The preservation unit 304 may be configured for rapid cryopreservation.

[0169] A cell processing system may comprise a processing unit and an analysis unit.The cell processing unit 401 and analysis unit 402 may be disposed separately. Alternatively, or in addition to, the cell processing unit and analysis unit may be integrated into a single cell processing and assay unit 403, as shown in FIG.4. The cell processing and assay unit 403 may be configured to provide various functions including, but not limited to, cell enrichment, cell isolation, washing, concentration, buffer exchange, de-clumping, or any combinations thereof. The cell processing and assay unit 403 may be configured to provide various product and release assays including, but not limited to, marker expression, phenotype, viability, concentration, potency, sterility, endotoxin, pyrogen, safety (e.g., vector copy number, replication competent virus, etc.), or any combinations thereof. The output (e.g., cells, media, etc.) from the cell processing and assay unit 403 may be provided to a bioreactor (e.g., solid state bioreactor) 404. The bioreactor 404 may be configured to provide various cellular processing functions including, but not limited to, cell culture, membrane transduction, expansion, gene delivery, cell selection (e.g., negative or positive selection), or any combinations thereof.

[0170] A cell processing system may be a benchtop system configured to perform variouscell processing processes, as shown in FIG.5A. The cell processing system 501 may provide cell isolation 502, activation 503, gene transfer 504, expansion 505, formulation 506, harvest 507, or any combination hereof. The integrated cell processing system 501 may be useful for research, preclinical cell therapy development, clinical research, or any combination thereof. The cell processing system 501 may provide several, non-limiting benefits including reduceWSGR Docket No.67566-702601 costs and labor, increase process repeatability, improve and reduce processing time to accelerate breakthroughs, improve outcomes, or any combination thereof. The cell processing system 501 may provide fully or partially automated functions performed in any order for hands-off or reduced touch operation.

[0171] Cell processing systems described herein may be usable for running a variety ofcell processing operations and workflows. An example cell processing workflow, as shown in FIG.5B, may include cell thawing 510, cell culture 511, cell engineering 512, assaying 513, cell selection 514, freezing 515, or any combination thereof. The various processes may include automated processes and non-automated processes. Processes may be performed in any order and some processes may be performed more than once while processing a population of cells. Thawing 510 may comprise using a thermal unit to alter the state of provided cells. The thermal unit may be integrated with a preservation unit. Cell culture 511 may be conducted in a bioreactor (e.g., solid state bioreactor) and may include culture and expansion. Cell engineering 512 may include, but is not limited to, membrane transduction (e.g., activation), gene delivery (e.g., electroporation, lipid nanoparticles (LPN), viral), or any combination thereof. Assaying 513 may include, but is not limited to, cytometry, imaging, molecular assays, cell-based assays, or any combinations thereof. Cell selection 514 may include, but is not limited to, in situ selection (e.g., with or without beads), ex situ selection, upstream selection, downstream selection, or any combinations thereof. The cell or biological product produced may be subjected to freezing 515 via the thermal unit.

[0172] The systems and methods described herein may provide continuous nutrientdelivery prior to, during, or after cell processing. FIG.6 schematically illustrates glucose levels, which may be an indicator of provided nutrients, as a function of time for example bioreactor configurations. In a static or batch configuration, glucose levels may begin high and decrease over time until depletion or near depletion occurs. In a batch fed configuration, glucose levels may begin high and exponentially decrease until a bolus of glucose is added returning the level to beginning or near beginning levels. This reduction and bolus cycle may repeat multiple times, for example, until the cell product is formed. In a continuous flow configuration, the glucose level may be maintained constant or otherwise closely controlled at a target level.

[0173] In an example, a cell processing system may be configured to provide fed batchworkflows. FIG.7 schematically illustrates an example workflow for producing a cell product from an example cell processing system. A workflow may include providing isolating a population of cells 701. The isolated cells may be mixed with a gel precursorWSGR Docket No.67566-702601 material 702. The mixture comprising the cells and the gel precursor may be loaded into a compartment, for example, a compartment of a cassette and polymerized 703 to encapsulate the cells in a 3D polymer matrix. Activation reagents may be provided 704 to the cells encapsulated within the 3D matrix. The cells may be cultured or expanded 705 within the 3D matrix for at least 1, 2, 3, 4, 5, 6, 8, 10, or more days. In an example, the cells may be cultured or expanded 705 within the 3D matrix for two to three days. Genes may be delivered to the expanded cells 706, for example, via providing a virus and media mixture to the cells. Cell may be cultured 707 during, or subsequent to, gene delivery to a target cell concentration or number of cells. Once the target concentration or number of cells is reached the 3D matrix may be liquified and recovered 708 from the compartment. The recovered cells may be harvested 709 as a cell product or for downstream processing.

[0174] A cell processing system, as described herein, may comprise multiple units andmay be configured to provide multiple processes, as shown in FIG.8. The cell processing system may be configured to receive liquid phase cellular material 801, for example, via a sterile interconnection. The system may comprise a preservation unit comprising a thermal unit configured to provide a controlled-rate thaw. The preservation unit may be configured to receive frozen cellular material and to thaw the cellular material. The thawed or liquid phase cells may be provided to a cell processing module 802 for de-clumping, enrichment, concentration adjustments, mixing with liquid gel precursors, or any combination thereof. The cell and liquid gel precursor mixture may be provided to a compartment of a bioreactor 803 for polymerization. The bioreactor 803 may be configured for perfusion of media, in situ cell selection, cell stimulation, gene delivery, or any combination thereof. The cells processed in the bioreactor may be provided to a cell processing unit 804. The cell processing unit 804 may be the same cell processing unit as 802 or a different cell processing unit. The cell processing unit 804 may be integrated with an analysis unit 805 and 806. The cell processing unit 704 and analysis unit 706 may configured for de-clumping, washing, concentration adjustment, material capture for release testing, release testing, buffer exchange for preservation, or any combinations thereof. The cells may be provided to a cryobag 807 or other vessel within a preservation unit for preservation (e.g., cryopreservation). Cell processing platforms

[0175] In an aspect, the present disclosure provides cell processing systems, devices, andmethods usable for cell processing. The described systems may include or provide for a variety of cell processing, cell culturing, or cell expansion applications. The described systems and methods may provide for two- or three-dimensional cell culture. In an example,WSGR Docket No.67566-702601 the described systems and methods provide for two-dimensional (2D) cell culture. In another example, the described systems and methods provide for three-dimensional (3D) cell culture. Two- and three-dimensional cell culture

[0176] In cell culture, cells may be categorized based on cell growth characteristics andrequirements, particularly in whether they attach or do not attach to surfaces. Adherent cells may be attached to a solid or semi-solid surface such as tissue culture plastic or microcarrier beads to grow and proliferate. These cells may be derived from organs where cells are naturally anchorage-dependent. Adherent cells may grow as a monolayer, attaching to the bottom of the culture dish, flask, or beads, or otherwise grow into three dimensional (3D) structures such as clusters or organoids. Adherent cells may be detached from the solid substrate using enzymatic or mechanical methods during or prior to subculturing, passaging, or harvesting. Suspension cells may not be attached to any surface for growth. Suspension cells may grow freely floating in the culture medium. Suspension cells may include cells derived from blood, such as lymphocytes or hematopoietic stem cells.

[0177] Both adherent and suspension cells may be cultured in two dimensional (2D)systems, where adherent cells survive and grow while adhered to a solid substrate (e.g., Petri dishes, flasks, or plates). For example, adherent cells may grow in a monolayer, while suspension cells may settle to the bottom surface due to gravity (e.g., in the absence of mixing) where they survive and grow. For adherent cell types, 2D culture approaches may lack scalability due to the linear relationship between maximum potential viable biomass and the available surface area for cells being cultured. In addition, the surface attachment may include more intricate handling during subculturing, passaging, or harvesting, and may require specific types of coated culture vessels for effective growth. Strategies to scale production by increasing the available surface area may utilize multi-layer plate or flask vessels which include multiple 2D surfaces. However, these types of 2D culture cell apparatus may have numerous challenges. For example, it may be challenging to uniformly distribute cells across or between the layers. To improve this process, procedures which change the orientation of the device (e.g., rotation) during cell seeding may be used to provide a more uniform distribution. It may be challenging to uniformly perfuse multi-layer vessels with media to support longer-term culture. It may also be challenging to provide uniform gas exchange (e.g., to provide a suitable supply of oxygen) by use of liquid- impermeable gas-permeable membranes, as each layer may include a gas exchange membrane to locally oxygenate cells. For suspension cell types, 2D culture approaches may also lack scalability and exhibit other biological challenges. Without a method of mixing orWSGR Docket No.67566-702601 keeping the cells in suspension, cells may settle to the bottom due to gravity. If the cell density is too high, the formation of a dense layer of settled cells can result in non-uniform delivery of nutrients and oxygen (whether nutrients or nutrients and gas are provided by excess media on top of the settled cells, or whether gas is provided by a gas-permeable liquid-impermeable membrane below the cell layer), resulting in increased variability or poor quality. Table 1 shows example differences between 2D and 3D cultures. Solid-state cell processing may provide widespread use of various cell types for therapeutic applications. For example, the systems and methods described herein may permit cell product generation from low yielding cell sources such as umbilical cord blood sources with small quantity of hematopoietic stem and progenitor cells (HSPCs). Generating cell products from low yielding cell sources in 2D may be difficult due to low expansion ability, loss of stemness features, diminished migration and engraftment, reduced capacity for long-term repopulation, or any combination thereof. The solid-state cell processing systems and methods described herein may provide a 880-fold increase in HSPCs, 10- to 100-fold increase in stemness marker genes, 10-fold increase in homing genes, 73-fold increase in long-term HSC frequency and prolonged in vivo duration of hematopoietic reconstitution. Table 1. Example differences between 2D and 3D cell culturing.WSGR Docket No.67566-702601

[0178] Stirred or mixed suspension cultures, which may utilize horizontal-impeller,paddle, and rocking-wave mixing mechanisms, can generate unfavorable hydrodynamic environments and stresses (e.g., shear stress) that negatively impact cell growth and quality maintenance. In perfused suspension cultures, cells may be lost in spent media during recirculation, media removal, or both. Use of a cell retention device in a fluid perfusion system, which may separate cells from spent media or retains cells in the bioreactor, such as by the process of alternating tangential flow (ATF) filtration in which cells are retained in the filter of the ATF while the spent media is removed, may reduce or prevent the loss of cells during perfusion. Alternative cell retention techniques include other filtration approaches including, but not limited to, passive flow-based retention (e.g., approaches which use inertial or hydrodynamic forces, such as by using a spiral flow or deterministic displacement configuration), and use of active flow-based retention (e.g., approaches which use acoustics or magnetic forces, such as by using an acoustic transducer to separate cells from spent media via acoustic particle focusing). These cell retention methods for suspension culture perfusion may cause reliability issues or reduce cell quality. For example, membrane-based and filter- based cell retention strategies can suffer from membrane fouling, flow-based cell retention strategies can suffer from clogging (especially microfluidic embodiments), and active cell retention strategies (e.g., acoustic focusing) can subject cells to unphysiological and harsh forces. Moreover, neither 2D nor suspension culture approaches provide the benefits of a 3D cell culture environment described herein.

[0179] Suspension bioreactors may use carrier particles, for example, microcarriers. Incell culture applications, microcarriers can comprise non-porous bead or particle substrates with a 2D surface onto which cells are localized or adhered. Particle substrates may comprise glass, polystyrene, microporous materials (e.g., cell-impenetrable, polymer materials such as gelatin, collagen, chitin, cellulose, acrylamide, or combinations thereof), or combinations thereof. These types of carrier particles may not provide the benefits of 3D cultureWSGR Docket No.67566-702601 environments described herein. Other carrier particle approaches may use porous or macroporous two and a half dimensional (2.5D) / 3D particles, which may provide a 3D culture environment (e.g., forming a cell-laden matrix, such particles being composed of materials including but not limited to cellulose, gelatin, dextran, collagen, acrylamide, and PEG), suitable for culture of adherent cells in a suspension bioreactor. Such substrates may be unsuitable for many types of cells, including but not limited to migratory cells, cells capable of mixed suspension-adherent culture, and non-adherent / suspension cells, such as T cells, which may not remain localized to the porous carriers during culture and may relocate to the suspension media phase. Regardless of carrier particle type (porous or non-porous), carrier particle approaches may have significant limitations. Cell loading onto or into carrier particles may be inefficient, relying on an optimized seeding density and environment providing for collision between cells and carrier particles to facilitate the attachment of cells. In addition, as there may be little or no communication between individual carrier particles, a random or stochastic cell seeding procedure may result in carriers being seeded with a highly variable initial number of cells. Carrier particles seeded with a greater initial number of cells may reach confluence, while other carrier particles either not seeded or seeded with a lesser initial number of cells may remain empty or do not reach confluence. Frequent passaging may overcome these particle-localized space limitations and heterogeneity. In addition, empty carrier particles may not contribute to the overall available surface area or volume of the culture system, reducing the effective manufacturing scale or yield of such systems below the theoretical maximum.

[0180] Although suspension bioreactors (which may be static or mixed, such as stirredand impeller bioreactors) may be suitable for carrier particle culture approaches, they may be configured to create an excess suspension media environment, in which the carrier particles comprise a fraction of the total bioreactor volume, for example, 16 grams (g) of carrier per Liter (L) of culture. Moreover, mixed or stirred bioreactors may expose cells, carrier particles, or both to shear and other physical stresses that may damage or detach cells, or otherwise degrade the carrier particles. Bioreactors configured for carrier particle cell culture may use batch, fed-batch, or perfusion feeding. For fed-batch or perfusion feeding, a method of separating and retaining carrier particles from spent media is used. Such methods may be subject to challenges similar to the challenges of retaining suspension cells in such bioreactors. Carrier particle culture approaches are also subject to carrier particle clumping and aggregation, which may negatively affect the supply of nutrients, oxygen, and other supportive factors to cells within clumps or aggregates. Clumps and aggregates may furtherWSGR Docket No.67566-702601 clog, impede, or reduce the efficiency of retention, feeding, and harvest procedures. When non-dissolvable or non-biodegradable carrier particles are used, cells may be harvested and separated from the carrier particles. However, inefficient cellular detachment methods may reduce harvest efficiency or cell yield, while harsh cellular detachment methods may reduce the viability or quality of resulting cell products. For use in humans, carrier particle contamination is a significant risk and may rely on additional procedures and quality control to ensure efficient removal and minimal-to-no carry over of carrier particles.

[0181] Packed and fixed-bed bioreactors may be suitable for carrier particle cultureapproaches. These types of bioreactors may face numerous limitations and challenges (in addition to challenges of clumping and aggregation, harvesting cells from carriers, and others described above) which limit scale-up, increase product variability, or reduce product quality. For example, it may be challenging to achieve a uniform distribution within the bed, such that the cell distribution, liquid flow, or liquid communication across and between any subsets or cross sections of the bed may result in, for example, nutrient gradients in the bed or aeration limitations. It can also be challenging to maintain a uniform cell distribution or fluid circulation throughout a fixed or packed bed, over time within a given batch and from batch to batch. Variation in bioreactor packing and preparation can also create variability in both processes and products. Because both cell distribution and fluid circulation directly impact bioreactor performance (e.g., aeration, nutrient supply, shear stresses on cells), this can result in challenges with manufacturing consistency and performance over time. When scaling up, changes in the dimensions or volume of the packed bed may rely on extensive, time- consuming empirical optimization of other system attributes such as perfusion flow rate.

[0182] Three-dimensional (3D) culture systems may grow cells in a 3D matrixenvironment. Three- dimensional culture processes performed manually in the lab environment by static culture, including through the use of spheroids, droplets, and gels or other 3D substrates in multi-well plates and flasks, may be unsuited for scalable manufacturing and bioprocessing. Alternatively, automated 3D cell culture systems can provide significant benefits.

[0183] Compared to 2D cultures, cells cultured in a 3D microenvironment may exhibitsubstantially different and often superior properties. For example, 2D cultures may be limited in depth to a monolayer, while 3D cultures may be distributed in 3D space. Cells cultured in 2D may be flatter, more elongated, or both, while cells cultured in 3D may have more natural and complex cell shapes. Cells in 2D culture may migrate along two dimensions, and can be accessed from a single dimension, while cells in 3D culture may migrate in 3D and can beWSGR Docket No.67566-702601 accessed from multiple dimensions. Cell junctions and cell-cell interactions in 2D cultures may be less common and less physiological compared to 3D cultures. Gene expression may be perturbed in 2D cell cultures, but more closely resemble in vivo expression patterns in 3D cultures. In 2D cultures, the effects of gravity and physical microforces may be minimal or absent, while 3D cultures may expose cells to more natural, in vivo-like gravitational and physical microforces. Additionally, 3D cultures may enable increased control over the microphysical environment (e.g., stiffness, elasticity, and presentation of factors to cells), whereas 2D cultures may provide little or no control over these parameters. The additional control over cell microenvironment provided by 3D culture approaches may be beneficial to reducing variability in products, both resulting from variability in source materials and by reducing variation introduced during production. A 3D matrix which further provides for the retention of cells during bioprocessing may have process advantages, both biological and process / automation, including but not limited to, providing a low-shear environment, reducing or eliminating use of cell retention devices during feeding or media perfusion, reduced susceptibility to contamination, and instrument platforms with simplified designs or fewer components, increasing reliability and reducing cost. For equivalent production throughput, 3D cultures may use a smaller working volume and overall volume than 2D cultures, due to increased cell concentrations (e.g., cell volume per reactor volume, cell number per reactor volume, or both).

[0184] Three-dimensional cell culture processes may be challenging to implement withexisting bioreactor designs, which includes static, hollow fiber, packed bed, and other bioreactor configurations. For example, it is challenging to culture cells in 3D while maintaining cellular localization to the 3D substrate and providing an adequate supply of culture components supporting cellular metabolism, such as media, nutrients, and dissolved oxygen. In a packed-bed reactor design, media may be substantially perfused through and around the cell-containing compartment (e.g., the packed bed). However, establishing a suitable rate of media flow through a 3D substrate to support cell growth may rely on unsuitably high fluid pressures or subject cells or the substrate to unsuitable forces negatively impacting the quality of cells or the quality and durability of the 3D substrate. Providing media flowing around a 3D substrate may not be a scalable delivery mechanism, as limited diffusion rates within a 3D substrate may limit the effective distance of perfusion, resulting in non-uniform perfusion or necrotic zones within the 3D substrate. The use of a 3D substrate that is porous enough to enable fluid perfusion at appropriate scale (e.g., woven fiber beds, aligned fiber beds, given the physical dimensions of the 3D substrate) may not provide aWSGR Docket No.67566-702601 suitable 3D microenvironment to capture the benefits of 3D cell culture (e.g. may be too microporous, such that cells are effectively in 2D culture attached to the scaffold, or in suspension culture in the interstitial spaces). In a hollow-fiber type perfusion bioreactor, a fraction of the intracapillary (IC) compartment, which comprises the space inside the hollow fibers as well as the head space on each end of the casing and the dead volume of the IC fluidic loop, is exposed to media perfusion by the extracapillary space of the casing. The rest of the volume may not be in fluidic communication with the extracapillary (EC) space. This may increase the challenges of cell seeding, gas exchange, and methods for providing nutrients to the cells. Because the 3D substrate substantially retains the cells, loading a cell- laden matrix in the IC may result in a substantial fraction of cells being retained in non- perfused IC spaces. Similarly, a fraction of the EC (which may comprise head spaces on each end of the casing and a dead volume of the EC fluidic loop) may be in fluidic communication with the IC. Loading a cell-laden matrix in the EC may similarly result in a substantial fraction of cells being retained in non-perfused EC spaces. Additionally, because hollow fibers may be distributed randomly within the casing, loading the EC with a cell-laden matrix may result in non-uniform perfusion by media flow in the IC. Cell processing systems

[0185] The manufacture of cell therapies and other valuable chemical and biological cellproducts may use cell processing (e.g., bioprocessing) beyond simple bioreactor culture, such as cell isolation and gene delivery. Bioreactor systems without integrated cell processing capabilities may involve the transfer of cellular biomass between different workflows and instrumentation platforms. Biomass transfer and processing between workflows and instrumentation may increase human interaction, increasing the labor used for production and therefore increasing product cost. In “open” configurations, the cellular biomass may be exposed to the environment during processing, leading to contamination, manufacturing failures, batch-to-batch variability, difficulty meeting regulatory standards including for sample tracing, and using larger footprints in highly controlled environments (e.g., cleanroom) or more highly trained personnel. In “closed” configurations, the systems may be designed to avoid exposure of the cellular biomass to the environment (e.g., through use of sterile barriers and connectors, through the incorporation of single-use technologies, or both), which may reduce the risk of contamination and increases flexibility in terms of facility design (e.g., less controlled) and personnel staffing (e.g., less trained). Closed configurations may use manual or automated methods of forming the numerous sterile interconnections to aseptically transfer the biomass between process workflows, which may increase the amountWSGR Docket No.67566-702601 of human labor used or the complexity of automated systems. Non-integrated manufacturing systems may use multiple instrument systems and platforms, creating challenges in scheduling and instrument availability when many products are being manufactured concurrently, as well as challenges in documentation and accurate record keeping. Transferring cells between platforms and processes may be harsh on cells when there is a change in cell concentration or buffer, and when cells are held in non-optimal conditions between processes (e.g., in a transfer vessel). Transferring cells within a closed process via the establishment and termination of closed sterile interconnects (e.g., sterile welding inputs and outputs at each operation or on each separate instrument platform, during consumable manufacturing, system setup, or system operation) may use complex fluidic or automation architectures or labor-intensive, complex procedures. Automated transfer of cells between processes, such as by using one or more robotic arms or robotic systems to physically transport the cellular biomass between instruments (or alternatively, to transport instrumentation between cellular biomasses), may be challenging to scale. Such robotic systems may not scale well due to their size and complexity and may further exhibit fundamental limits in terms of scaling multiple parallel processes (e.g., the number of concurrent operations that can be supported by a single robot arm may be limited by time to perform each operation and physical reach of the robot arms or motion systems). For these reasons, closed integrated systems may improve consistency, purity, and safety while helping to lower overall manufacturing costs by reducing labor and facility requirements. Closed integrated systems which perform more, substantially all, or all processes involved in cell processing may be scalable by simply increasing the number of systems used in manufacturing.

[0186] Another challenge for cell processing platforms and technologies is the integrationwith cryopreserved input materials and the generation of cryopreserved output materials. When bioprocessing from a cryopreserved source material, the frozen sample may be submerged in a water bath at 37°C until a visual determination is made that the ice has melted (e.g., wet thawing). This method may be simple and low cost but may rely on specialized technicians to be executed successfully and with consistency. Variability in user behavior and laboratory settings can cause batch-to-batch variation. Untrained operators might choose slightly different, and often subjective, cryobag angles, agitation speeds, and end-of-thaw criteria, producing a spectrum of inconsistent results. While sufficient in a research setting, water baths present challenges in clinical and cleanroom environments. In addition to the risk of process and product variability, the presence of warm water may introduce contaminationWSGR Docket No.67566-702601 risk that is unacceptable in controlled settings. The integration of an automated thawing module functionally connected to a bioprocessing system may solve these challenges.

[0187] When cell processing a cryopreserved product, the cell sample or other materialmay be suspended in a specialized buffer suitable for cryopreservation, and the temperature may be lowered to freezing using an optimized procedure to minimize damage to the cells or product. Cell cryopreservation buffers may be at least mildly cytotoxic, such that extended cell exposure to the liquid buffer may result in loss of post-thaw viability and other quality attributes. The integration of an automated cryopreservation module functionally connected to the cell processing system may minimize the time in which cells are exposed to cryopreservation buffer prior to freezing, while also reducing the timing and complexity of manufacturing, as the product may be stable once frozen and can be collected from the system at a convenient time.

[0188] As described herein, integrated bioprocessing systems (e.g., integrated cellprocessing systems) may provide various benefits over other bioprocessing or cell processing systems. For example, the described systems may provide simpler fluidics, such as fewer valves, tubing, and interconnects, which may provide for smaller and lower cost instrumentation, simpler consumables, and ease of sterilization. The described systems and methods may provide for a reduced routing of cells between processes as compared to other bioprocessing or cell processing systems. The systems and methods described herein may enhance cellular phenotypes and provide expanded applications as compared to other bioprocessing systems. For example, systems and methods described herein may provide for healthier cells due to reduced processing and handling. In some embodiments, three dimensional (3D) matrices may be used to replicate natural tissue extracellular matrix to provide superior phenotypes. The described systems and methods may be useful for processing adherent or suspension cells.

[0189] The cell processing systems described herein may be useful for immunoassaysand target discovery, cell research and cell and gene therapy (CGT) development, clinical cell therapy, induced pluripotent stem cell (iPSC) and adherent cell bioprocessing, continuous biologics manufacturing, or any combination thereof. Immunoassays and target discovery applications may include, but is not limited to, massively multiplex ribonucleic acid (RNA)- based artificial antigen presenting cells (APC) for antigen screening and immunoreactivity validation or scalable receptor and target screening for autoimmune research and fully-human chimeric antigen receptor (CAR) development. Cell research and CGT development applications may include, but is not limited to, multiplex RNA cell engineering to understandWSGR Docket No.67566-702601 phenotypes and functional consequences of gene expression heterogeneity, development of phenotypic enhancements for cell and gene therapies, cell therapy process development, or any combination thereof. Clinical cell therapy applications may include, but is not limited to, multiplex RNA cell phenotype engineering and CAR delivery, safer profile due to transient kinetics and reduced or no risk of oncogenic integration, focus on oncology and autoimmune disease areas, long-term delivery of synthetic immune systems, or combinations thereof. IPSC and adherent cell bioprocessing applications may include, but are not limited to, bioreactor (e.g., solid-state) methods of scalable iPSC expansion and developmental programming, bioreactor (e.g., solid-state) methods of scalable production of adherent cell types such as islet cells and cardiomyocytes, research and clinical use, or combinations thereof. The systems and methods described herein may provide continuous biologics manufacturing for high density cell line culture in three-dimensional (3D) matrix with continuous media perfusion and collection of secreted proteins for purification. Three-dimensional matrices and methods of use

[0190] In an aspect, the present disclosure provides systems for cell processing. Asystem for cell processing may include a plurality of three-dimensional (3D) matrices and a plurality of fluid flow paths. A 3D matrix of the plurality of 3D matrices may be configured to retain or may retain a population of cells. A fluid flow path of the plurality of fluid flow paths may be independent from and adjacent to the 3D matrix. The fluid flow path may be configured to deliver or may deliver a fluidic material to the 3D matrix to process the population of cells into a cell product.

[0191] In another aspect, the present disclosure provides methods for cell processing. Amethod for cell processing may include providing a population of cells, a plurality of 3D matrices, and a plurality of fluid flow paths. The population of cells may be retained within a 3D matrix of the plurality of 3D matrices. A fluid flow path of the plurality of fluid flow paths may be independent from and adjacent to the 3D matrix. The method may include using the fluid flow path to deliver fluidic material to the 3D matrix. The method may include processing the population of cells into a cell product in the presence of the fluidic material delivered to the 3D matrix.

[0192] In another aspect, the present disclosure provides methods for cell processing. Amethod for cell processing may include providing a 3D matrix retaining, containing, encapsulating, immobilizing, or any combination thereof a plurality of cells in a cell processing unit. Within the cell processing unit, the plurality of cells may be automatically processed within the 3D matrix to produce a plurality of processed cells. Within the cellWSGR Docket No.67566-702601 processing unit, the processed cells may be automatically separated from the 3D matrix. The method may further include collecting the plurality of processed cells.

[0193] In another aspect, the present disclosure provides systems for cell processing. Asystem for cell processing may include a cell processing unit and one or more computer processors. The cell processing unit may be configured to retain or may retain a 3D matrix. The 3D matrix may be configured to encapsulate or may encapsulate a plurality of cells. The one or more computer processors may be operatively coupled to the cell processing unit. The one or more computer processors may be individually or collectively programmed to automatically (i) process the plurality of cells within the 3D matrix to produce a plurality of processed cells, (ii) direct the cell processing unit to separate the plurality of processed cells from the 3D matrix, (iii) direct the cell processing unit to collect the plurality of processed cells, or any combination thereof.

[0194] In another aspect, the present disclosure provides systems for manufacturing a cellproduct. A system may include a 3D matrix configured to retain, contain, encapsulate, or immobilize or that retains, contains, encapsulates or immobilizes a population of cells at a density of greater than 80 million cells per cubic centimeter (cells / cm3). The system may be capable of processing cells of the population of cells, with aid of the 3D matrix, into a cell therapy product.

[0195] In another aspect, the present disclosure provides methods for manufacturing acell therapy product. A method for manufacturing a cell therapy product may include providing a population of cells at a density of greater than 80 million cells / cm3retained, contained, encapsulated, or immobilized within a 3D matrix and, with the aid of the 3D matrix, processing cells of the population of cells into the cell therapy product.

[0196] In another aspect, the present disclosure provides bioreactors for processing apopulation of cells. A bioreactor for processing a population of cells may include a 3D matrix configured to retain, contain, encapsulate, or immobilize the population of cells. The bioreactor may have a volume of at most about two cubic meters (m3). The bioreactor may be configured to process or may process, with the aid of the 3D matrix and at high reproducibility, the population of cells into a cell product.

[0197] In another aspect, the present disclosure provides methods for processing apopulation of cells within a bioreactor. A method for processing a population of cells may include providing the population of cells retained, contained, encapsulated, or immobilized within a 3D matrix of a bioreactor. The bioreactor may have a volume of at most about 2 m3.WSGR Docket No.67566-702601 The method may include processing the population of cells into a cell product with aid of the 3D matrix and at high reproducibility.

[0198] In another aspect, the present disclosure provides systems for cell therapymanufacturing. A system for cell therapy manufacturing may include a 3D matrix configured to retain, contain, encapsulate, or immobilized or that retains, contains, encapsulates, or immobilizes a population of cells at a density of at least about 5 million cells / cm3. The system may be capable of processing cells or may process cells of the population within the 3D matrix for a time period of at most 48 hours to generate a cell product. The cell produce may be usable to generate a cell therapy.

[0199] In another aspect, the present disclosure provides methods for manufacturing acell therapy. A method for manufacturing a cell therapy may include providing a population of cells at a density of at least about 5 million cells / cm3retained, contained, encapsulated, or immobilized within a 3D matrix. The method may include processing the cells of the population of cells within the 3D matrix for a time period of at most about 48 hours to generate a cell product. The cell product may be used to generate a cell therapy.

[0200] 3D matrices may provide several benefits for cell processing over solution phasecell processing. Fluid, nutrient, or reagent transfer in bulk and microfluidic bioreactors may be challenging, as shown in FIG.9A. Bulk and microfluidic bioreactors may transfer cells and fluids from one process to another thereby increasing cell handling and the likelihood of cell damage. Further, bulk and microfluidic bioreactors may use multiple wash and separation processes that may subject the cells to shear stresses that may damage the cells. In an alternative configuration, cells may be processed within a 3D matrix, as shown in FIG. 9B. Processing cells within a 3D matrix may reduce the shear stresses on the cells by permitting fluid, nutrient, or reagent transfer via tangential or perfusion flow of fluids. The 3D matrix and compartment retaining the 3D matrix may be configured such that all or substantially all the fluid flows along a surface of the 3D matrix such that nutrients and reagents within the fluid diffuse into the 3D matrix to contact the cells encapsulated therein. The cells may be subjected to multiple processes within the 3D matrix, thereby reducing cell handling. Tangential or perfusion flow of nutrients, reagents, or both may permit levels of those nutrients or reagents within the 3D matrix to be highly controlled to permit cells to be processed at high cell densities (e.g., ~10-100 million cells / cm3).

[0201] The 3D substrates utilized in cell-matrix formulations may comprise a porous,permeable solid, or mixtures thereof, suspended in an interstitial fluid predominantly consisting of water, and also comprising cells in suspension in the interstitial aqueous liquid,WSGR Docket No.67566-702601 adhered to the 3D substrate and in contact with the interstitial aqueous liquid, or both. The 3D substrate may comprise a three-dimensional structure with a defined or undefined structure described as, but not limited to, a matrix, mesh, network, or otherwise as a physically interconnected system of molecules such as polymers. The structure of the 3D substrate may support the structural integrity of the 3D cell culture environment, providing an interstitial space in which cells may reside, grow, proliferate, or any combination thereof, and facilitating the transmission of nutrients and other essential dissolved materials, as well as material reagents and constructs for genetic engineering. The configuration of the interstitial space, characterized by the organization and porosity of the 3D substrate, as well as the physical properties of the substrate such as hydrophilicity, cell adhesion properties, and biofouling properties, may allow for the diffusion of water-soluble substances, making it an ideal medium for cellular activities. The configuration of the interstitial space and structural properties of the 3D substrate may further allow for active exchange of the interstitial fluid, such as by use of a fluid flow which may partially, substantially, or totally displace a first interstitial fluid with a second interstitial fluid. In some embodiments, the 3D substrate additionally provides for the retention of cells within the 3D substrate during cell culture or passive or active exchange of interstitial fluid. Three-dimensional substrates may include, but are not limited to, hydrogel matrices, porous solid matrices, inverse opal matrices, and composite matrices (e.g., interpenetrating matrices).

[0202] Hydrogel matrices, for example, those used in aqueous environments for cellculture, may comprise hydrophilic polymers. The hydrogel matrices may comprise all or substantially all hydrophilic polymers. The hydrogel matrices may comprise greater than or equal to about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more hydrophilic polymers. The hydrogel matrices may comprise from about 20% to 30%, 20% to 40%, 20% to 50%, 20%, to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 98%, 30% to 40%, 30% to 50%, 30%, to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 98%, 40% to 50%, 40%, to 60%, 40% to 70%, 40% to 80%, 40% to 90%, 40% to 95%, 40% to 98%, 50%, to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 60% to 70%, 60% to 80%, 60% to 90%, 60% to 95%, 60% to 98%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 98%, 80% to 90%, 80% to 95%, 80% to 98%, 90% to 95%, 90% to 98%, or 95% to 98% hydrophilic polymers.

[0203] Hydrophilic polymers, while hydrophilic, or water-absorbent, may form a 3Dmatrix which itself is insoluble in water. The polymer matrix may swell in the presence of water without dissolving. This swelling may occur because the hydrophilic (water-attracting)WSGR Docket No.67566-702601 nature of the polymer chains allows them to absorb and retain a significant amount of water, which can be many times their dry weight. Despite their affinity for water, these polymers may be chemically or physically crosslinked, which prevents them from dissolving in the water. Crosslinking between and among polymers comprising the hydrogel may impart stability to the hydrogel matrix. Crosslinking may involve forming links between and among the individual polymer chains, thereby forming a three-dimensional network structure. This network can further comprise within its structure or volume other structural materials (such as in the cases of hybrid gel materials and matrices formed of interpenetrating networks), functional molecules (e.g., biomolecules), water molecules, and other particles, such as cells. Crosslinks can be formed through chemical bonds (e.g., covalent crosslinking) or through physical interactions. Physical interactions may include physical crosslinking such as ionic bonds, hydrogen bonds, hydrophobic interactions, polyelectric complexation, electrostatic interactions, condensation, or any combination thereof. The ratio of chemical and physical crosslinking may alter the mechanical strength, degradation rate, biological interactions, or any combination thereof of the hydrogel matrix.

[0204] Crosslinks may be stable or reversible under given conditions (e.g., stimulus), asdescribed elsewhere herein. The insolubility and crosslinked nature of the hydrogel matrix in an aqueous environment may provide stable and supportive scaffold for cells. Such a 3D substrate may mimic the extracellular matrix in which cells naturally reside. A 3D substrate may further provide structural support and a medium or substrate through which nutrients and other soluble factors can diffuse or be presented to or otherwise be available for contact with cells. Additionally, a hydrogel's physical structure and properties, including porosity (e.g., the size and distribution of its pores), stiffness, elasticity, may be tailored or engineered as described herein, including for the purposes of regulating cell migration, proliferation, differentiation, phenotype, gene expression, or any combination thereof.

[0205] Hydrogels may be formed from natural polymers, modified polymers, syntheticpolymers, or any combination thereof. Hydrogel materials may be selected for biocompatibility, biodegradability, mechanical strength, ease of modification, or any combination thereof. The material used for the 3D matrix may be modified or altered to be compatible with a given type of cell. Natural polymers may include, but are not limited to, fibrinogen, hyaluronic acid, collagen, Matrigel, gelatin, chitosan, alginate, cellulose, heparin, dextran, keratin, agarose, silk fibroin, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide polymers, or any combination thereof. Synthetic polymers may include, but are not limited to, Poly(ethylene glycol) (PEG), Poly(vinyl alcohol) (PVA), Poly(acrylic acid)WSGR Docket No.67566-702601 (PAA), Poly(lactic-co-glycolic acid) (PLGA), Poly(lactic acid) (PLA), Poly(glycolic acid) (PGA), Poly(caprolactone) (PCL), Poly(vinyl acetate) (PVA), Poly(ethyleneimine) (PEI), Poly(vinylpyrrolidone) (PVP), Poly(N-isopropylacrylamide) (PNIPAM), Polysiloxanes, Polyurethanes, poly(ethylene glycol) diacrylate, poly(ethylene glycol) dimethacrylate, derivatives thereof, or any combination thereof. The 3D matrix may comprise a single polymer or may comprise a mixture of polymers configured for nutrient diffusion, mechanical support of cells, biocompatibility, or any combination thereof.

[0206] Polymers (e.g., hydrogel polymers) may or may not be chemically modified. Inan example, polymers (e.g., hydrogel polymers) are chemically modified. For example, collagen may be provided as a chemically modified form of the natural protein collagen, such as, methacrylated collagen (often abbreviated as collagen-MA or methacryloyl collagen), in which collagen may be functionalized with methacrylate groups. The process of methacrylation may involve the introduction of methacryloyl groups into the amino acid residues of the collagen molecule, for example, using methacrylic anhydride. This chemical modification may occur mainly at the lysine residues and the N-terminal groups of the collagen. Using methacrylated collagen may be beneficial because of its ability to undergo rapid thermal gel-sol transition (e.g., exhibiting a reversible transition from a liquid to a gel with increasing temperature) and photocrosslinking when exposed to light (e.g., ultraviolet light) in the presence of a photoinitiator. The methacrylate groups may form covalent bonds with each other, creating a crosslinked network. Chemical modification may not alter the biocompatibility and biodegradability of natural collagen, making it an excellent scaffold material that can support cell adhesion, proliferation, and differentiation. Hydrogels formed from methacrylated collagen may have tunable mechanical properties, which may be adjusted to match the properties of a native tissue environment. The degree of substitution of methacrylate groups can be varied, altering the density of the crosslinking and the physical properties of the resulting hydrogels, such as their thermal responsiveness, stiffness, degradation rate, and pore size. In another example, a PEG polymer may be prepared as a block copolymer with Poly(N-isopropylacrylamide) (PNIPAM), also known as thermogelling PEG–PNIPAM block copolymers. PEG-PNIPAM block copolymers exhibit thermally responsive gel-sol transition (e.g., exhibiting a reversible transition from a liquid to a gel with increasing temperature). Each polymer segment may provide specific attributes that contribute to the overall behavior of the copolymer. PEG is a hydrophilic polymer that may be biocompatible and non-immunogenic polymer. PEG may be used in biomedical applications. In copolymers, PEG may serve as a segment that stabilizes and increases theWSGR Docket No.67566-702601 solubility of the copolymer in aqueous environments. PNIPAM may exhibit thermoresponsive behavior. Below its Lower Critical Solution Temperature (LCST), which may be around 32°C, PNIPAM is hydrophilic and soluble in water. Above this temperature, PNIPAM may become hydrophobic and insoluble, causing the polymer to precipitate or form a gel. In these copolymers, PEG and PNIPAM may be linked in a block copolymer architecture, where each polymer forms a distinct block within the copolymer chain. The ratio of each polymer component may be controlled, thereby affecting the properties of the copolymer. The copolymer may comprise PNIPAM and secondary polymer. The ratio of PNIPAM to secondary polymer (e.g., PNIPAM:PEG) may be from about 1000:1 to about 1:1000. The ratio of PNIPAM to secondary polymer may be selected based on target mechanical properties, chemical properties, responsivity, synthesis route, copolymer composition, or any combination thereof. The block copolymer structure may allow PEG- PNIPAM materials to leverage the water solubility and biocompatibility of PEG along with the temperature-sensitive gelation properties of PNIPAM. Other LCST polymers, which may be incorporated into stimulus-responsive copolymers, include but are not limited to poly((meth)acrylamide)s: poly(N,N-diethylacrylamide), poly(2- carboxyisopropylacrylamide), poly(N-(L)-(1-hydroxymethyl) propylmeth- acrylamide, poly(N-acryloyl-N’-propilpiperazine), poly(N-ethylacrylamide), poly(N-methyl- N-ethylacrylamide), poly(N-n-propylacrylamide), poly(N-ethylmethacrylamide), poly(N-methyl-N-isopropylacrylamide), poly(N-isopropylmethacrylamide), poly(N-n-propyl-methacrylamide), poly(N-methyl-N-isopropylacrylamide), poly(N-cyclopropylacrylamide),poly(N-cyclopropymethacrylamide), poly(N,N-bis(2- methoxyethyl) acrylamide), poly(N-(3-methoxypropyl) acrylamide), poly(ethoxypropylacrylamide), poly(aminomethoxypropylacrylamide), or combinations thereof. Alternatively, other polymers may be used or incorporated into the 3D matrix to provide stimuli (e.g., chemical, physical, thermal, or any combination thereof) responsive polymers. Other temperature- responsive polymers include, but are not limited to, Poly(N-vinylcaprolactam), poly(2-ethyl- 2-oxazoline), poly(vinyl methyl ether), poly(2-isopropyl-2-oxazoline), poly((2- dimethylamino) ethyl methacrylate), poly(propylene oxide), and poly(N-acryloylpiperidine). Other pH responsive polymers include, but are not limited to, Poly(acrylic acid), poly(2-ethyl acrylic acid), poly(N,N-dimethyl aminoethyl methacrylate), poly(vinyl imidazole), Polyaspertate, polylysine, and polyhistidine. Other light responsive polymers include, but are not limited to, Poly(ethylene oxide-methacrylate) with pyrene pendant, nitrobenzyl and dithiodipropionic acid modified polyetherimide, poly-N-isopropylacrylamide-spiropyran,WSGR Docket No.67566-702601 poly[N-isopropylacrylamide-b-sodium 2-(acrylamido)−2-methylpropane sulfonate- spiropyran]. Other glucose-responsive polymers include, but are not limited to, Poly[(2- dimethylamino) ethyl methacrylate-co-3- acrylamidephenylboronic acid]. Stimulus responsive polymers, including LCST polymers such as PNIPAM and others listed above, may be synthesized as copolymers with other polymers, including but not limited to, poly(amino acids); polysaccharides such as alginate, cellulose, chitosan, guar gum and dextran; proteins; and synthetic polymers including poly(esters), poly(caprolactone), Polyethylenimine (PEI), poly(ethylene glycol). Poly(ε-caprolactone) (PCL), acrylic acid (AA) and acrylate monomers such as 2-hydroxyethyl methacrylate (HEMA) and poly(ε- caprolactone) dimethacrylate (PCLDMA), and propylacrylic acid (PAA). Examples of such copolymers include, but are not limited to, PEG-PNIPAM, alginate / N-isopropylacrylamide (NIPAM), PNIPAM / poly(sodium acrylate), and methacryloylchitosan / PNIPAM. Copolymers may comprise two or more components or block constituents, such as diblock copolymer, triblock copolymer, or blended copolymers. Polymers and copolymers may be designed and synthesized with a myriad of topologies / morphologies, such as linear polymers (homopolymers, di / ter / multi-block copolymers, and organic / inorganic hybrid polymers) and non-linear polymers (star polymers, (hyper)-branched polymers). Polymers and copolymers may be synthesized using various methods including but not limited to free radical polymerization (FRP), atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT), and graft copolymerization.

[0207] Alternatively, or in addition to, 3D substrates for cell processing may includeporous solids. These matrices can be made from a wide range of materials, including but not limited to plastics (e.g., polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polycarbonate, acrylic, cyclic olefin copolymers, cyclic olefin polymers, or any combinations thereof), ceramics, glass, metals, natural fibers, or any combinations thereof. Unlike hydrogels, these materials may have defined, micro-scale porosity with precise control over pore size and distribution. This porosity may be created through methods like sintering, foaming, leaching, weaving, 3D printing, or a combination thereof. Solid porous matrices may be more rigid and stable than hydrogels and may not necessarily swell or change shape in the presence of water or biological fluids, which can be advantageous for certain applications requiring dimensional stability. Solid porous materials may also be modified to alter physical, chemical, or biological properties. Functionalization and modification of porous solid 3D scaffold materials may include all methods of modifying these materials for 2D cell culture applications, including but not limited to plasma surfaceWSGR Docket No.67566-702601 modification, self-assembled monolayer deposition, covalent grafting, surface coating or absorption, or any combination thereof. For example, plastic and other solid scaffold materials may be treated to modify the surface energy, surface tension, or wettability, such as by plasma treatment or oxidation reactions forming carbonyl (-C=O-). Carboxyl (HOOC-), hydroperoxide (HOO-) and hydroxyl (HO-) groups. As another example, materials may be treated to affect protein binding, protein deposition, biofouling, and cell adhesion, such as surface hydrolysis of polyester scaffolds to promote protein adsorption for improving cell attachment. Surface functionalization can be performed by exposing surfaces to RF plasma. Many gases can be excited and used to functionalize surfaces for a wide variety of applications. Surface functionalization techniques may include using air plasma, oxygen plasma, and ammonia plasma as well as other exotic gases. Each gas can have varying effects on a substrate. For example, ammonia plasma and n-heptylamine vapor treatments can be used to attach amine functional groups. Surfaces may be treated with thin polysaccharide films or coatings, including through covalent attachment, such as by using oxidized polysaccharides which form Shiff base linkages with amine-functionalized materials.

[0208] Alternatively, or in addition to, 3D substrates may comprise Inverse Opal(IOPAL) materials that mimic the ordered porous architecture of opals, but with an inverted morphology. To form IOPAL 3D substrates, a first sacrificial material (e.g., the opal template) may be deposited, packed, or otherwise formed into a 3D spatial configuration (e.g., by using a mold). For example, the opal template may be formed of monodisperse microspheres (e.g., made of polystyrene, silica, polymethyl methacrylate, etc.) that are packed into a dense structure, such as through sedimentation, vertical deposition, or centrifugation, which may allow the spheres to naturally order into a 3D configuration. The 3D structure may comprise a substantially regular structure (e.g., a face-centered cubic (FCC) lattice). A 3D substrate precursor material may be infiltrated into the interstices of the opal template to fill the voids without disturbing the 3D structure. The 3D substrate material may be subsequently formed into a 3D substrate, such as through sol-gel processes or polymerization. The sacrificial template may then be removed, such as by dissolution in a suitable solvent (e.g., toluene for polystyrene) or by another stimulus-responsive template chemistry (e.g., by using a temperature sensitive template material and subsequently heating or cooling to liquify the template facilitating removal from the IOPAL structure). The removal of the opal template may leave behind a porous structure with an ordered array of voids, which may mirror the original arrangement of the template. In some examples, the removal of the sacrificial porogen creates a network of interstitial spaces in the exactWSGR Docket No.67566-702601 geometric arrangement of the original template, resulting in interconnected voids in the inverse opal structure that may be uniform and highly ordered. Due to their ability to form 3D structures with highly controlled porosity and structural regularity, IOPAL materials can provide uniform diffusion and other desirable material properties. IOPAL substrates may comprise a variety of materials including, but not limited to, hydrogels, porous solids, or any combination thereof.

[0209] In an example, the 3D matrix may comprise a composite 3D substrate. Composite3D substrates may be formed from mixtures of 3D substrate materials or interpenetrating 3D matrices. A composite 3D substrate may comprise any suitable mixture of hydrogels, polymers, non-hydrogel substrates (e.g., porous solids, 3D scaffolds, particles, or any combination thereof), or any combination thereof, which may support cell culture. To support cell culture, 3D substrates may provide a physical 3D space for cells, a supply of liquid media, dissolved gasses, or other reagents and compositions supporting cell culture. The various components, or a portion thereof, making up the composite 3D matrix may be physically crosslinked, may physically associate, or may not be physically associate. The 3D substrate may comprise one or more interpenetrating networks, such as a first mechanically strong, nonreversible network interpenetrated with a second network featuring reversible crosslinks. The use of composite 3D substrates or mixture of 3D substrate materials can provide advantages such as including separate materials to serve select functions. In another example, a 3D substrate may comprise a first material which provides for cell retention, and a second material which provides for compatible cellular growth microenvironments, such separate materials being configured separately and used together to form a 3D substrate usable for cell retention and growth. Other examples include, but are not limited to, interpenetrating hydrogel matrices, such as those formed by the combinations of thermally- responsive hydrogel materials (e.g., methacrylated collagen and PEG-PNIPAM) and separately, a porous solid matrix such as polystyrene, which features a large pore size (e.g., greater than 100 micrometers (µm)), filled with a separate hydrogel matrix such as collagen gel. 3D substrate properties

[0210] Physical properties of 3D substrates for cell culture may include, but are notlimited to, stiffness, softness, rigidity, swelling, porosity, stability, optical properties, dimensional properties, or any combinations thereof. The physical characteristics of the 3D substrate may be modified or otherwise engineered by altering the composition of the matrix, the method of forming the matrix, the soluble environment of the matrix, or any combinationWSGR Docket No.67566-702601 thereof. Methods for modifying or tuning the physical properties of a 3D substrate may include changing the crosslinking density (e.g., by altering the crosslinking potential of one or more underlying components), changing the proportions of components (e.g., by altering the percent of crosslinking components), changing the molecular weight of polymers or substrates, adding components (e.g., by adding nanomaterials, including degradable, removable, or sacrificial porogens which may not be a component of the formed matrix, but otherwise alter the distribution and network architecture of the matrix), changing the soluble environment (e.g., such as pH, ionic strength, etc.), or any combination thereof.

[0211] A 3D substrate may provide a high surface area to volume ratio to efficientlyprovide nutrients, gas exchange, and other reagents like viral particles to the 3D cell culture substrate. 3D matrices or substrates with higher surface area to volume ratio may have a high interfacial area as compared to the volume of the 3D substrate. 3D substrates with high surface area to volume ratios may provide more uniform cellular environments than 3D substrates with low surface area to volume ratio. For example, 3D matrices or substrates with higher surface area to volume ratios because fluidic materials such as nutrients and reagents may have shorter distances to diffuse or transport into the 3D matrix as compared to a similar matrix with a lower surface area to volume ration. Shorter diffusion or transport distance may provide smaller concentration gradients across the 3D substrate and more uniform cellular environment throughout the 3D matrix or substrate. A 3D matrix or substrate may have a surface area to volume ratio of greater than or equal to about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, or more. A 3D matrix or substrate may have a surface area to volume ratio from 0.1 to 0.5, 0.1 to 1, 0.1 to 2, 0.1 to 3, 0.1 to 4, 0.1 to 5, 0.1 to 10, 0.1 to 15, 0.1 to 20, 0.5 to 1, 0.5 to 2, 0.5 to 3, 0.5 to 4, 0.5 to 5, 0.5 to 10, 0.5 to 15, 0.5 to 20, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 2 to 3, 2 to 4, 2 to 5, 2 to 10, 2 to 15, 2 to 20, 3 to 4, 3 to 5, 3 to 10, 3 to 15, 3 to 20, 4 to 5, 4 to 10, 4 to 15, 4 to 20, 5 to 10, 5 to 15, 5 to 20, 10 to 15, 10 to 20, or 15 to 20.

[0212] 3D matrices or substrates may comprise any shape. For example, a 3D matrixmay be a slab, sphere, fiber, or any other shape as described elsewhere herein. In an example, a 3D matrix is a slab. In another example, a 3D matrix comprises a fiber shape. In another example, a 3D matrix comprises a spherical shape. A 3D matrix or substrate may have one dimension that is substantially smaller than any other dimension. For example, a 3D matrix may be a slab and the thickness of the slab may be substantially smaller than the length or width of the slab. In another example, the 3D matrix may be a sphere and the diameter may be substantially smaller than the circumference. In another example, the 3DWSGR Docket No.67566-702601 matrix may be a fiber and the diameter of the fiber may be substantially smaller than the length of the fiber. During cell processing, fluid may flow in a direction normal or substantially normal to the small dimension (e.g., the fluid flow may be tangential to a long dimension of the 3D matrix). For example, fluid flow may be parallel to a long dimension of the slab or along a surface of the slab disposed at approximately a 90-degree, 80-degree, 70- degree, or 60-degree angle from the short dimension (e.g., the fluid flows along the length or width of the slab). In an example, the 3D matrix may comprise a fiber and the fluid may flow along a length of the fiber. In another example, the 3D matrix may comprise a sphere, and the fluid may flow along an outer circumference of the sphere. In The 3D matrix may have a small dimension that is less than or equal to about 20 millimeters (mm), 10 mm, 8 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 900 µm, 800 µm, 700 µm, 600 µm, 500 µm, 400 µm, 300 µm, 200 µm, 100 µm, 50 µm, 25 µm, 10 µm, or less. In an example, the 3D matrix may have a small dimension of less than or equal to about 10 mm. In an example, the 3D matrix may have a small dimension of less than or equal to about 1 mm. In an example, the 3D matrix may have a small dimension of less than or equal to about 500 µm. In an example, the 3D matrix may have a small dimension of less than or equal to about 200 µm. In an example, the 3D matrix may have a small dimension of less than or equal to about 100 µm. In an example, the 3D matrix may have a small dimension of less than or equal to about 10 µm. In an example, the 3D matrix may comprise a slab configuration with a small dimension of less than or equal to about 10 mm. In an example, the 3D matrix may comprise a slab configuration with a small dimension of less than or equal to about 1 mm. In an example, the 3D matrix may comprise a slab configuration with a small dimension of less than or equal to about 500 µm. In an example, the 3D matrix may comprise a slab configuration with a small dimension of less than or equal to about 200 µm. In an example, the 3D matrix comprises a fiber configuration and the fiber comprises a small dimension (e.g., diameter) that is less than or equal to about 10 mm. The small dimension may be from about 10 µm to 25 µm, 10 µm to 50 µm, 10 µm to 100 µm, 10 µm to 200 µm, 10 µm to 200 µm, 10 µm to 300 µm, 10 µm to 400 µm, 10 µm to 500 µm, 10 µm to 600 µm, 10 µm to 700 µm, 10 µm to 800 µm, 10 µm to 900 µm, 10 µm to 1 mm, 10 µm to 2 mm, 10 µm to 3 mm, 10 µm to 4 mm, 10 µm to 5 mm, 10 µm to 6 mm, 10 µm to 8 mm, 10 µm to 10 mm, 10 µm to 20 mm, 25 µm to 50 µm, 25 µm to 100 µm, 25 µm to 200 µm, 25 µm to 200 µm, 25 µm to 300 µm, 25 µm to 400 µm, 25 µm to 500 µm, 25 µm to 600 µm, 25 µm to 700 µm, 25 µm to 800 µm, 25 µm to 900 µm, 25 µm to 1 mm, 25 µm to 2 mm, 25 µm to 3 mm, 25 µm to 4 mm, 25 µm to 5 mm, 25 µm to 6 mm, 25 µm to 8 mm, 25 µm to 10 mm, 25 µm to 20 mm, 50 µm to 100 µm, 50 µm to 200WSGR Docket No.67566-702601 µm, 50 µm to 200 µm, 50 µm to 300 µm, 50 µm to 400 µm, 50 µm to 500 µm, 50 µm to 600 µm, 50 µm to 700 µm, 50 µm to 800 µm, 50 µm to 900 µm, 50 µm to 1 mm, 50 µm to 2 mm, 50 µm to 3 mm, 50 µm to 4 mm, 50 µm to 5 mm, 50 µm to 6 mm, 50 µm to 8 mm, 50 µm to 10 mm, 50 µm to 20 mm, 100 µm to 200 µm, 100 µm to 200 µm, 100 µm to 300 µm, 100 µm to 400 µm, 100 µm to 500 µm, 100 µm to 600 µm, 100 µm to 700 µm, 100 µm to 800 µm, 100 µm to 900 µm, 100 µm to 1 mm, 100 µm to 2 mm, 100 µm to 3 mm, 100 µm to 4 mm, 100 µm to 5 mm, 100 µm to 6 mm, 100 µm to 8 mm, 100 µm to 10 mm, 100 µm to 20 mm, 200 µm to 300 µm, 200 µm to 400 µm, 200 µm to 500 µm, 200 µm to 600 µm, 200 µm to 700 µm, 200 µm to 800 µm, 200 µm to 900 µm, 200 µm to 1 mm, 200 µm to 2 mm, 200 µm to 3 mm, 200 µm to 4 mm, 200 µm to 5 mm, 200 µm to 6 mm, 200 µm to 8 mm, 200 µm to 10 mm, 200 µm to 20 mm, 300 µm to 400 µm, 300 µm to 500 µm, 300 µm to 600 µm, 300 µm to 700 µm, 300 µm to 800 µm, 300 µm to 900 µm, 300 µm to 1 mm, 300 µm to 2 mm, 300 µm to 3 mm, 300 µm to 4 mm, 300 µm to 5 mm, 300 µm to 6 mm, 300 µm to 8 mm, 300 µm to 10 mm, 300 µm to 20 mm, 400 µm to 500 µm, 400 µm to 600 µm, 400 µm to 700 µm, 400 µm to 800 µm, 400 µm to 900 µm, 400 µm to 1 mm, 400 µm to 2 mm, 400 µm to 3 mm, 400 µm to 4 mm, 400 µm to 5 mm, 400 µm to 6 mm, 400 µm to 8 mm, 400 µm to 10 mm, 400 µm to 20 mm, 500 µm to 600 µm, 500 µm to 700 µm, 500 µm to 800 µm, 500 µm to 900 µm, 500 µm to 1 mm, 500 µm to 2 mm, 500 µm to 3 mm, 500 µm to 4 mm, 500 µm to 5 mm, 500 µm to 6 mm, 500 µm to 8 mm, 500 µm to 10 mm, 500 µm to 20 mm, 600 µm to 700 µm, 600 µm to 800 µm, 600 µm to 900 µm, 600 µm to 1 mm, 600 µm to 2 mm, 600 µm to 3 mm, 600 µm to 4 mm, 600 µm to 5 mm, 600 µm to 6 mm, 600 µm to 8 mm, 600 µm to 10 mm, 600 µm to 20 mm, 700 µm to 800 µm, 700 µm to 900 µm, 700 µm to 1 mm, 700 µm to 2 mm, 700 µm to 3 mm, 700 µm to 4 mm, 700 µm to 5 mm, 700 µm to 6 mm, 700 µm to 8 mm, 700 µm to 10 mm, 700 µm to 20 mm, 800 µm to 900 µm, 800 µm to 1 mm, 800 µm to 2 mm, 800 µm to 3 mm, 800 µm to 4 mm, 800 µm to 5 mm, 800 µm to 6 mm, 800 µm to 8 mm, 800 µm to 10 mm, 800 µm to 20 mm, 900 µm to 1 mm, 900 µm to 2 mm, 900 µm to 3 mm, 900 µm to 4 mm, 900 µm to 5 mm, 900 µm to 6 mm, 900 µm to 8 mm, 900 µm to 10 mm, 900 µm to 20 mm, 1 mm to 2 mm, 1 mm to 3 mm, 1 mm to 4 mm, 1 mm to 5 mm, 1 mm to 6 mm, 1 mm to 8 mm, 1 mm to 10 mm, 1 mm to 20 mm, 2 mm to 3 mm, 2 mm to 4 mm, 2 mm to 5 mm, 2 mm to 6 mm, 2 mm to 8 mm, 2 mm to 10 mm, 2 mm to 20 mm, 3 mm to 4 mm, 3 mm to 5 mm, 3 mm to 6 mm, 3 mm to 8 mm, 3 mm to 10 mm, 3 mm to 20 mm, 4 mm to 5 mm, 4 mm to 6 mm, 4 mm to 8 mm, 4 mm to 10 mm, 4 mm to 20 mm, 5 mm to 6 mm, 5 mm to 8 mm, 5 mm to 10 mm, 5 mm to 20 mm, 6 mm to 8 mm, 6 mm to 10 mm, 6 mm to 20WSGR Docket No.67566-702601 mm, 8 mm to 10 mm, 8 mm to 20 mm, or 10 mm to 20 mm. In an example, the small dimension is from about 10 µm to 100 µm.

[0213] A 3D matrix may be configured such that fluid may flow along a surface of the3D matrix. In an example, the 3D matrix may comprise a fiber-like or plate-like configuration, as shown in FIG.10A. Alternatively, or in addition to, the 3D matrix may comprise a spherical-like configuration. The 3D matrix may comprise a surface area that is significantly larger than a volume of the 3D matrix to permit diffusion and transport of nutrients and reagents into the 3D matrix. In an example, the thickness or diameter of the 3D matrix may be substantially less than other dimensions of the 3D matrix. 3D matrices comprising large surface area to volume ratios may permit the use of small or large media volumes, as shown in FIG.10B, depending upon cell product being produced. In an example, the system may be configured for low fluid volumes. Low fluid volumes may permit high flow rate and more frequent batch feeds. Lower fluid volume operations may be useful short process runs, such as zero to three or zero to five days. Low fluid volume configuration may minimize the amount of activation and genetic payload reagents used, saving costs and increasing reagent efficiency. Alternatively, the 3D matrix and system may be configured for large or larger media volumes. Larger media volumes may provide for lower fluid flow rates and less frequent batch feeds. Configurations with larger fluid volumes may be useful for longer process runs, for example, process runs of longer than five days.

[0214] The amount of cell product produced may depend on the volume available for cellprocessing. The volume available for cell processing may be the volume of a single 3D matrix or the combined volume of a plurality of 3D matrices. In an example, the volume available for cell processing is the volume of a single 3D matrix. In another example, the volume available for cell processing is the volume of a plurality 3D matrices. In another example, a cell processing system may have a plurality of 3D matrices and each 3D matrix may perform a separate cell process such that the volume available for generating the cell product from each process is the volume of a single 3D matrix. In another example, a cell processing system may have a plurality of 3D matrices each performing the same cell processing operations to generate a single cell product from the plurality of 3D matrices. In another example, a subset of the plurality of 3D matrices may perform a first operation or series of operations to generate a first cell product and another subset of the plurality of 3D matrices may perform a second operation or series of operations to generate another cell product. The volume of a 3D matrix may be at least about 1 milliliter (mL), 2 mL, 3 mL, 5WSGR Docket No.67566-702601 mL, 10 mL, 25 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1000 mL, or more. The volume of a 3D matrix may be less than or equal to about 1000 mL, 900 mL, 800 mL, 700 mL, 600 mL, 500 mL, 400 mL, 300 mL, 200 mL, 150 mL, 100 mL, 75 mL, 50 mL, 25 mL, 10 mL, 5 mL, 3 mL, 2 mL, 1 mL, or less. The volume of a 3D matrix may be from about 1 mL to 2 mL, 1 mL to 3 mL, 1 mL to 5 mL, 1 mL to 10 mL, 1 mL to 25 mL, 1 mL to 50 mL, 1 mL to 75 mL, 1 mL to 100 mL, 1 mL to 150 mL, 1 mL to 200 mL, 1 mL to 300 mL, 1 mL to 400 mL, 1 mL to 500 mL, 1 mL to 600 mL, 1 mL to 700 mL, 1 mL to 800 mL, 1 mL to 900 mL, 1 mL to 1000 mL, 2 mL to 3 mL, 2 mL to 5 mL, 2 mL to 10 mL, 2 mL to 25 mL, 2 mL to 50 mL, 2 mL to 75 mL, 2 mL to 100 mL, 2 mL to 150 mL, 2 mL to 200 mL, 2 mL to 300 mL, 2 mL to 400 mL, 2 mL to 500 mL, 2 mL to 600 mL, 2 mL to 700 mL, 2 mL to 800 mL, 2 mL to 900 mL, 2 mL to 1000 mL, 3 mL to 5 mL, 3 mL to 10 mL, 3 mL to 25 mL, 3 mL to 50 mL, 3 mL to 75 mL, 3 mL to 100 mL, 3 mL to 150 mL, 3 mL to 200 mL, 3 mL to 300 mL, 3 mL to 400 mL, 3 mL to 500 mL, 3 mL to 600 mL, 3 mL to 700 mL, 3 mL to 800 mL, 3 mL to 900 mL, 3 mL to 1000 mL, 5 mL to 10 mL, 5 mL to 25 mL, 5 mL to 50 mL, 5 mL to 75 mL, 5 mL to 100 mL, 5 mL to 150 mL, 5 mL to 200 mL, 5 mL to 300 mL, 5 mL to 400 mL, 5 mL to 500 mL, 5 mL to 600 mL, 5 mL to 700 mL, 5 mL to 800 mL, 5 mL to 900 mL, 5 mL to 1000 mL, 10 mL to 25 mL, 10 mL to 50 mL, 10 mL to 75 mL, 10 mL to 100 mL, 10 mL to 150 mL, 10 mL to 200 mL, 10 mL to 300 mL, 10 mL to 400 mL, 10 mL to 500 mL, 10 mL to 600 mL, 10 mL to 700 mL, 10 mL to 800 mL, 10 mL to 900 mL, 10 mL to 1000 mL, 25 mL to 50 mL, 25 mL to 75 mL, 25 mL to 100 mL, 25 mL to 150 mL, 25 mL to 200 mL, 25 mL to 300 mL, 25 mL to 400 mL, 25 mL to 500 mL, 25 mL to 600 mL, 25 mL to 700 mL, 25 mL to 800 mL, 25 mL to 900 mL, 25 mL to 1000 mL, 50 mL to 75 mL, 50 mL to 100 mL, 50 mL to 150 mL, 50 mL to 200 mL, 50 mL to 300 mL, 50 mL to 400 mL, 50 mL to 500 mL, 50 mL to 600 mL, 50 mL to 700 mL, 50 mL to 800 mL, 50 mL to 900 mL, 50 mL to 1000 mL, 75 mL to 100 mL, 75 mL to 150 mL, 75 mL to 200 mL, 75 mL to 300 mL, 75 mL to 400 mL, 75 mL to 500 mL, 75 mL to 600 mL, 75 mL to 700 mL, 75 mL to 800 mL, 75 mL to 900 mL, 75 mL to 1000 mL, 100 mL to 150 mL, 100 mL to 200 mL, 100 mL to 300 mL, 100 mL to 400 mL, 100 mL to 500 mL, 100 mL to 600 mL, 100 mL to 700 mL, 100 mL to 800 mL, 100 mL to 900 mL, 100 mL to 1000 mL, 150 mL to 200 mL, 150 mL to 300 mL, 150 mL to 400 mL, 150 mL to 500 mL, 150 mL to 600 mL, 150 mL to 700 mL, 150 mL to 800 mL, 150 mL to 900 mL, 150 mL to 1000 mL, 200 mL to 300 mL, 200 mL to 400 mL, 200 mL to 500 mL, 200 mL to 600 mL, 200 mL to 700 mL, 200 mL to 800 mL, 200 mL to 900 mL, 200 mL to 1000 mL, 300 mL to 400 mL, 300 mL to 500 mL, 300 mL to 600 mL, 300 mL to 700 mL, 300 mL to 800 mL, 300 mL toWSGR Docket No.67566-702601 900 mL, 300 mL to 1000 mL, 400 mL to 500 mL, 400 mL to 600 mL, 400 mL to 700 mL, 400 mL to 800 mL, 400 mL to 900 mL, 400 mL to 1000 mL, 500 mL to 600 mL, 500 mL to 700 mL, 500 mL to 800 mL, 500 mL to 900 mL, 500 mL to 1000 mL, 600 mL to 700 mL, 600 mL to 800 mL, 600 mL to 900 mL, 600 mL to 1000 mL, 700 mL to 800 mL, 700 mL to 900 mL, 700 mL to 1000 mL, 800 mL to 900 mL, 800 mL to 1000 mL, or 900 mL to 1000 mL. In an example, a 3D matrix has a volume from about 5 mL to 100 mL.

[0215] The combined volume of a plurality of 3D matrix may be at least about 10 mL, 25mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 8 L, 10 L, or more. The combined volume of a 3D matrix may be less than or equal to about 10 L, 8 L, 6 L, 5 L, 4 L, 3 L, 2 L, 1 L, 900 mL, 800 mL, 700 mL, 600 mL, 500 mL, 400 mL, 300 mL, 200 mL, 150 mL, 100 mL, 75 mL, 50 mL, 25 mL, 10 mL, or less. The combined volume of a 3D matrix may be from about 10 mL to 25 mL, 10 mL to 50 mL, 10 mL to 75 mL, 10 mL to 100 mL, 10 mL to 150 mL, 10 mL to 200 mL, 10 mL to 300 mL, 10 mL to 400 mL, 10 mL to 500 mL, 10 mL to 600 mL, 10 mL to 700 mL, 10 mL to 800 mL, 10 mL to 900 mL, 10 mL to 1 L, 10 mL to 2 L, 10 mL to 3 L, 10 mL to 4 L, 10 mL to 5 L, 10 mL to 6 L, 10 mL to 8 L, 10 mL to 10 L, 25 mL to 50 mL, 25 mL to 75 mL, 25 mL to 100 mL, 25 mL to 150 mL, 25 mL to 200 mL, 25 mL to 300 mL, 25 mL to 400 mL, 25 mL to 500 mL, 25 mL to 600 mL, 25 mL to 700 mL, 25 mL to 800 mL, 25 mL to 900 mL, 25 mL to 1 L, 25 mL to 2 L, 25 mL to 3 L, 25 mL to 4 L, 25 mL to 5 L, 25 mL to 6 L, 25 mL to 8 L, 25 mL to 10 L, 50 mL to 75 mL, 50 mL to 100 mL, 50 mL to 150 mL, 50 mL to 200 mL, 50 mL to 300 mL, 50 mL to 400 mL, 50 mL to 500 mL, 50 mL to 600 mL, 50 mL to 700 mL, 50 mL to 800 mL, 50 mL to 900 mL, 50 mL to 1 L, 50 mL to 2 L, 50 mL to 3 L, 50 mL to 4 L, 50 mL to 5 L, 50 mL to 6 L, 50 mL to 8 L, 50 mL to 10 L, 75 mL to 100 mL, 75 mL to 150 mL, 75 mL to 200 mL, 75 mL to 300 mL, 75 mL to 400 mL, 75 mL to 500 mL, 75 mL to 600 mL, 75 mL to 700 mL, 75 mL to 800 mL, 75 mL to 900 mL, 75 mL to 1 L, 75 mL to 2 L, 75 mL to 3 L, 75 mL to 4 L, 75 mL to 5 L, 75 mL to 6 L, 75 mL to 8 L, 75 mL to 10 L, 100 mL to 150 mL, 100 mL to 200 mL, 100 mL to 300 mL, 100 mL to 400 mL, 100 mL to 500 mL, 100 mL to 600 mL, 100 mL to 700 mL, 100 mL to 800 mL, 100 mL to 900 mL, 100 mL to 1 L, 100 mL to 2 L, 100 mL to 3 L, 100 mL to 4 L, 100 mL to 5 L, 100 mL to 6 L, 100 mL to 8 L, 100 mL to 10 L,150 mL to 200 mL, 150 mL to 300 mL, 150 mL to 400 mL, 150 mL to 500 mL, 150 mL to 600 mL, 150 mL to 700 mL, 150 mL to 800 mL, 150 mL to 900 mL, 150 mL to 1 L, 150 mL to 2 L, 150 mL to 3 L, 150 mL to 4 L, 150 mL to 5 L, 150 mL to 6 L, 150 mL to 8 L, 150 mL to 10 L, 200 mL to 300 mL, 200 mL to 400 mL, 200 mL to 500 mL, 200 mL to 600 mL, 200 mL to 700 mL, 200 mL to 800 mL, 200 mL to 900WSGR Docket No.67566-702601 mL, 200 mL to 1 L, 200 mL to 2 L, 200 mL to 3 L, 200 mL to 4 L, 200 mL to 5 L, 200 mL to 6 L, 200 mL to 8 L, 200 mL to 10 L, 300 mL to 400 mL, 300 mL to 500 mL, 300 mL to 600 mL, 300 mL to 700 mL, 300 mL to 800 mL, 300 mL to 900 mL, 300 mL to 1 L, 300 mL to 2 L, 300 mL to 3 L, 300 mL to 4 L, 300 mL to 5 L, 300 mL to 6 L, 300 mL to 8 L, 300 mL to 10 L, 400 mL to 500 mL, 400 mL to 600 mL, 400 mL to 700 mL, 400 mL to 800 mL, 400 mL to 900 mL, 400 mL to 1 L, 400 mL to 2 L, 400 mL to 3 L, 400 mL to 4 L, 400 mL to 5 L, 400 mL to 6 L, 400 mL to 8 L, 400 mL to 10 L, 500 mL to 600 mL, 500 mL to 700 mL, 500 mL to 800 mL, 500 mL to 900 mL, 500 mL to 1 L, 500 mL to 2 L, 500 mL to 3 L, 500 mL to 4 L, 500 mL to 5 L, 500 mL to 6 L, 500 mL to 8 L, 500 mL to 10 L, 600 mL to 700 mL, 600 mL to 800 mL, 600 mL to 900 mL, 600 mL to 1 L, 600 mL to 2 L, 600 mL to 3 L, 600 mL to 4 L, 600 mL to 5 L, 600 mL to 6 L, 600 mL to 8 L, 600 mL to 10 L, 700 mL to 800 mL, 700 mL to 900 mL, 700 mL to 1 mL, 700 mL to 2 mL, 700 mL to 3 mL, 700 mL to 4 mL, 700 mL to 5 mL, 700 mL to 6 mL, 700 mL to 8 mL, 700 mL to 10 mL, 800 mL to 900 mL, 800 mL to 1 L, 800 mL to 2 L, 800 mL to 3 L, 800 mL to 4 L, 800 mL to 5 L, 800 mL to 6 L, 800 mL to 8 L, 800 mL to 10 L, 900 mL to 1 L, 900 mL to 2 L, 900 mL to 3 L, 900 mL to 4 L, 900 mL to 5 L, 900 mL to 6 L, 900 mL to 8 L, 900 mL to 10 L, 1L to 2 L, 1 L to 3 L, 1 L to 4 L, 1 L to 5 L, 1 L to 6 L, 1 L to 8 L, 1 L to 10 L, 2 L to 3 L, 2 L to 4 L, 2 L to 5 L, 2 L to 6 L, 2 L to 8 L, 2 L to 10 L, 3 L to 4 L, 3 L to 5 L, 3 L to 6 L, 3 L to 8 L, 3 L to 10 L, 4 L to 5 L, 4 L to 6 L, 4 L to 8 L, 4 L to 10 L, 5 L to 6 L, 5 L to 8 L, 5 L to 10 L, 6 L to 8 L, 6 L to 10 L, or 8 L to 10 L. In an example, a plurality of 3D matrix has a combined volume from about 100 mL to 10 L.

[0216] The efficiency of material transport and diffusion of nutrients and reagents from afluid flowing over or adjacent to the 3D matrix may be dependent upon the interfacial area between the fluid or flowing fluid and the 3D matrix. The interfacial area between the fluid (e.g., fluid flow path or fluid compartment) and the 3D matrix may be greater than or equal to about 1 square centimeters (cm2), 2 cm2, 3 cm2, 4 cm2, 5 cm2, 6 cm2, 8 cm2, 10 cm2, 15 cm2, 20 cm2, 30 cm2, 50 cm2, or greater. The interfacial area between the fluid (e.g., fluid flow path or fluid compartment) and the 3D matrix may be from about 1 cm2to 2 cm2, 1 cm2to 3 cm2, 1 cm2to 4 cm2, 1 cm2to 5 cm2, 1 cm2to 6 cm2, 1 cm2to 8 cm2, 1 cm2to 10 cm2, 1 cm2to 15 cm2, 1 cm2to 20 cm2, 1 cm2to 30 cm2, 1 cm2to 50 cm2, 2 cm2to 3 cm2, 2 cm2to 4 cm2, 2 cm2to 5 cm2, 2 cm2to 6 cm2, 2 cm2to 8 cm2, 2 cm2to 10 cm2, 2 cm2to 15 cm2, 2 cm2to 20 cm2, 2 cm2to 30 cm2, 2 cm2to 50 cm2, 3 cm2to 4 cm2, 3 cm2to 5 cm2, 3 cm2to 6 cm2, 3 cm2to 8 cm2, 3 cm2to 10 cm2, 3 cm2to 15 cm2, 3 cm2to 20 cm2, 3 cm2to 30 cm2, 3 cm2to 50 cm2, 4 cm2to 5 cm2, 4 cm2to 6 cm2, 4 cm2to 8 cm2, 4 cm2to 10 cm2, 4 cm2to 15 cm2, 4 cm2WSGR Docket No.67566-702601 to 20 cm2, 4 cm2to 30 cm2, 4 cm2to 50 cm2, 5 cm2to 6 cm2, 5 cm2to 8 cm2, 5 cm2to 10 cm2, 5 cm2to 15 cm2, 5 cm2to 20 cm2, 5 cm2to 30 cm2, 5 cm2to 50 cm2, 6 cm2to 8 cm2, 6 cm2to 10 cm2, 6 cm2to 15 cm2, 6 cm2to 20 cm2, 6 cm2to 30 cm2, 6 cm2to 50 cm2, 8 cm2to 10 cm2, 8 cm2to 15 cm2, 8 cm2to 20 cm2, 8 cm2to 30 cm2, 8 cm2to 50 cm2, 10 cm2to 15 cm2, 10 cm2to 20 cm2, 10 cm2to 30 cm2, 10 cm2to 50 cm2, 15 cm2to 20 cm2, 15 cm2to 30 cm2, 15 cm2to 50 cm2, 20 cm2to 30 cm2, 20 cm2to 50 cm2, or 30 cm2to 50 cm2. In an example, the interfacial area is greater than or equal to about 5 cm2.

[0217] Processing cells within a 3D matrix, as shown in FIG. 11A, may provide forimproved processing or cell culture conditions that may improve the health of produced cells and reliability of cell manufacturing process. A 3D matrix may retain cells within a cell compartment, create a favorable biophysical microenvironment mimicking natural tissue extra cellular matrix (e.g., matrix stiffness), create high local concentrations of secreted factors, provide a programmable substrate for cell transduction and signaling, or any combinations thereof. The 3D matrix may provide for tunable physical characteristics, as shown in FIG.11B. For example, 3D matrices that comprise a dense, soft matrix may deform as cells proliferate within the 3D matrix. 3D matrices that comprise porous, stiff matrices may permit cell proliferation within the matrix with small amounts of deformation. 3D matrices that comprise stiff, dense matrices may not permit cell proliferation.

[0218] The physical stiffness, softness, or rigidity of 3D substrates may alter the ability ofcells to grow, proliferate, or otherwise be processed within the 3D matrices described herein. Physical stiffness, softness, or rigidity of 3D substrates may be described and measured in terms of mechanical parameters such as stiffness, elastic modulus, and viscoelasticity. The elastic modulus (e.g., Young’s Modulus) is a measure of the stiffness of a material. It may be defined as the ratio of stress (force per unit area) to strain (deformation in response to stress). For 3D substrates such as hydrogels, the elastic modulus may be determined using compression or tensile tests, where a sample is deformed under a given load, and the resulting deformation is measured. The modulus may then be calculated from the slope of the stress- strain curve in the elastic region. The shear modulus may describe the material's response to shear stress (parallel to the surface) and is particularly relevant for materials like hydrogels that can undergo significant shear deformation. Shear modulus may be measured using rheometry, where a hydrogel sample is subjected to oscillatory shear, and the resulting shear stress and strain are used to calculate the modulus. The bulk modulus may describe a material's response to (uniform) hydrostatic pressure. For isotropic materials, shear modulus (G), elastic modulus (E), and bulk modulus (K) may be related to each other as a function ofWSGR Docket No.67566-702601 a material's Poisson's ratio (u) as shown by the equations: E = 2G(1+u) = 3K(1-2u). In some embodiments, the 3D matrix may be configured to have an elastic modulus or other physical descriptor resembling or in the range of those exhibited by natural tissue extracellular matrix (ECM), e.g., of brain tissue (E ∼1.4-1.9 kPa), or another type of tissue. The 3D matrix may have an elastic modulus of greater than or equal to about 0.1 kilopascals (kPa), 0.25 kPa, 0.5 kPa, 0.75 kPa, 1 kPa, 1.25 kPa, 1.5 kPa, 1.75 kPa, 2 kPa, 2.5 kPa, 3 kPa, 4 kPa, 5 kPa, 6 kPa, 7 kPa, 8 kPa, 9 kPa, 10 kPa, or more. The 3D matrix may have an elastic module of less than or equal to about 10 kPa, 9 kPa, 8 kPa, 7 kPa, 6 kPa, 5 kPa, 4 kPa, 3 kPa, 2.5 kPa, 2 kPa, 1.75 kPa, 1.5 kPa, 1.25 kPa, 1 kPa, 0.75 kPa, 0.5 kPa, 0.25 kPa, 0.1 kPa, or less. The 3D matrix may have an elastic modulus from about 0.1 kPa to 0.25 kPa, 0.1 kPa to 0.5 kPa, 0.1 kPa to 0.75 kPa, 0.1 kPa to 1 kPa, 0.1 kPa to 1.25 kPa, 0.1 kPa to 1.5 kPa, 0.1 kPa to 1.75 kPa, 0.1 kPa to 2 kPa, 0.1 kPa to 2.5 kPa, 0.1 kPa to 3 kPa, 0.1 kPa to 4 kPa, 0.1 kPa to 5 kPa, 0.1 kPa to 6 kPa, 0.1 kPa to 7 kPa, 0.1 kPa to 8 kPa, 0.1 kPa to 9 kPa, 0.1 kPa to 10 kPa, 0.25 kPa to 0.5 kPa, 0.25 kPa to 0.75 kPa, 0.25 kPa to 1 kPa, 0.25 kPa to 1.25 kPa, 0.25 kPa to 1.5 kPa, 0.25 kPa to 1.75 kPa, 0.25 kPa to 2 kPa, 0.25 kPa to 2.5 kPa, 0.25 kPa to 3 kPa, 0.25 kPa to 4 kPa, 0.25 kPa to 5 kPa, 0.25 kPa to 6 kPa, 0.25 kPa to 7 kPa, 0.25 kPa to 8 kPa, 0.25 kPa to 9 kPa, 0.25 kPa to 10 kPa, 0.5 kPa to 0.75 kPa, 0.5 kPa to 1 kPa, 0.5 kPa to 1.25 kPa, 0.5 kPa to 1.5 kPa, 0.5 kPa to 1.75 kPa, 0.5 kPa to 2 kPa, 0.5 kPa to 2.5 kPa, 0.5 kPa to 3 kPa, 0.5 kPa to 4 kPa, 0.5 kPa to 5 kPa, 0.5 kPa to 6 kPa, 0.5 kPa to 7 kPa, 0.5 kPa to 8 kPa, 0.5 kPa to 9 kPa, 0.5 kPa to 10 kPa, 0.75 kPa to 1 kPa, 0.75 kPa to 1.25 kPa, 0.75 kPa to 1.5 kPa, 0.75 kPa to 1.75 kPa, 0.75 kPa to 2 kPa, 0.75 kPa to 2.5 kPa, 0.75 kPa to 3 kPa, 0.75 kPa to 4 kPa, 0.75 kPa to 5 kPa, 0.75 kPa to 6 kPa, 0.75 kPa to 7 kPa, 0.75 kPa to 8 kPa, 0.75 kPa to 9 kPa, 0.75 kPa to 10 kPa, 1 kPa to 1.25 kPa, 1 kPa to 1.5 kPa, 1 kPa to 1.75 kPa, 1 kPa to 2 kPa, 1 kPa to 2.5 kPa, 1 kPa to 3 kPa, 1 kPa to 4 kPa, 1 kPa to 5 kPa, 1 kPa to 6 kPa, 1 kPa to 7 kPa, 1 kPa to 8 kPa, 1 kPa to 9 kPa, 1 kPa to 10 kPa, 1.25 kPa to 1.5 kPa, 1.25 kPa to 1.75 kPa, 1.25 kPa to 2 kPa, 1.25 kPa to 2.5 kPa, 1.25 kPa to 3 kPa, 1.25 kPa to 4 kPa, 1.25 kPa to 5 kPa, 1.25 kPa to 6 kPa, 1.25 kPa to 7 kPa, 1.25 kPa to 8 kPa, 1.25 kPa to 9 kPa, 1.25 kPa to 10 kPa, 1.5 kPa to 1.75 kPa, 1.5 kPa to 2 kPa, 1.5 kPa to 2.5 kPa, 1.5 kPa to 3 kPa, 1.5 kPa to 4 kPa, 1.5 kPa to 5 kPa, 1.5 kPa to 6 kPa, 1.5 kPa to 7 kPa, 1.5 kPa to 8 kPa, 1.5 kPa to 9 kPa, 1.5 kPa to 10 kPa, 1.75 kPa to 2 kPa, 1.75 kPa to 2.5 kPa, 1.75 kPa to 3 kPa, 1.75 kPa to 4 kPa, 1.75 kPa to 5 kPa, 1.75 kPa to 6 kPa, 1.75 kPa to 7 kPa, 1.75 kPa to 8 kPa, 1.75 kPa to 9 kPa, 1.75 kPa to 10 kPa, 2.5 kPa to 2.5 kPa, 2.5 kPa to 3 kPa, 2.5 kPa to 4 kPa, 2.5 kPa to 5 kPa, 2.5 kPa to 6 kPa, 2.5 kPa to 7 kPa, 2.5 kPa to 8 kPa, 2.5 kPa to 9 kPa, 2.5 kPa to 10 kPa, 3 kPa to 4 kPa, 3 kPa to 5 kPa, 3 kPa to 6 kPa, 3 kPa to 7 kPa, 3 kPa to 8 kPa, 3 kPa to 9 kPa,WSGR Docket No.67566-702601 3 kPa to 10 kPa, 4 kPa to 5 kPa, 4 kPa to 6 kPa, 4 kPa to 7 kPa, 4 kPa to 8 kPa, 4 kPa to 9 kPa, 4 kPa to 10 kPa, 5 kPa to 6 kPa, 5 kPa to 7 kPa, 5 kPa to 8 kPa, 5 kPa to 9 kPa, 5 kPa to 10 kPa, 6 kPa to 7 kPa, 6 kPa to 8 kPa, 6 kPa to 9 kPa, 6 kPa to 10 kPa, 7 kPa to 8 kPa, 7 kPa to 9 kPa, 7 kPa to 10 kPa, 8 kPa to 9 kPa, 8 kPa to 10 kPa, or 9 kPa to 10 kPa.

[0219] The viscoelasticity of the 3D matrix may be tuned or altered for select cellprocessing applications. Viscoelasticity may describe materials that exhibit both viscous and elastic characteristics when deformed. Hydrogels may display significant viscoelastic behavior such that the hydrogel may dissipate energy (e.g., like a viscous fluid) and recover the original shape (e.g., like an elastic solid). Dynamic mechanical analysis (DMA) may be used to measure viscoelasticity, where a 3D matrix such as a hydrogel may be subjected to a sinusoidal stress and the resulting strain may be measured. The phase difference between stress and strain may provide insights into the material's viscoelastic properties. Viscoelasticity and relaxation time, independent of stiffness, may impact cellular behaviors. For example, in hydrogels that exhibit stress relaxation, each force or strain a cell applies to the matrix over time may initially be resisted with a certain stiffness, defined by the initial elastic modulus, followed by a decrease in resistance over time. Stress relaxation, for example, may be measured by a stress relaxation test (τ1 / 2). Stress relaxation timescales may be less than or equal to about 1 week, 1 day, 1 hour, 1 minute, 1 second, or less. Stress relaxation timescales may be greater than or equal to about 1 second, 1 minute, 1 hour, 1 day, 1 week, or more.

[0220] The swelling properties of 3D matrices may be tuned or otherwise modified forselect cell processing applications. Swelling properties may be described and measured in terms of swelling ratio, e.g., the amount of water or buffer a 3D substrate (e.g., a hydrogel or other polymer matrix) can absorb and retain. Swelling may be an indicator of the network hydrophilicity as well as of the relative crosslinking density. 3D matrices with stiffer networks may exhibit lower swelling and matrices with more flexible networks may exhibit greater swelling. Swelling ratio may indirectly influence mechanical softness and flexibility. The swelling ratio may be a measure of how much a matrix swells beyond an initial state, or the ratio of its swollen volume to its dry volume. For example, to determine a swelling ratio, dry material may be weighed or its volume measured, submerged in water or other suitable aqueous buffer until equilibrium swelling is reached, then reweighed or its volume re- measured to determine the swelling ratio (e.g., Q1a = Vs / Vd; Q1 volume swelling ratio from dry, Vs volume saturated, Vd volume dry; Q1b = (Vs - Vd ) / Vd); Q1c = Ms / Md; Q1c mass swelling ratio from dry, Msmass saturated, Mdmass dry; Q1d= (Ms- Md) / Md)).WSGR Docket No.67566-702601 Alternatively, the swelling ratio may be determined by weighing or determining the volume of a solvated or hydrated matrix in an initial state (e.g., an initial hydrogel of 2% collagen w / v hydrogel), submerging the matrix in water or other suitable aqueous buffer until equilibrium swelling is reached, then reweighing or re-measuring the volume to determine the swelling ratio (e.g., Q2a = Vs / Vi; Q2 volume swelling from initial state; Vs volume saturated; Vi initial volume; Q2b = (Vs – Vi) / Vi); Q2c = Ms / Mi; Q1c mass swelling ratio from initial, Ms mass saturated, Miinitial mass; Q2d= (Ms– Mi) / Mi)). In some embodiments, the 3D matrix does not swell substantially (e.g., swelling ratio Q2d of less than about 50% ). The swelling ratio of the 3D matrix may be greater than or equal to about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, or more. The swelling ratio of the 3D matrix may be less than or equal to about 500%, 400%, 300%, 250%, 200% 175%, 150%, 125%, 100%, 75%, 50%, 25%, or less. The swelling ratio of the 3D matrix may be from about 25% to 50%, 25% to 75%, 25% to 100%, 25% to 125%, 25% to 150%, 25% to 175%, 25% to 200%, 25% to 250%, 25% to 300%, 25% to 400%, 25% to 500%, 50% to 75%, 50% to 100%, 50% to 125%, 50% to 150%, 50% to 175%, 50% to 200%, 50% to 250%, 50% to 300%, 50% to 400%, 50% to 500%, 75% to 100%, 75% to 125%, 75% to 150%, 75% to 175%, 75% to 200%, 75% to 250%, 75% to 300%, 75% to 400%, 75% to 500%, 100% to 125%, 100% to 150%, 100% to 175%, 100% to 200%, 100% to 250%, 100% to 300%, 100% to 400%, 100% to 500%, 125% to 150%, 125% to 175%, 125% to 200%, 125% to 250%, 125% to 300%, 125% to 400%, 125% to 500%, 150% to 175%, 150% to 200%, 150% to 250%, 150% to 300%, 150% to 400%, 150% to 500%, 175% to 200%, 175% to 250%, 175% to 300%, 175% to 400%, 175% to 500%, 200% to 250%, 200% to 300%, 200% to 400%, 200% to 500%, 250% to 300%, 250% to 400%, 250% to 500%, 300% to 400%, 300% to 500%, or 400% to 500%. In an example, the swelling ratio of the 3D matrix is less than or equal to about 100%.

[0221] In some embodiments, the 3D volume of the matrix may be constrained by theconfiguration of the vessel, such as a flow cell or compartment thereof, such that the 3D matrix is prevented from absorbing additional water and swelling beyond the defined volume of the container. In some embodiments, the 3D volume of the matrix may be variable, exhibiting swelling over time or in response to the addition of solvents or other reagents. For example, the 3D matrix may be a programmably swellable matrix which undergoes a change in swelling properties in response to an external stimulus. The external stimulus may be a chemical stimulus, thermal stimulus, electromagnetic stimulus, physical stimulus, or any combination thereof. In some embodiments, the vessel containing the 3D matrix may not physically constrain or otherwise limit swelling of the 3D volume of physical dimensions ofWSGR Docket No.67566-702601 the 3D matrix. For example, the container retaining the 3D matrix may comprise a deformable vessel. The deformable vessel may be internally configured such that 3D matrix swelling occupies internal spaces otherwise occupied by fluid or gas when the 3D matrix is in a non-swollen state, and which become displaced by the 3D matrix upon swelling.

[0222] The porosity of 3D substrates may be tuned or otherwise modified for a given cellprocessing application. The porosity of 3D substrates may be determined by a number of methodologies, including through microscopy (e.g., by stimulated emission depletion (STED) microscopy, atomic force microscopy (AFM), or cryogenic scanning electron microscopy (Cryo-SEM)) to visualize and measure the morphology and size distribution of pores. Porosity may also be quantified by measuring the pore volume fraction, e.g., porosity e = Vp / Vt× 100; Vp= pore volume = (Ww- Wd) / p (Ww= weight wet, Wd= weight dry, p = fluid density); Vt = total volume).3D matrix porosity can influence the swelling behavior and other mechanical properties of the 3D matrix. For example, lower swelling and higher modulus may be correlated with a smaller porosity. The mesh size, or molecular porosity, of the matrix may be on the nanometer to micrometer scale and can influence nutrient flux throughout the matrix and cell retention, proliferation, growth, and other aspects of cellular state or phenotype. In some embodiments, the matrix porosity is on average smaller than a cell, but large enough to allow the diffusion of macromolecules for maintenance of cellular viability. In some embodiments, the matrix porosity is on average larger than a cell, such as to facilitate cellular proliferation and migration within the 3D matrix, while still retaining cells. The average pore diameter may be constant throughout the 3D matrix or may vary throughout the 3D matrix. The 3D matrix may have a porosity of greater than or equal to about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In an example, the 3D matrix has a porosity of greater than or equal to about 80%. The 3D matrix may have a porosity of less than or equal to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less. The 3D matrix may have a porosity from about 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 40% to 90%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 60% to 70%, 60% to 80%, 60% to 90%, 70% to 80%, 70% to 90%, or 80% to 90%.

[0223] The 3D matrix or substrate may have an average pore size that permits nutrients,reagents, and other materials to diffuse or otherwise move through the 3D matrix while retaining or substantially retaining cells within the 3D matrix. In some examples, the 3D matrix comprises a porous, solid matrix having interconnected pores. The average pore sizeWSGR Docket No.67566-702601 of the interconnected pores may be sufficiently large to permit growth of cells (e.g., lymphocytes or other target cells) within the 3D matrix. The 3D matrix or substrate may have an average pore size of less than or equal to about 1000 µm, 900 µm, 800 µm, 700 µm, 600 µm, 500 µm, 400 µm, 300 µm, 200 µm, 100 µm, 75 µm, 50 µm, 25 µm, 10 µm, 5 µm, 1 µm, 0.5 µm, 0.1 µm, or less. In an example, the 3D matrix or substrate has an average pore size of less than or equal to about 400 µm. The 3D matrix or substrate may have an average pore size of greater than or equal to about 0.1 µm, 0.5 µm, 1 µm, 5 µm, 10 µm, 25 µm, 50 µm, 75 µm, 100 µm, 200 µm, 300 µm, 400 µm, 500 µm, 600 µm, 700 µm, 800 µm, 900 µm, 1000 µm, or greater. The 3D matrix or substrate may have an average pore size from about 0.1 µm to 0.5 µm, 0.1 µm to 1 µm, 0.1 µm to 5 µm, 0.1 µm to 10 µm, 0.1 µm to 25 µm, 0.1 µm to 50 µm, 0.1 µm to 75 µm, 0.1 µm to 100 µm, 0.1 µm to 200 µm, 0.1 µm to 300 µm, 0.1 µm to 400 µm, 0.1 µm to 500 µm, 0.1 µm to 600 µm, 0.1 µm to 700 µm, 0.1 µm to 800 µm, 0.1 µm to 900 µm, 0.1 µm to 1000 µm, 0.5 µm to 1 µm, 0.5 µm to 5 µm, 0.5 µm to 10 µm, 0.5 µm to 25 µm, 0.5 µm to 50 µm, 0.5 µm to 75 µm, 0.5 µm to 100 µm, 0.5 µm to 200 µm, 0.5 µm to 300 µm, 0.5 µm to 400 µm, 0.5 µm to 500 µm, 0.5 µm to 600 µm, 0.5 µm to 700 µm, 0.5 µm to 800 µm, 0.5 µm to 900 µm, 0.5 µm to 1000 µm, 1 µm to 5 µm, 1 µm to 10 µm, 1 µm to 25 µm, 1 µm to 50 µm, 1 µm to 75 µm, 1 µm to 100 µm, 1 µm to 200 µm, 1 µm to 300 µm, 1 µm to 400 µm, 1 µm to 500 µm, 1 µm to 600 µm, 1 µm to 700 µm, 1 µm to 800 µm, 1 µm to 900 µm, 1 µm to 1000 µm, 5 µm to 10 µm, 5 µm to 25 µm, 5 µm to 50 µm, 5 µm to 75 µm, 5 µm to 100 µm, 5 µm to 200 µm, 5 µm to 300 µm, 5 µm to 400 µm, 5 µm to 500 µm, 5 µm to 600 µm, 5 µm to 700 µm, 5 µm to 800 µm, 5 µm to 900 µm, 5 µm to 1000 µm, 10 µm to 25 µm, 10 µm to 50 µm, 10 µm to 75 µm, 10 µm to 100 µm, 10 µm to 200 µm, 10 µm to 300 µm, 10 µm to 400 µm, 10 µm to 500 µm, 10 µm to 600 µm, 10 µm to 700 µm, 10 µm to 800 µm, 10 µm to 900 µm, 10 µm to 1000 µm, 25 µm to 50 µm, 25 µm to 75 µm, 25 µm to 100 µm, 25 µm to 200 µm, 25 µm to 300 µm, 25 µm to 400 µm, 25 µm to 500 µm, 25 µm to 600 µm, 25 µm to 700 µm, 25 µm to 800 µm, 25 µm to 900 µm, 25 µm to 1000 µm, 50 µm to 75 µm, 50 µm to 100 µm, 50 µm to 200 µm, 50 µm to 300 µm, 50 µm to 400 µm, 50 µm to 500 µm, 50 µm to 600 µm, 50 µm to 700 µm, 50 µm to 800 µm, 50 µm to 900 µm, 50 µm to 1000 µm, 75 µm to 100 µm, 75 µm to 200 µm, 75 µm to 300 µm, 75 µm to 400 µm, 75 µm to 500 µm, 75 µm to 600 µm, 75 µm to 700 µm, 75 µm to 800 µm, 75 µm to 900 µm, 75 µm to 1000 µm, 100 µm to 200 µm, 100 µm to 300 µm, 100 µm to 400 µm, 100 µm to 500 µm, 100 µm to 600 µm, 100 µm to 700 µm, 100 µm to 800 µm, 100 µm to 900 µm, 100 µm to 1000 µm, 200 µm to 300 µm, 200 µm to 400 µm, 200 µm to 500 µm, 200 µm to 600 µm, 200 µm to 700 µm, 200 µm to 800 µm, 200 µm to 900 µm, 200 µm to 1000 µm,WSGR Docket No.67566-702601 300 µm to 400 µm, 300 µm to 500 µm, 300 µm to 600 µm, 300 µm to 700 µm, 300 µm to 800 µm, 300 µm to 900 µm, 300 µm to 1000 µm, 400 µm to 500 µm, 400 µm to 600 µm, 400 µm to 700 µm, 400 µm to 800 µm, 400 µm to 900 µm, 400 µm to 1000 µm, 500 µm to 600 µm, 500 µm to 700 µm, 500 µm to 800 µm, 500 µm to 900 µm, 500 µm to 1000 µm, 600 µm to 700 µm, 600µm to 800 µm, 600µm to 900 µm, 600µm to 1000 µm, 700µm to 800 µm, 700µm to 900 µm, 700µm to 1000 µm, 800µm to 900 µm, 800µm to 1000 µm, or 900µm to 1000 µm. In an example, the 3D matrix or substrate comprises a pore size from about 0.5 µm to 400 µm.

[0224] The stability of 3D substrates may be tuned or otherwise modified for select cellprocessing applications. Certain 3D substrate materials may be substantially chemically or biologically stable. For example, porous solid matrices formed of materials such as glass, ceramics, or plastics can be considered stable over indefinite timescales because they are not subject to substantial chemical or biological material degradation under cell processing use cases (e.g., cell culture conditions). In some embodiments, the 3D substrate is substantially stable at macroscale and over relevant timescales (e.g., hours, days, or weeks). For example, 3D matrices comprising natural polymeric hydrogels, such as collagen and fibrin, may degrade in the presence of cells by the activity of cell-mediated proteases including matrix metalloproteinases (MMPs).3D hydrogel matrices comprising synthetic polymers can also be engineered with peptide crosslinkers, which may be designed to degrade in response to cell- mediated protease activity.3D matrices comprising degradable moieties may be stable at macroscale despite these biological processes. For example, such degradation processes may remodel the 3D matrix locally around cells, thereby permitting cellular proliferation, cellular migration, or both within the local 3D environment. During local remodeling the macroscale matrix may remain substantially structurally intact (e.g., gelled). In some embodiments, the 3D matrix may be substantially degraded during cell culture processes. For example, after a given time the 3D matrix may no longer be substantially structurally intact. In some embodiments, matrix materials may provide “self-healing” properties or other dynamic reorganization properties over time. The “self-healing” properties may permit the 3D matrix to autonomously recover an original state after damage or degradation. 3D matrices configured for dynamic reorganization may permit polymer disentanglement, disruption of weak physical interactions, rearrangement of reversible interactions, degradation processes, or any combination thereof.

[0225] The optical properties of 3D matrices may be tuned or otherwise modified forselect cell processing applications. For example, the optical properties of 3D matrices used inWSGR Docket No.67566-702601 3D cell culture may provide in-process monitoring and analysis. In an example, the optical properties of a 3D substrate may determine which imaging and spectroscopic techniques may be effectively applied to observe and measure the biological and physical processes occurring within the 3D substrate during cell culture. In some embodiments, 3D matrices (e.g., hydrogel matrices) with high transparency may be used to enable optical techniques such as measurement of optical density and backscatter. To measure optical density, light with one or more specific wavelength(s) may be emitted into a 3D substrate and the intensity of light transmitted through the 3D substrate may be measured by a detector. A 3D substrate containing relatively more cells or denser cells may transmit less light. Using the Lambert Beer law, the transmitted light signal may be used to calculated the optical density. To measure backscatter, light with one or more specific wavelength(s) may be emitted into a 3D substrate, and a sensor close to the light source may detect the amount or intensity of light scattered back by cells and other particles present in the 3D substrate. A 3D substrate containing relatively more cells or denser cells may backscatter more light. Measuring optical density (OD) and backscatter can be a useful in situ process analytical tool in 3D cell culture, for example, when assessing cell growth, proliferation, or metabolic activity within the 3D matrix. Such measurements may be used to estimate the concentration of cells or other substances in a solution by measuring the absorbance of light at specific wavelengths. In some embodiments, 3D substrates which do not absorb significantly at the measurement wavelength or scatter light, which can interfere with accurate readings, are used to enable optical density and backscatter measurements. In some embodiments, such as those utilizing thicker or more opaque hydrogels, calibration or correction factors can be used to account for reduced light penetration in optical density measurements. In some embodiments, 3D matrices with high transparency may be used to permit the use of microscopy techniques, including high-resolution methods like confocal microscopy. Optical transparency may provide minimal light scattering and absorption, allowing for clear imaging of cells and structures within a 3D matrix. In some embodiments, a 3D matrix such as a hydrogel may exhibit optical scattering properties, particularly with thicker or more densely crosslinked hydrogels. Adjusting the refractive index of the hydrogel to match that of the immersion medium or the cells may help reduce scattering. Matching the refractive index of the 3D substrate with that of the surrounding medium (usually buffer or culture medium) may reduce refraction at interfaces within the imaging field, enhancing image clarity and depth penetration. A matched refractive index may provide for deep imaging, allowing for more effective use of techniques such as multi-photon microscopy. Certain 3D substrates, such asWSGR Docket No.67566-702601 porous solids and hydrogels derived from natural polymers, may exhibit autofluorescence, which can interfere with fluorescence microscopy. In some embodiments, 3D matrices with low autofluorescence may be used to permit fluorescence imaging techniques, or configuration of an imaging modality using specific wavelengths of excitation and emission (e.g., wavelengths where autofluorescence is reduced or minimal for a certain material) can minimize the negative impact of autofluorescence on optical sensing.

[0226] The 3D matrix may be tuned or otherwise modified for optical homogeneity topermit accurate spectroscopic measurements. Inhomogeneities may lead to variable light pathlengths and affect the accuracy of spectroscopic data, such as with Near-Infrared (NIR) and Raman spectroscopy. In some embodiments, a 3D substrate is selected for compatibility with specific spectroscopic techniques. In an example, a 3D matrix is configured to permit Near-Infrared (NIR) Spectroscopy, which can penetrate deeper into 3D matrices such as hydrogels (compared to visible light) making it useful for monitoring cellular processes, water content, solvent interactions, and other bulk properties of a cell-laden 3D substrate. In another example, a 3D matrix is configured to permit Raman spectroscopy, a measurement technique sensitive to molecular vibrations that can provide detailed information about the chemical environment within a 3D substrate. The optical properties of the 3D matrix may be selected or designed to enhance the quality of the Raman signals. The 3D matrix may incorporate or otherwise couple with optical sensors that respond to changes in pH, temperature, specific biomolecules, or any combinations thereof. These sensors may provide real-time, in situ monitoring of the microenvironment which may provide monitoring of dynamic cellular processes. In some embodiments, 3D matrix compositions may support the propagation of light with minimal distortion for technologies like optical coherence tomography (OCT) or photonic crystal-based sensing. Another optical consideration may be the stability of the 3D substrate under light exposure. Some 3D substrates, such as hydrogels, may be susceptible to photodegradation, especially under intense or prolonged light exposure used in certain imaging techniques. This may affect both the structural integrity of the hydrogel and the viability of encapsulated cells. In some embodiments, a 3D substrate which is not susceptible to photodegradation under relevant exposure conditions may be used. As described herein, in some embodiments, the 3D matrix may be optically clear, substantially optically clear, light scattering, opaque, and have other photonic properties such as refractive index and light scattering. In some embodiments, 3D matrices are optically transparent, substantially optically transparent, or otherwise optically suitable for imaging, such as to permit in situ cell imaging, including by microscopy methods such as widefield, brightfield,WSGR Docket No.67566-702601 light sheet, confocal microscopy, or any combination thereof. In some embodiments, 3D matrices are optically suitable for spectroscopy, such as to permit in situ process analytics using NIR spectroscopy or Raman spectroscopy. Electrically conductive 3D substrates

[0227] The 3D matrix may be configured for or may provide electrical conductance.Electrical conductance may be useful for processing cell types that exhibit phenotypically relevant electrochemistry or electrophysiology. In some embodiments, electrical conductance of 3D substrates may permit in situ electropermeabilization within a 3D matrix, such as for the purpose of electroporation cell transfection and genetic engineering. In some embodiments, an electrically conductive matrix can be used to incorporate electrical signals as biorelevant physical cues. Electrical biorelevant cues may be useful for the culture and study of electrochemically active cell types such as neurons and cardiomyocytes. These cells may be highly responsive to electrical stimuli, which play a role in their function, development, and communication. Incorporating electrical cues into cell culture environments may enhance physiological relevance and thereby support the formation and maintenance of relevant cellular phenotypes. Direct electrical stimulation may be applied by delivering electrical pulses directly to the 3D substrate, culture medium, or through electrodes embedded in, around, or throughout the cell culture substrate. This method may mimic the natural electrical activity that cells, such as neurons and cardiomyocytes, experience in vivo. The stimulation may be configured in various ways (e.g., amplitude, frequency, pulse duration, or combinations thereof) to match physiological conditions as closely as possible. For neurons, this can promote axon growth and synapse formation, while for cardiomyocytes, it can enhance the maturation, alignment, contraction strength, or any combination thereof.

[0228] In some embodiments, the material(s) comprising the 3D substrate, and the 3Dsubstrate itself, may be a weak electrical conductor(s). For example, collagen hydrogel matrices may have electrical conductivity that is generally very low, for example, close to that of an insulator. In another example, PEG is a non-ionic polymer and does not conduct electricity. A 3D substrate formed from PEG hydrogels may be non-conductive unless modified. In some embodiments, the 3D substrate may be considered a volume conductor, or material that allows electric current to pass through its entire volume (e.g., in contrast to surface conductors, where current flows primarily along the surface). Volume conductors may permit current flow through their three-dimensional mass. A 3D substrate may have an electrical conductivity of greater than or equal to about 10-10Siemens per meter (S / m), 10-9S / m, 10-8S / m, 10-7S / m, 10-6S / m, 10-5S / m, 10-4S / m, 10-3S / m, 10-2S / m, 10-1S / m, 100S / m,WSGR Docket No.67566-702601 101S / m, or more. A 3D substrate may have an electrical conductivity of less than or equal to about 101S / m, 100S / m, 10-1S / m, 10-2S / m, 10-3S / m, 10-4S / m, 10-5S / m, 10-6S / m, 10-7S / m, 10-8S / m, 10-9S / m, 10-10S / m, or less. A 3D substrate, depending on composition, may have an electrical conductivity from about 10-10to 10-9S / m, 10-10to 10-8S / m, 10-10to 10-7S / m, 10-10to 10-6S / m, 10-10to 10-5S / m, 10-10to 10-4S / m, 10-10to 10-3S / m, 10-10to 10-2S / m, 10-10to 10-1S / m, 10-10to 100S / m, 10-10to 101S / m, 10-9to 10-8S / m, 10-9to 10-7S / m, 10-9to 10-6S / m, 10-9to 10-5S / m, 10-9to 10-4S / m, 10-9to 10-3S / m, 10-9to 10-2S / m, 10-9to 10-1S / m, 10-9to 100S / m, 10-9to 101S / m, 10-8to 10-7S / m, 10-8to 10-6S / m, 10-8to 10-5S / m, 10-8to 10-4S / m, 10-8to 10-3S / m, 10-8to 10-2S / m, 10-8to 10-1S / m, 10-8to 100S / m, 10-8to 101S / m, 10-7to 10-6S / m, 10-7to 10-5S / m, 10-7to 10-4S / m, 10-7to 10-3S / m, 10-7to 10-2S / m, 10-7to 10-1S / m, 10-7to 100S / m, 10-7to 101S / m, 10-6to 10-5S / m, 10-6to 10-4S / m, 10-6to 10-3S / m, 10-6to 10-2S / m, 10-6to 10-1S / m, 10-6to 100S / m, 10-6to 101S / m, 10-5to 10-4S / m, 10-5to 10-3S / m, 10-5to 10-2S / m, 10-5to 10-1S / m, 10-5to 100S / m, 10-5to 101S / m, 10-4to 10-3S / m, 10-4to 10-2S / m, 10-4to 10-1S / m, 10-4to 100S / m, 10-4to 101S / m, 10-3to 10-2S / m, 10-3to 10-1S / m, 10-3to 100S / m, 10-3to 101S / m, 10-2to 10-1S / m, 10-2to 100S / m, 10-2to 101S / m, 10-1to 100S / m, 10-1to 101S / m, or 100to 101S / m. In an example, the 3D matrix or substrate has a conductivity from about 10-5to 10-1S / m.

[0229] Incorporating ionic solutions or 3D matrix components with ionic or conductivecomponents may increase matrix conductivity. In an example, the 3D matric comprises ionic or conductive components that increase matrix conductivity. The cell-laden 3D substrate environment may comprise a conductive material or biomaterial, featuring ionic or electrical conductivity, such as by the presence of mobile ion species as dissolved electrolytes, which may be present in cell culture medias and buffers. For example, cell culture media and buffers may have an electrical conductivity around ~1.5 to 2.0 S / m, similar to extracellular fluid. The conductivity of such media may be dependent upon the concentration of dissolved ions, such as from added salts (e.g., NaCl, KCl, CaCl2), nutrients, and buffering agents. The conductivity of cell culture media may aid in maintaining osmotic balance and pH and also permit various cell functions that depend on ion transport and electrical gradients. The conductivity parameter may provide that an applied electrical field effectively interacts with the cells without causing excessive heat or electrolytic effects. In some embodiments, the 3D substrate may provide electrical and electrochemical properties via integration of conductive materials into the 3D substrate, or usage of a 3D substrate comprising electrically conductive materials, including, but not limited to, gold (e.g., ~4.1 × 107S / m), carbon nanotubes (e.g., ~105S / m), graphene (e.g., ~106S / m), conductive polymers (e.g., polypyrrole, ~10-100 S / cm,WSGR Docket No.67566-702601 PEDOT:PSS, ~0.1-1 kS / cm), or any combination thereof. These materials can conduct electrical signals, provide localized electrical stimulation, facilitate in situ electroporation, or any combination thereof.

[0230] In some embodiments, the 3D substrate is filled with an ionic solution acting as anelectroporation or membrane electropermeabilization, buffer. The electrical conductivity of electroporation buffers can vary and may be tailored to the specific application and cell type. Lower conductivity buffers (around 0.1 to 1.0 S / m) may be used to reduce joule heating during the electroporation process. Some electroporation processes may use lower or higher conductivities, depending on the field strength and pulse duration used. Electroporation buffers may include phosphate buffers or solutes such as sucrose to maintain osmolarity with minimal ionic strength. The composition and electrical conductivity may be modified to maximize pore formation in the cell membrane while minimizing cell death. Electroporation efficiency may depend on the conductivity and permittivity of the media, cell membrane, cell cytosol, and by cell size and shape. In some embodiments, the electrical conductivity of the 3D substrate and interstitial fluids may be tuned to a specific system configuration and biological application.

[0231] As described elsewhere herein, the apparatus and systems for cell processing mayfurther incorporate electrical elements which may couple to or are otherwise in electrical communication with the 3D substrate, ionic solutions, or both to provide electrical interfaces (e.g., stimulation, sensing) with processed cells or to provide membrane electropermeabilization. Programmable 3D substrates

[0232] A 3D matrix may be functionalized, as shown in FIG. 12A. Functionalization ofa 3D matrix may provide for a structural microenvironment that enhances or otherwise improves signal transduction. For example, a 3D matrix may be functionalized with cytokines, antibodies, or other components useful for cell processing. Functionalization may be static such that the functionalization does not change during cell processing. Alternatively, the functionalization may change during cell processing such that the 3Dmatrix may be programmable. A programmable matrix, as shown in FIG. 12B, maycomprise chemical tethering moieties. The chemical tethering moieties may be configured to permit tethering of biomolecular factors, such as cytokines and antibodies, to the 3D matrix. Functional biomolecules may be removably coupled to the 3D matrix such that, during cell processing, the functional biomolecules may be released from the 3D matrix and removed via washing.WSGR Docket No.67566-702601

[0233] In some embodiments, the 3D matrix is a programmable 3D matrix.Programmable 3D matrices may physically interact with cells by virtue of comprising a 3D local environment in which living cells reside (e.g., with physical properties such as stiffness and porosity). The 3D substrate may further comprise cell-interacting aspects with designed biological properties. Such cell-interacting aspects may include, but are not limited to, factors related to cell adhesion and membrane signal transduction.

[0234] In some embodiments, the 3D substrate comprises features which determinecellular adhesivity or attachment.3D matrix materials may provide complex and mechanistically diverse adhesivity interactions with cells. Cellular surface proteins and the cell membrane may interact with a 3D substrate via steric, electrostatic, hydrophobic, hydrogen bonding, or other interactions. Transmembrane proteins and cell surface receptors, such as integrins and other proteoglycans, may bind to ligands (e.g., individually or synergistically) on or in 3D substrate (e.g., when cognate adhesion moieties are provided by a certain 3D substrate composition), thereby providing cellular adhesion, mechanical interactions with cells, cell signaling, or a combination thereof. The density of various cell adhesion moieties may depend on the composition of a matrix and may be tuned to promote a cellular phenotype, morphology, motility, or other cellular characteristic. In certain embodiments, substrates without intrinsic cell-interaction moieties may still permit cell adhesion through the cellular production of extracellular matrix and other proteins secreted by cells within the matrix, which may form a pericellular matrix with which the cells can interact and onto which the cells can adhere over time. In some embodiments, the matrix is substantially non-adhesive to cells. In some embodiments, the matrix is substantially adhesive to cells.

[0235] The 3D matrix or 3D substrate may be functionalized to generate a functionalized3D matrix or substrate. Functionalizing the 3D matrix may permit the 3D matrix to be stimuli-responsive. Alternatively, or in addition to, functionalizing the 3D matrix may permit the 3D matrix to interact with the plurality of cells, for example, to activate or otherwise stimulate cells of the plurality of cells. Alternatively, or in addition to, functionalizing the 3D matrix may permit the matrix to be a substrate for the plurality of cells or to otherwise provide a given cellular environment to the plurality of cells. The 3D matrix or substrate may be functionalized using pre-functionalized polymer precursors to generate the 3D matrix. For example, pre-functionalized polymer precursors may be provided to a cell chamber in tandem with or in replacement of non-functionalized polymer precursors. The pre-functionalized polymer precursors may be incorporated into the 3D substrate upon polymerization of theWSGR Docket No.67566-702601 polymer precursors. In an example, cells and pre-functionalized polymer precursors may be provided simultaneously such that formation of the functionalized 3D matrix occurs concurrently entrapment or encapsulation of the plurality of cells. Alternatively, or in addition to, the 3D matrix or substrate may be formed, and functionalization may occur subsequent to polymerization of the 3D matrix or substrate. In an example, functionalizing the 3D matrix comprises coupling or immobilizing one or more biomolecules to the 3D matrix. The one or more biomolecules may be proteins, peptides, cytokines, antibodies, nucleic acid molecules, or any combinations thereof. In an example, the one or more biomolecules may be usable for membrane cell transduction. The one or more biomolecules may remain conjugated or cognate to the 3D matrix during cell processing. Alternatively, or in addition to, the functionalized biomolecules may be released from the 3D matrix or substrate and, in some examples, replaced with other biomolecules.

[0236] In some embodiments, the 3D substrate comprises cell-interacting factors. Suchfactors may provide one or more functions, including for example, without limiting other possible functions, mimicking a tissue microenvironment, simulating or providing an extracellular matrix composition, providing membrane signal transduction, modulating cellular properties including cell adhesion, proliferation, migration, differentiation (e.g., including immune cell stimulation), survival, providing stimulatory, co-stimulatory, pro- survival, or pro-phenotype cellular cues, or any combinations thereof. Cell-interacting factors may include, but are not limited to, matrix proteins, glycoproteins, glycosaminoglycans, proteoglycans, ECM-sequestered growth factors, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), as well as other secreted proteins, or any combinations thereof. For example, peptides may be incorporated into a 3D substrate to mimic the specific molecules, sequences, or moieties found in a natural extracellular matrix that interact with cell receptors, influencing cell adhesion, migration, and fate. RGD or peptides with related motifs may be used to permit cell attachment via the adhesive sequence arginine–glycine–aspartic acid naturally found in the extracellular matrix protein fibronectin, which may enhance cell adhesion by interacting with integrin receptors. IKVAV Peptides (Ile-Lys-Val-Ala-Val) may promote neuronal cell adhesion and neurite outgrowth. YIGSR Peptides (Tyr-Ile-Gly-Ser-Arg) may be used to influence cell attachment and proliferation. As another class of examples, growth factors may be incorporated within the 3D substrate to promote cell proliferation, differentiation, tissue regeneration, or any combination thereof. Fibroblast Growth Factors (FGFs) may be used in neural stem cell cultures to promote proliferation. Vascular Endothelial Growth Factor (VEGF) may be usedWSGR Docket No.67566-702601 to promote angiogenesis. Bone Morphogenetic Proteins (BMPs) may be incorporated for bone tissue engineering to stimulate osteogenic differentiation. In some embodiments, the 3D substrate may comprise signal transduction factors that include, but are not limited to, antibodies, such as those which bind CD3 and CD28 for T cell stimulation, or co-stimulatory factors, such as cytokines and interleukins, including, but not limited to, IL-2 and IL-10. Such factors may be used to modulate cellular immune responses or promote specific pathways of immune lineage cell differentiation, regeneration, and function. In some embodiments, hormones can be used within the 3D substrate to influence hormonal pathways and support the growth and function of hormone-responsive cell types.

[0237] Cell-interacting factors may be coupled to a 3D matrix. Alternatively, or inaddition to, cell-interacting factors may be non-covalently associated with the 3D matrix via electrostatic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, or any combination thereof. Cell-interacting factors may be covalently coupled to the 3D matrix via crosslinking, immobilization, binding, on any combination thereof. Cell-interacting factors may be encapsulated or otherwise physically retained within the 3D matrix. Cell-interacting factors may be irreversibly coupled to the 3D matrix. Alternatively, or in addition to, the 3D matrix may be configured for controlled release of cell-interacting factors.

[0238] In some embodiments, one or more cell-interacting factors are provided viacovalent binding or crosslinking to a 3D substrate, which may stabilize the molecules within the 3D matrix or provide a uniform or homogenous and controlled signal (e.g., to present such molecules to cells at a particular average concentration within the 3D substrate, such as an equivalent milligram per milliliter (mg / mL) of a solution-phase formulation).3D matrix functionalization may be tuned considering the selection of 3D substrate material, crosslinking density, functionalization strategy, or any combination thereof to provide a select bioactive composition for cellular contact and interaction. Alternatively, the 3D matrix may be an inactivated composition, for example, comprising an antibody which may be crosslinked to the 3D substrate in such a manner as to prevent binding or the otherwise targeted biological interaction. Functionalization chemistry may include, but are not limited to, covalent bonding chemistries, photocrosslinking, enzymatic crosslinking, Michael Addition, or any combination thereof. Carbodiimide chemistry may be used for crosslinking proteins to various substrates and involves the use of carbodiimides, such as 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC). EDC may activate carboxyl groups to react with amine groups, forming stable amide bonds. This chemistry may be used in conjunction withWSGR Docket No.67566-702601 N-hydroxysuccinimide (NHS) to improve efficiency. Glutaraldehyde crosslinking may be effective for crosslinking proteins through their amine groups, forming Schiff bases. This chemistry has the benefit of its simplicity and the robustness of the resulting crosslinks. Click chemistry, including copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain- promoted azide-alkyne cycloaddition (SPAAC), may be highly specific, efficient, and work under mild or biocompatible conditions, making them useful for coupling sensitive biomolecules. Incorporating photocrosslinkable moieties such as benzophenone, aryl azide, or cinnamoyl groups into a biomolecule or polymer may permit crosslinking upon exposure to ultraviolet (UV) or visible light. Photocrosslinking may provide spatial and temporal control over the crosslinking process. A thiol-ene reaction, a UV-induced reaction between thiol groups and alkenes, may generate thioether bonds. Enzymatic crosslinking may include examples such as transglutaminase, an enzyme which catalyzes the formation of covalent bonds between glutamine and lysine residues in proteins, and laccase and peroxidase enzymes, which may catalyze the oxidation of phenolic compounds to form crosslinks, such as those used in the modification of lignin or other natural polymers. Thiol-Michael Addition reactions between a thiol group and an electron-deficient double bond, such as acrylates or maleimides, may have the benefit of cytocompatibility and reactivity under physiological conditions. Certain conjugation chemistries may involve the chemical modification or conjugation of carbohydrates (sugars) to proteins or on carbohydrate-modified proteins. Chemoenzymatic bioconjugation may use enzymes, for example, glycosyltransferases, to attach sugars with specific functional groups to proteins. Utilizing glycosyltransferases to transfer a sugar molecule with a pre-attached functional group (e.g., biotin, or other conjugation group) to targeted sites on a protein to provide highly specific bioconjugation that permits retention of protein functionality for sensitive proteins such as antibodies. These sugars or functionalized sugars may serve as linkers for subsequent conjugation operations. Sugars on proteins may be modified to introduce azides or alkynes, and then used in copper- catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC) to attach various functional groups. Glycosylation sites on proteins may be used to attach polyethylene glycol (PEG) molecules that have been functionalized to react with sugars. This may involve using a linker that can react with the hydroxyl groups of the sugars to form stable bonds. Carbohydrates on the protein surface may be chemically modified to introduce aldehyde or ketone groups, which may be used to form oxime or hydrazone linkages with aminooxy- or hydrazine-functionalized molecules. Other methods of 3D matrix bioconjugation may be used to prepare a biofunctional 3D substrate.WSGR Docket No.67566-702601

[0239] In some embodiments, protein-protein interactions are used to immobilizebioactive proteins, leveraging specific and high-affinity binding pairs to create functional 3D substrates. For example, the biotin-streptavidin interaction, one of the strongest non-covalent interactions in nature, has a remarkably high affinity (Kd≈ 10-15M). Biotinylated proteins can be immobilized in hydrogels that contain streptavidin or vice versa. In another example, collagen-binding peptides (CBP) can be used to link peptides via specific affinity for collagen molecules. This targeted protein interaction can be leveraged for protein immobilization by incorporating functionalized CBPs into a 3D matrix comprising collagen. The His-Tag and Ni-NTA (Nitrilotriacetic Acid) interaction may use short sequences of histidine residues added to proteins, which can bind to nickel ions on Ni-NTA groups integrated within a 3D substrate. This interaction may further be used for reversible immobilization as described elsewhere herein. Incorporating Protein A or G into a 3D substrate can facilitate the immobilization of antibodies. Protein A and Protein G have high affinity for the Fc region of IgG-type antibodies. In some embodiments, multivalent linkers may be employed to increase functionalization efficiency and control the density of cell-interacting factors within a 3D substrate. For example, four-arm, six-arm, eight-arm, ten-arm, twelve-arm, and other multi- armed PEG molecules, which may be provided in particular molecular weights or distributions thereof, further comprising chemical conjugation groups (e.g., 4-arm PEG Succinimidyl Carboxymethyl Ester), can be used to create stable linkages between 3D substrates and cell-interacting factors.

[0240] In some embodiments, one or more cell-interacting factors are provided via non-covalent binding to a 3D substrate. Transient binding, such as between positively and negatively charged polymers, proteins, or other biomolecules, can retain factors via electrostatic interactions. Hydrophobic interactions, such as those driven through self- assembly of amphiphilic molecules, which aggregate in aqueous environments to minimize exposure of their hydrophobic segments, can retain factors within a 3D substrate. In some embodiments, transient binding of one or more cell-interacting factors to a 3D substrate may be stabilized by additional crosslinking, such as by using the chemistries described elsewhere herein.

[0241] In some embodiments, the 3D matrix may be configured with a controlled releasemechanism that provides cell-interacting factors within the 3D matrix. The controlled release mechanism may include encapsulation, cleavable linkers, ligand-binding interactions, or any combination thereof. In some embodiments, the cell-interacting factor(s) are one or more proteins. In some embodiments, the cell-interacting factor(s) are viral vectors. In someWSGR Docket No.67566-702601 embodiments, the cell-interacting factors comprise both proteins and viral vectors. Protein encapsulation may provide controlled release of bioactive factors into a culture environment or within a biological system such as a 3D matrix. Protein encapsulation may provide for the localized and sustained release of proteins and other factors that can, for example, direct cell behavior or modulate immune responses. Controlled release mechanisms may involve entrapping proteins or other factors within a carrier material, such as a hydrogel, nanoparticle, microsphere, liposome, or combination thereof. The carrier material may protect the molecule from degradation and permit controlled release through various mechanisms. In a diffusion-controlled release system, the protein or other factor may diffuse out of an encapsulating material over time. Diffusion may be driven by a concentration gradient. The rate of diffusion can be adjusted by modifying the porosity and crosslinking density of the encapsulation material. In a degradation-controlled release system, the encapsulating material may be biodegradable and, as it breaks down, the encapsulated proteins or other cell- interacting factors may be gradually released. The degradation rate can be tuned by the selection of materials or crosslinking agents in encapsulation materials.

[0242] In a stimulus responsive release system, the encapsulating material may bedesigned to respond to specific stimuli (e.g., pH, temperature, enzyme activity, or any combination thereof) present in the environment. The stimulus may trigger the release of protein(s) or cell-interacting factor(s). For example, proteins can be encapsulated within alginate beads using ionic crosslinking with calcium ions. The release rate may be controlled by the ionic strength and pH of the medium, which alter the stability of the alginate matrix. In another example, a temperature-sensitive hydrogel, such as poly(N-isopropylacrylamide) (PNIPAM) may undergo a phase transition at a given temperatures. The phase transition may release encapsulated proteins in response to a temperature changes. In another example, PLGA microspheres formed from Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer which degrades over time, may release encapsulated proteins upon hydrolytic degradation and other processes which degrade the polymer matrix. Polymer composition and molecular weight may alter or modify the degradation rate of such encapsulation materials, and thus the release kinetics. In another example, dendrimers or other branched synthetic polymers can encapsulate proteins or other cell-interacting factors within their structure, such that the release can be controlled by the design of the polymer, including its size and surface functionality.

[0243] In an example, proteins or other cell-interacting factors may be provided to the 3Dmatrix in liposomes. Liposomes may be spherical vesicles with a lipid bilayer that canWSGR Docket No.67566-702601 encapsulate hydrophilic proteins or cell-interacting factors within their aqueous core. The liposomes may be introduced within the 3D substrate environment and release a payload over time via liposomal destabilization and leeching. Modifications to the liposome structure, such as the inclusion of pH-sensitive or thermosensitive lipids, can further allow for the release of proteins from liposomal formulations in response to environmental changes. In another example, hydroxyapatite (HA) particles can be used for the sustained release of cell- interacting factors such as viral particles within a 3D substrate. Hydroxyapatite can adsorb proteins and other biological molecules onto its surface due to its high surface area and porous structure, or by encapsulation of these molecules within its matrix, depending on formulation. Viral particles can be bound to HA particles, allowing for their gradual release as the HA degrades or as the particles desorb from the surface. The release rate can be adjusted by modifying the particle size, porosity, and surface characteristics of HA. For example, smaller particles or higher porosity particles may provide faster release rates than larger particles or particles with lower porosity. Viral particles and other cell-interacting factors can be encapsulated within biodegradable fibers, with release rates tailored by the fiber composition and structure. Viral particles can be encapsulated within fibers produced by electrospinning and other methods. These fibers may provide a high surface area and can be engineered to degrade over targeted timeframes, releasing cell-interacting factors such as viral particles. Combining different polymers or incorporating nanoparticles into the fibers can further refine the release profiles and enhance the stability of the factors such as viral particles. Other types of nanoparticles, such as silica nanoparticles, and protein-based matrices, such as those formed from gelatin, collagen, and silk, can adsorb factors such as viral particles onto their surface or encapsulate them within their porous structure, providing protection and a controlled release mechanism. Each controlled-release mechanism or encapsulation method may offer unique advantages and can be selected based on the specific requirements of the application, such as the targeted release profile, the stability of the factor(s), and the environmental conditions of the cell processing system. These strategies may permit precise control over the spatial and temporal availability of bioactive factors, enhancing the effectiveness of cell processing systems.

[0244] In some embodiments, elastin-like polypeptides (ELPs) may be used for theimmobilization, controlled release, or both of cell-interacting factors within a 3D substrate. ELPs may comprise repeating pentapeptide sequences (Val-Pro-Gly-Xaa-Gly, where Xaa can be any amino acid except proline) that exhibit Lower Critical Solution Temperature (LCST) behavior. Below the LCST, ELPs may be soluble in water. Above the LCST ELPs mayWSGR Docket No.67566-702601 undergo a transition to become insoluble and aggregated in water. This property can be harnessed to control the solubility and localization of ELP-tagged proteins within 3D matrices. By adjusting the environmental temperature to just above the LCST, ELPs can be induced to form aggregates, effectively capturing and immobilizing the linked proteins within the matrix. ELPs can be chemically modified to include reactive groups (such as acrylates). Through copolymerization or crosslinking reactions, ELPs can be integrated into a 3D matrix structure. This integration provides that when the ELP undergoes a phase transition, the linked protein(s) may be retained within the matrix, allowing for controlled spatial distribution. Proteins of interest can be genetically fused to ELP sequences. This fusion may permit proteins to inherit the phase transition properties of the ELP, providing responsive and reversible immobilization within 3D matrices. ELPs can be used to encapsulate proteins within microspheres or nanoparticles that are then embedded in a 3D substrate. Upon temperature change, these particles undergo a transition that can control the release rate of the encapsulated proteins. ELPs can also contribute to or increase the mechanical reinforcement of hydrogels. By behaving somewhat like natural elastin, they can impart elasticity and durability to a 3D matrix, which may be beneficial for creating scaffolds that mimic a natural extracellular matrix.

[0245] The linkages between or spatial colocalization of the cell-interacting factor(s) anda 3D matrix may be formed external to a cell processing system or on demand within the cell processing system. In some embodiments, a pre-functionalized 3D substrate is provided, such as a functionalized porous solid support material contained within a suitable consumable apparatus. In some embodiments, a 3D matrix precursor solution, which comprises polymers functionalized with cell-interacting factor(s), is provided, optionally mixed with a population of cells and allowed to gel, forming a functionalized cell-interacting 3D substrate. In some embodiments, a stock 3D matrix precursor solution comprising polymer(s) bearing attachment moieties may be provided such that upon mixing with one or more additional reagent(s) (e.g., reagents including cell-interacting factors), a functionalized cell-interacting 3D matrix or functionalized cell-interacting 3D matrix precursor solution (e.g., which upon gelation forms a functionalized cell-interacting 3D matrix) is formed. In some embodiments, a 3D substrate bearing attachment moieties or a 3D matrix precursor solution comprising attachment moieties may be provided and subsequently an aqueous reagent comprising at least one cell-interacting factor is provided and brought into contact with the 3D substrate, thereby forming linkages between the 3D substrate and the cell-interacting factor(s).WSGR Docket No.67566-702601

[0246] In some embodiments, a 3D matrix precursor solution comprising a controlledrelease formulation of cell-interacting factor(s) is provided. The 3D matrix precursor solution may be mixed with cells and permitted to gel, thereby forming a functionalized 3D substrate with controlled-release of cell-interacting factors. In some embodiments, a stock 3D matrix precursor solution is provided, and separately, a controlled release formulation of cell- interacting factor(s) is provided, such that upon mixing and gelation, a 3D substrate comprising controlled-release of cell-interacting factors is generated. In some embodiments, a 3D substrate, or a 3D matrix precursor solution is provided and subsequently an aqueous reagent comprising at least one controlled-release formulation of cell-interacting factor(s) is provided and brought into contact with the 3D substrate, thereby forming a 3D substrate comprising controlled-release cell-interacting factor(s).

[0247] In some embodiments, a functional aspect of a 3D substrate, such as a linkagebetween the 3D substrate and a cell-interacting factor, may be reversed, removed, cleaved, or any combination thereof. Linkages may be generated from any reversible conjugation or 3D deposition method, or reversible controlled-release formulation. These reversible linkages may respond to specific stimuli (e.g., chemical, thermal, physical, enzymatic, etc.) or incorporate mechanisms for competitive displacement. Cleavable linkers may be stable until exposed to certain internal or external stimuli, after which they cleave and release the conjugated molecule. Stimuli responsive linkers may include, but are not limited to, pH- sensitive linkers (e.g., hydrazone), enzyme-responsive linkers (e.g., enzyme cleavable peptide linkers), redox-responsive linkers (e.g., linkers comprising disulfide bonds configured to cleave in a reducing environment), and photo-cleavable linkers (e.g., O-Nitrobenzyl derivatives which cleave upon UV or near-UV light exposure). In an example, the linker is hydrazone which may be stable at physiological pH and cleaves in acidic environments. In another example, the linker is a peptide linker configured to be cleaved by matrix metalloproteinases (MMPs). Alternatively, a competitive displacement mechanism may be used in which binding of a bioconjugated molecule may be reversed by introducing a competitive molecule that has a higher affinity for the binding partner to displace the first molecule. For example, a cell-laden 3D matrix functionalized with one or more cell- interacting factors may be provided. Contacting the cell-laden 3D matrix with a reagent or other stimulus which reverses a linkage between the cell-interacting factors and the matrix may releases the factor(s) from the 3D matrix to remove the factors from the 3D microenvironment. Released factors may be removed from the 3D microenvironment by washing, diffusion, fluid flow, or any combination thereof.WSGR Docket No.67566-702601

[0248] In some embodiments, a combination of conjugation and cleavage chemistries areused to temporally program signaling cascades using a 3D matrix to deliver cellular cues with specified timing, such as for a specified duration. These capabilities can be used to precisely control complex stimulatory, co-stimulatory, pro-survival, and pro-phenotype cellular cues to provide biological controls over cellular processes and products. For example, a cell-laden 3D matrix substrate comprising one or more functionalization moieties is provided. An aqueous reagent comprising one or more component(s) may be introduced into and brought into contact with the 3D matrix such that the functional component is incorporated into or onto the matrix via the functionalization moieties (e.g., by binding, chemical bonding, ionic bonding, electrostatic interaction, etc.). The 3D substrate may be contacted with a second reagent or other stimulus which reverses the linkage between the first component(s) and the matrix functionalization moiety. Reversing the linkage may release the first component(s) such that it may be removed from the 3D microenvironment via washing, diffusion, fluid flow, or any combination thereof. A third reagent comprising one or more functional component(s) may be introduced into and brought into contact with the 3D substrate such that the functional component is incorporated into or onto the 3D matrix via the first or a second functionalization moiety. Combination or combinatorial functionalization may provide dynamic or iterative temporal control and programming of the of 3D microenvironment. Dynamic or iterative temporal control may permit the use of multiplexed or concurrent cellular signaling workflows. The use of two or more orthogonal functionalization moieties or chemistries further permits temporally dynamic and controllable programs for cellular membrane signal transduction.

[0249] In some embodiments, the 3D substrate may be sterilized prior to use in cellularprocessing. Sterilizing the 3D substrate may reduce or prevent contamination during cell processing and may permit clinical-regulatory suitability of using 3D matrices in cell processing workflows.3D substrates may be sterilized before cell processing (e.g., cell culturing) using temperature (e.g., autoclave), gamma or germicidal UV irradiation, ethylene oxide exposure, ethanol treatment, dense carbon dioxide gas sterilization, other methods, or combinations thereof. For workflows including a gel-sol transition, the precursor solution(s) may be sterilized before matrix formation. Sterilization of the precursor may include, but is not limited to, filtering (e.g., of lower viscosity solutions), gamma or germicidal UV irradiation of the solution or dry polymer, temperature (e.g., autoclave), or any combination thereof. The sterilization method used may be selected based on compatibility and cell processing application. For example, sterilization methods that do not degrade, denature, orWSGR Docket No.67566-702601 otherwise alter the physical and biological properties of the 3D substrate material(s) may be used.3D substrate materials used in the manufacture of clinical products, such as for human use, may be subject to rigorous safety and performance standards. For example, a 3D substrate material may be selected for biocompatibility properties or capacity for facile or substantially complete removal (e.g., within an acceptance criteria) from a formulated product. A 3D substrate material which may carry over into a formulated product may be selected from a group of materials which does not evoke harmful immune reactions, cause toxicity, or lead to infection. Alternatively, a 3D substrate material may be selected or designed based on its capacity for facile or substantially complete removal from a formulated product. A 3D substrate material may be selected from a group of materials which do not or substantially do not carry over to product formulation. For example, the 3D substrate material may be removed during cell washing. A 3D substrate material may be selected for nonspecific interactions with cellular product. For example, materials may be selected that have little or no nonspecific interactions with the cellular product to reduce carry over of the material into the product. A 3D substrate material may be selected for the ability to remove the material from the formulated product. For example, the material may be easily washed away, degraded, or both to reach an acceptable level within the cell product. A 3D substrate may be selected or manufactured to minimize the presence of endotoxins, which can cause severe inflammatory responses, and may be subject to regulated limits in formulated clinical products. A 3D substrate material may be selected from a group of materials which can be manufactured consistently to provide uniformity in produce attributed or clinical outcomes. Uniformity may be subject to regulatory approval. The specific for these attributes can vary based on the intended application and regulatory guidelines. Reversible gel-sol 3D substrates

[0250] In some embodiments, a 3D substrate is formed using a matrix composition whichundergoes stimulus-responsive or reversible gel-sol transition. For example, a stimuli- responsive hydrogel may be used which responds to physical or biochemical stimuli. Physical or biochemical stimuli may include, but are not limited to, temperature, light, electromagnetic fields, acoustic waves, pH, ions, crosslinkers, other reagents, or any combination thereof.

[0251] Thermoresponsive 3D matrices may exhibit changes in their physical state orproperties in response to temperature changes. This behavior may be governed by critical temperatures that define transitions in a 3D matrix’s solubility and structure. The 3D matrix may exhibit a first phase at temperatures below the Lower Critical Solution TemperatureWSGR Docket No.67566-702601 (LCST) and a second phase at temperatures above the Upper Critical Solution Temperature (UCST). LCST may be the temperature below which a polymer is soluble in a given solvent and above which it becomes insoluble. When the temperature is raised above the LCST, hydrophilic-hydrophobic balance may shift towards hydrophobicity due to changes in polymer-solvent interactions. The shift towards hydrophobicity may cause the polymer to aggregate and precipitate out of solution or form a gel. This behavior often results from an increase in hydrophobic interactions within the polymer chains that overcomes the polymer- solvent interactions as temperature rises. For polymers like Poly(N-isopropylacrylamide) (PNIPAM), the hydrogen bonding with water that maintains solubility at lower temperatures is disrupted at higher temperatures, leading to phase separation. Such materials may exhibit reversible phase transitions to permit re-solubilization of the matrix substrate upon reduction in temperature. This temperature-controlled gel-sol transition may permit facile harvesting of cell products. Polymers which may undergo thermally responsive gel-sol transition include, but are not limited to, gelatin, collagen, cellulose, chitosan, starch, carrageenan, hyaluronic acid, xantham, xyloglucan, elastin, dextran, or combinations thereof. Additional chemical modifications may alter the thermal responsiveness of polymeric hydrogel materials. For example, methacrylated collagen may be capable of reversible thermal gel-sol transitions where the thermal gelation of natural collagen may be thermally irreversible.

[0252] Other classes of stimulus-responsive 3D substrate materials which may be usedinclude materials that are pH-responsive, chemical-responsive, enzyme-responsive, electro- responsive, photo-responsive, or combinations thereof. pH responsive polymer materials may comprise polymers with pendant acidic or basic groups which either accept or donate protons in response to changes in pH, resulting in gel-sol transition. Chemically responsive polymer materials may use reversible chemical bonds to mediate gel-sol transition. Examples of materials and mechanisms with reversible chemical bonds include but are not limited to: those which feature disulfide bonds, such as in the PEGSSDA crosslinker; ionic condensation of alginate, which is crosslinked in the presence of cations such as Ca2+and reversed by chelators such as EDTA; and reversible chemical bonds that may be broken by introduction of a competitive molecular species, e.g. FLAG-tag-based interactions which may be broken by introduction of competing FLAG peptides. Enzyme-responsive materials, such as those composed of naturally derived materials such as collagen (collagenase), fibrin (nattokinase), and HA (hyaluronidase) may permit gel-sol transition and cell liberation by degradation of the hydrogel matrix in the presence of, or upon addition of, or under the activity of, enzymes in a suitable buffer and reaction environment. When using enzyme-responsive materials, careWSGR Docket No.67566-702601 may be taken not to disrupt cell surface receptors through extended enzyme treatment. Use of a specific enzyme, such as collagenase, may provide benefits over non-specific proteases. Electro-sensitive hydrogels may be responsive to electric fields, which affect changes in their internal polymeric binding, structure, or both. For example, polyelectrolyte polymer materials may contain ionizable groups in the polymer backbone or side chains. Examples include synthetic polymers such as polyvinyl alcohol, polypyrrole, polyaniline, polythiophene, acrylic acid / vinyl sulfonic acid, and sulfonated polystyrene. Light-sensitive polymer materials may respond to particular wavelengths of light. For example, a gel matrix comprising deoxycholic acid-modified b-cyclodextrin derivative and an azobenzene- branched poly(-acrylic acid) copolymer may convert from gel to solution phase upon exposure to a first light (e.g., 355 nanometer (nm) wavelength light) and transition from solution phase to gel upon exposure to a second light (e.g., 450 nm light) irradiation. As described herein, other embodiments of 3D matrix substrates do not undergo gel-sol transition, such as in the case of a static or pre-formed matrix, such as a porous solid matrix.

[0253] In some embodiments, a liquid 3D matrix precursor solution is provided, mixedwith cells, and subjected to gelation (e.g., via application of a stimulus) to form a 3D matrix. For example, a precursor solution, such as of a PEG-PNIPAM polymer, or of a collagen or collagen-based polymer, may be provided, which is in a liquid phase at 4°C, but undergoes a gel-sol transition when raised to 37°C, a suitable temperature for cell processing. In another example, a precursor solution, such as of an alginate or alginate-based polymer, may be provided, which is in a liquid phase until mixed with a suitable cation source (e.g., Ca2+, Br2+, or Sr2+salt solution). The precursor solution may be mixed with cells and loaded into a cell processing compartment (e.g., compartment of a bioreactor). A salt solution may be added to the compartment resulting in gelation of the 3D matrix. Subsequently, a stimulus may be provided which reverses the gel-sol transition. For example, the compartment temperature may be lowered to 4°C, resulting in gel-sol transition of thermally-responsive hydrogel matrices, or a ion chelator such as EDTA may be introduced to an alginate-based chemically- responsive hydrogel matrix, resulting in gel-sol transition. The liquid comprising the solubilized gel matrix material and cells may be harvested, such as by removing the liquid mixture from the compartment and washing or otherwise isolating the cells for downstream use as a liquid suspension. In some embodiments, a 3D substrate comprising a stimulus- responsive gel-sol transition property is provided, and cells may be seeded onto or into the 3D matrix. Stimulus may be provided which reverses the gel-sol transition facilitating the harvest of cells from the solubilized gel matrix material.WSGR Docket No.67566-702601 Alternate 3D substrate configurations

[0254] In some embodiments, a 3D substrate or cell-matrix may not comprise amacroscale monolithic matrix composition. For example, the 3D substrate may comprise other configurations, including liquid-like solids, colloidal solutions, packed bed, and dense suspensions (e.g., colloidal mixture configurations), which may also provide a matrix- or 3D- like environment and function at least in part in retaining cells. In some embodiments, the 3D substrate resembles a colloidal mixture, in which the matrix substrate comprises suspended, dispersed insoluble particles, including porous particles such as beads, gel beads, or gels, and ribbon-like and fibrous particles. Such configurations, despite not forming a monolithic hydrogel or monolithic 3D matrix, may exhibit hydrogel or matrix-like properties when densely suspended in a fluid medium such as cell culture medium. In some embodiments, a colloidal formulation of 3D substrates may be suspended within a porous matrix, such as a hydrogel, effectively forming an interpenetrating network of 3D matrix substrates supporting cell processing. The compositions of matter, chemistries, and methods described herein relating to 3D matrices, programmable substrates, membrane signal transduction, biological properties, reversible gel-sol, and other features described elsewhere herein may be utilized in the described non-monolithic matrix configurations.

[0255] In some embodiments, a solid-state bioreactor or cell processing system may beconfigured with a cell compartment which does not comprise a monolithic 3D substrate, such as a monolithic porous solid scaffold or a monolithic 3D matrix such as a hydrogel. In some embodiments, a solid-state bioreactor or cell processing system may be configured to permit maintenance of a cell compartment and optionally a perfusion compartment. For example, a 3D substrate may comprise a dense colloidal solution of particles, ribbons, or other substrates that may support cell culture and provide a cell processing environment similar to the systems and methods described elsewhere herein. For example, a system comprising a non- monolithic substrate may further comprise integrated sensing, perfusion flow, in situ genetic engineering, or any combination thereof. Such a configuration may make use of a perfusion flow configuration which does not substantially disturb or suspend the 3D substrate environment, such as a low flow rate or a flow configuration which applies minimal disruptive fluidic forces (e.g., shear) to the 3D substrate. A solid-state bioreactor or cell processing system may be configured with a semi-permeable membrane interface between the cell and perfusion or fluid flow compartments, which may retain or substantially retain both the cells and 3D substrate in the cell compartment (e.g., a pore or filtration size that, for example, neither microgels or fibers forming a 3D substrate nor the cells can pass through).WSGR Docket No.67566-702601 In such a configuration, nutrient supply and other reagents may be primarily delivered via diffusion from the perfusion compartment, by secondary flows directed through a cell compartment, or both. In such a configuration, nutrient supply and other reagents may be provided via other solid-state bioreactor configurations providing described herein to provide nutrients and other materials to the 3D substrate, such as those using vessels including flasks and bags.

[0256] A 3D matrix may be in contact with fluid within the fluid flow path or may beseparated from the fluid flow path, as shown in FIG.13. The 3D matrix may be separated from the fluid flow pathway by a membrane. The membrane may be porous such that reagents and waste may diffuse through the membrane. Alternatively, the interface between the 3D matrix and the fluid flow path may be a liquid interface such that the fluid flowing in the fluid flow path directly contacts the 3D matrix. In another example, the interface between the fluid flow path and the 3D matrix may be a thin gel layer. The thin gel layer may be a hydrogel. The thin gel interface may be configured such that reagents and waste may diffuse through the thin gel to or from the fluid. As described herein, the 3D matrix may be a stimulus-responsive 3D matrix. The 3D matrix may be configured to retain a variety of cellular substrates. For example, cells may be directly embedded within the 3D matrix. In another example, cells may be disposed in or on two dimensional (2D) carriers (e.g., ribbons), 3D carriers (e.g., gel beads), 2D beads (e.g., magnetic beads), microporous scaffolds (e.g., polystyrene mesh), or any combination thereof. Carriers or scaffolds comprising cells may then be encapsulated or embedded within the 3D matrix. In some examples, carriers or scaffolds comprising cells may be disposed in a solution and not encapsulated or embedded within a 3D matrix. In such examples, the carriers or scaffolds may be retained within the cell compartment by a membrane disposed between the cell compartment and the fluid flow pathway. Cell loading

[0257] Various systems and methods may be used to provide components into a cellcompartment or 3D matrix within a cell compartment. Systems and methods may provide that cells are localized or substantially localized in the 3D substrate, cell compartment, or both. Alternatively, cells may be localized or substantially localized within a region or area of a bioreactor or cell processing system in fluid communication with cell culture medium or perfusion flow. In some embodiments, one or more liquid 3D substrate precursor(s) are provided, mixed with one or more cell source(s), introduced to the solid-state bioreactor or a compartment thereof, and permitted to or directed to form a 3D substrate containing,WSGR Docket No.67566-702601 embedding, or encapsulating cells. For example, a solid-state bioreactor may be configured with, or configured to interface with a cell processing system with, a mixing vessel (which may function to mix cells with a liquid 3D matrix precursor solution and, in some examples, other reagents continuously or in batches). In another example, a solid-state bioreactor may be configured with, or configured to interface with a cell processing system with, a mixing module in a flow configuration such that a 3D matrix solution(s), cells, and, in some examples, other reagents are brought together and mixed in a continuous or semi-continuous manner as they are transferred within or into a bioreactor, cell processing unit, or compartment thereof. Methods may provide that the cell-laden 3D matrix is localized or substantially localized to a region or compartment of a bioreactor, such as a region or compartment configured to be in fluid communication with a cell culture media or perfusion flow. In an example, a sacrificial material, such as 3D matrix material not containing cells, another liquid, or a gas, may be introduced into the fluidic system and used to push the cell- laden matrix volume into a targeted position within the bioreactor. In another example, a negative pressure may be applied to pull the cell-laden matrix volume into a targeted position within the bioreactor (e.g., as shown in FIG.15). As another example, an extrusion, deposition, or printing method may be used to place a cell-laden matrix volume in a targeted position within the bioreactor. These and other methods may be used in any combination. The solid-state bioreactor may be configured with fluidic paths and systems which minimize dead volumes of liquid cell-matrix mixtures.

[0258] Cells may be loaded into or onto a 3D substrate using any retention,encapsulation, or adhesion methods. In an example, a plurality of cells is encapsulated or entrapped within the 3D matrix. In an example, the 3D matrix or substrate is formed in presence of the plurality of cells to generate a 3D matrix encapsuling the plurality of cells. The 3D matrix encapsulating the plurality of cells may be generated internal to or otherwise within a compartment (e.g., cell compartment) as described elsewhere herein. Alternatively, the 3D matrix encapsulating or entrapping the plurality of cells may be generated external to the compartment and, subsequently, loaded into the cell compartment. In an example, the cell processing unit may be configured to receive a pre-formed 3D matrix with or within the plurality of cells. In an example, the plurality of cells may be mixed with matrix precursors in a solution and the solution may be provided to a compartment of the cell processing system or bioreactor and polymerized in place. Polymerizing the 3D matrix in place may comprise applying a stimulus to the matrix or polymer precursors. In an example, the stimulus may be applied to the matrix or polymer precursor in a compartment of the cell processing system orWSGR Docket No.67566-702601 bioreactor to generate the 3D matrix encapsulating the plurality of cells in situ. As described elsewhere herein, the stimulus may be a thermal stimulus, chemical stimulus, enzymatic stimulus, or any combination thereof. In another example, the 3D matrix may be generated in absence of a plurality of cells, either internal or external to a compartment, and the plurality of cells may be added to the pre-formed 3D matrix such that the cells adhere to the 3D matrix, migrate into the 3D substrate, or both.

[0259] In some embodiments, cells may be loaded or otherwise deposited into apreformed 3D cell culture substrate. For example, a cell source in suspension may be introduced to the bioreactor comprising a 3D substrate such that cells are passively deposited, or permitted to or directed to, actively penetrate the 3D substrate to become substantially retained in the 3D matrix. In another example, a solid-state bioreactor may be configured with a 3D substrate cell compartment and perfusion compartment oriented such that cells introduced to the perfusion compartment, as through a fluid flow, settle onto and penetrate into the 3D substrate by gravity. In some embodiments, the 3D substrate component of the solid-state bioreactor or cell processing system may be configured to entrap cells within the 3D matrix environment. For example, a 3D substrate may be provided in the solid-state bioreactor apparatus which is configured with a matrix environment promoting cellular retention or cellular migration into the 3D substrate. In some embodiments, the 3D substrate is functionalized (e.g., statically or programmatically) with factors that promote cellular interactions and such factors may promote the specific retention of cells in the matrix, or the specific migration of cells into the matrix where they are substantially retained. In some embodiments, substrate functionalization may be configured to promote retention or migration of cells of a specific type or nature. For example, a 3D substrate of a solid-state bioreactor may be configured with a 3D microenvironment that attracts or retains cells of a specific type due to the nature of the 3D microenvironment. In another example, a 3D substrate of a solid-state bioreactor may be configured with a 3D microenvironment that supports the viable culture of a specific type of cell, such that other types of cells are unable to maintain a viable cell state or proliferate. In some embodiments, a solid-state bioreactor may be configured with a stimulus-responsive 3D substrate capable of attracting or retaining cells upon a stimulus which modulates one or more properties of the 3D substrate. For example, the solid-state bioreactor may be configured with a stimulus-responsive 3D matrix which modulates the porosity of the substrate. Subsequent to bringing a cell suspension in contact with the 3D substrate in a highly porous configuration, such as by the addition or seeding of cells, a stimulus (e.g., a crosslinker) may be applied which causes a reduction inWSGR Docket No.67566-702601 porosity of the 3D substrate, thereby causing the cells to become entrapped or substantially retained within the substrate.

[0260] Cells may be loaded into a pre-formed matrix or, alternatively, a matrix may begenerated in presence of a plurality of cells, as shown in FIG.14. Cells may be loaded into a pre-formed matrix by flowing cells into a compartment comprising a pre-formed 3D matrix. One side of the compartment may comprise a membrane or other interface configured to retain the 3D matrix. The cells may adhere to the 3D matrix and migrate into and proliferate within the 3D matrix. The cells may settle into the 3D matrix due to gravity. In another example, the cells may be mixed with polymer or gel precursors. The polymer or gel precursors may be stimuli responsive such that the 3D matrix is formed or degraded by the application of a stimulus (e.g., thermal, chemical, or enzyme stimulus). The 3D matrix may be a thermoresponsive matrix that is a liquid or semi-liquid at low temperatures and solid at higher temperatures. The cells may be embedded or encapsulated in the matrix upon formation of the matrix.

[0261] The 3D matrix may be generated in a cell compartment. Alternatively, as shownin FIG.15, the 3D matrix may be pre-formed and loaded into a cell compartment. A preformed 3D matrix may be loaded via a pressure differential or application of a mechanical force. In some examples, the cell compartment includes a sacrificial 3D matrix that is pushed out via the non-sacrificial 3D matrix or pulled out via application of negative pressure.

[0262] In some embodiments, a solid-state bioreactor comprises a plurality of hollowfibers within a casing and cell-laden 3D substrate is substantially localized to the region of the intracapillary space (IC) in fluid communication with the extracapillary space (EC) of the casing for perfusion. In some embodiments, a solid-state bioreactor may be configured with a hollow fiber structure with significantly reduced head space and fluidic system dead volume compared to existing hollow fiber bioreactors. In some embodiments, a solid-state bioreactor configured with a hollow fiber design may be configured with features that permit localizing the cell-matrix substantially into the fibers (and not in the head space and dead volume), such as those described above. Alternatively, a hollow fiber configuration that provides a substantially spatially uniform, defined, or adequate distribution of hollow fibers within a casing may permit loading the EC with a cell-laden 3D substrate that is in substantially uniform fluid communication with perfused media in the IC. Such a configuration may also utilize modified head space, dead volume, or loading procedure to maximally localize the cell-matrix to the perfused compartment of the EC. Cell culture and expansionWSGR Docket No.67566-702601

[0263] In some embodiments, a solid-state bioreactor is configured to culture cells toproduce cellular biomass, cellular products, or both. In an example, the cell product is a cell therapy product. Cellular biomass growth can be divided into several distinct phases. Biomass growth can comprise an initial lag phase, characterized by a low growth rate while cells are adapting to a new environment inside the bioreactor. During an exponential growth (or log) phase, cell division may continue at a constant or near constant rate. As nutrients and free space are depleted and by-products accumulate, the rate of cellular biomass growth mayslow, and the culture may enter a stationary phase or cellular biomass may decline. Theduration of each phase may be dependent on a number of factors including the initial seeding density, expansion or proliferation rate, culture media, cell type, or other bioreactor and cellular factors. A solid-state bioreactor may be usable for various cell culture methods. In some embodiments, a perfusion method that minimizes environmental variation throughout biomass growth and provides a targeted cellular environment may be used. For example, in a continuous perfusion configuration, fresh media is provided and spent medium is removed continuously, at specific or variable rate to maintain a fixed total volume in the perfusion fluidic system. Alternatively, the total volume of the fluidic system may be variable, for example, by using a fluid reservoir within the fluidic system that can accommodate variable volumes. In some embodiments, a solid-state bioreactor is configured to perform a static batch culture process such that the bioreactor is loaded with a fixed working volume of culture media used for the duration of the culture period, and media is not replaced during the period of cell incubation. While such a configuration may provide cell culture for a specified duration or may produce lower cell densities and production yields compared to batch-fed and perfusion processes, such a configuration may be suitable for certain applications including short-term culture during which other cell bioprocessing operations are used. For example, a solid-state bioreactor may be provided in a batch static configuration for a rapid cell therapy production process (e.g., T-Charge, FasT-CAR), in which cells are not substantially expanded, or are not permitted to substantially proliferate during manufacturing, but instead are maintained in a viable state during cell selection, activation, gene engineering, and other bioprocess operations. In such a configuration, a perfusion compartment may not exhibit flow (e.g., is configured with a static media volume). In such a configuration, the bioreactor may be further configured with a gas-exchange capability (e.g., via using a gas- permeable membrane as described elsewhere herein), such as to continuously supply oxygen to the static media. In an example, a solid-state bioreactor may be configured with a perfusion compartment that provides fluid flow, such as for the purpose of recirculation or gasWSGR Docket No.67566-702601 exchange without providing fresh media to the volume of cell media in the perfusion fluidic compartment or the fluidic system of the perfusion compartment. In some embodiments, a solid-state bioreactor is configured to perform a batch-fed or fed-batch culture process such that fresh media is provided during bioreactor operation to replace a volume of spent media or to dilute the spent media with fresh media in a non-continuous manner. For example, in a batch-fed configuration, an equal volume of spent media may be removed from the perfusion fluidic system to each volume of fresh media introduced to the perfusion fluidic system, such that the perfusion fluidic system maintains a constant total volume up to the working volume of the perfusion system (e.g., substantially all the media may be replaced during each feeding). In a fed-batch configuration, fresh media may be added non-continuously or continuously over time, but spent media may not be removed, increasing the total volume of the perfusion fluidic system over time. Such a configuration may be permitted by using a reservoir in line with the perfusion fluidic system, including fixed-volume and deformable reservoir vessel types, which may be configured to hold a variable amount of culture media or other fluid. For example, a solid-state bioreactor configured with a fixed volume fluid reservoir may hold a variable amount of liquid over time and increases or decreases in fluid volume may be accommodated by the displacement of gas (e.g., air). In another example, such a configuration may be permitted by the use of a deformable vessel within the solid- state bioreactor, such as a perfusion compartment capable of expanding upon addition of fluid (e.g., in an accordion configuration). Washing and buffer exchange

[0264] In some embodiments, a solid-state bioreactor or cell processing system isconfigured to perform fluid exchange operations, such as to substantially replace a first fluid within the bioreactor cell processing unit or compartment thereof with a second fluid. For example, a fluid exchange operation may be used to substantially replace a first interstitial fluid within a 3D substrate with a second fluid. Such an operation may provide buffer exchange or to wash cells retained within the bioreactor. During cell processing media exchange may be frequently performed to change or substantially change a media condition (e.g., to replace the media with an electroporation buffer suitable for in situ electroporation), or to substantially remove a factor, such as an activation reagent or viral particles. Fluid exchange may purge or replace the perfusion media or fluid present in the 3D substrate and cell compartment. Fluid exchange may be performed iteratively to substantially exchange a first liquid from a cell compartment as it is diluted by diffusive mixing with the second liquid provided in a perfusion compartment. In configurations with two or more perfusionWSGR Docket No.67566-702601 compartments in fluid communication with a cell compartment, differences in pressure or flow rate between the two or more compartments can be used to accelerate wash-out and reagent exchange of the cell compartment and 3D substrate. For example, flow rate or fluid pressure in one fluid flow path may be less than or significantly less than flow rate or fluid pressure in another fluid flow path such that fluid from the higher flow rate or fluid pressure is driven through the cell compartment or 3D matrix to provide fluid exchange.

[0265] In some embodiments, a solid-state bioreactor is configured to provide one ormore methods for retaining cells within the bioreactor to permit efficient cell washing and buffer exchange in the presence and absence of the 3D substrate. For example, the 3D matrix may provide cell retention. Alternatively, or in addition to, the solid-state bioreactor may comprise one or more membranes, cell trapping features, or other cell capture mechanisms to retain the cells in absence of the 3D substrate (e.g., before formation or after dissolution or disintegration). For example, a solid-state bioreactor or cell processing system may be configured with a 3D substrate which provides for cell retention during operation, and one or more semi-permeable membrane interface(s) between a 3D substrate (cell compartment) and one or more media or perfusion compartment(s). During solid-state cell culture, the 3D substrate may function to retain cells and provide a 3D environment for cell culture and growth, while the semi-permeable membrane interface functions to retain the 3D substrate, and, in some cases, to provide for additional cell retention, such as by use of a membrane with a filter size cutoff unsuitable for cells to pass through. In some embodiments, a 3D substrate may be configured to provide a reversible gel-sol transition in response to a stimulus such that cells may be washed in situ within a solid-state bioreactor in such a configuration. For example, upon a gel-sol stimulus, such as a change in temperature, addition of a chemical factor or other reagent, or other stimulus of gel-sol transition, the 3D substrate may transition from a substantially solid or semi-solid 3D substrate into a substantially liquid or solubilized state; upon such a 3D substrate gel-sol transition, cells contained within the 3D substrate may become suspension-state surrounded and suspend in the liquified or solubilized matrix. For hybrid 3D substrates, such as those which comprise a porous solid scaffold interpenetrated with a hydrogel scaffold onto which cells attach or with which the cells primarily interact and grow, a stimulus-responsive gel-sol transition may affect a subset of the 3D substrate while providing a cell culture transition, for example, from 3D culture to effectively suspension culture within the porous solid. Subsequently, buffers or media such as wash buffers or cryopreservation buffers may be pumped into and throughout the bioreactor or cell processing system, as described herein, while the semi-permeableWSGR Docket No.67566-702601 membrane acts to retain the cells in the same volume, space, or compartment such that cells may be in a suspension in the liquified 3D matrix or otherwise not attached to residual solid 3D substrate after such gel-sol transition. A solid-state bioreactor in such a configuration may permit in situ washing or buffer exchange. Washed or buffer exchanged cells may subsequently be collected from the cell compartment, such as through a dedicated port connected to the cell compartment, or by subsequently moving, removing, or degrading the membrane or interface between the cell and other compartment(s). In some embodiments, a solid-state bioreactor or cell processing system may be configured to perform an in situ clinical formulation operation using one or more in situ washing or buffer exchange procedures to suspend the cells in a suitable formulation buffer for harvest and downstream clinical use. Cell harvesting

[0266] In some embodiments, a solid-state bioreactor is configured to perform a cellharvest operation. In some embodiments, cells are harvested from the solid-state bioreactor at a fixed time, on demand, or once they reach a target number, concentration, phenotype, or other relevant metric, such as provided by one or more sensor(s) and feedback systems within the bioreactor or bioprocessing system as described elsewhere herein. The 3D matrix may be dissolved, and the plurality of cells, or population thereof, may be collected to generate the cell product. In some embodiments, a solid-state bioreactor is configured with a stimulus- responsive gel-sol 3D substrate which facilitates cell harvest. The stimulus may be a thermal stimulus, chemical stimulus, enzymatic stimulus, or any combination thereof. In an example, application of a single stimulus may permit the plurality of cells to be released from the 3D matrix. In another example, application of at least two stimuli permits the plurality of cells to be released from the 3D matrix. Application of a single stimulus may release a select amount of cells and application of a second, different stimulus may release another amount of cells from the 3D matrix. By inducing a phase transition of the 3D substrate from solid-phase or semi-solid phase to liquid-phase (in whole or in part), such as through the application of a stimulus, cells may be harvested by collecting suspension-phase cells or cells suspended in the solubilized matrix. For example, a solid-state bioreactor may be configured to culture cells in a thermally-responsive reversible gel-sol material, such as methacrylated collagen or a polyethylene glycol (PEG)-Poly(N-isopropylacrylamide) (PNIPAAm) block copolymer. Upon lowering the temperature of the bioreactor or cell compartment, the 3D substrate may undergo a phase transition to a solubilized or liquid state and cells previously attached to the 3D substrate may be transitioned to suspension-phase. The liquified substrate containing theWSGR Docket No.67566-702601 cells may be harvested from the bioreactor, such as by using fluid flow. In some embodiments, an permeable or semi-permeable interface may be disposed adjacent to the cell compartment or perfusion compartment and the washing and buffer exchange methods described elsewhere herein may be used to partially or substantially remove the liquid matrix components from the cells while the cells are retained in the compartment, e.g., to facilitate cell harvest by reducing viscosity, or to facilitate the exchange of cells from the matrix reagent into a buffer suitable for downstream processing, including but not limited to cryopreservation buffers and formulation buffers.

[0267] In some embodiments, the solid-state bioreactor or cell processing system isconfigured to enable the harvest of cells directly from a 3D substrate. For example, fluid flow in or through the cell compartment may be configured to remove cells from the 3D substrate, such as by applying fluid force(s) capable of disrupting cell interactions with the 3D substrate to wash cells out of and from the 3D substrate. In some embodiments, the solid-state bioreactor or cell processing system may be configured with a programmable or stimulus- responsive 3D substrate which facilitates cell harvest. For example, by using a 3D substrate with the property of stimulus-responsive modulation of mass transfer properties, such as porosity or swelling, the harvest of cells being cultured within and retained by a 3D substrate may be facilitated by a stimulus which causes the 3D substrate to swell or increase porosity, such that cells are no longer substantially retained by the 3D substrate or can be removed by fluid flow. In an example, by using a stimuli-responsive 3D substrate configured to modulate cell attachment or migration properties of the 3D matrix, the harvest of cells being cultured within and retained by a 3D substrate may be facilitated by a stimulus which causes the 3D substrate to interact differently with the cells, such that cells may no longer be retained or substantially retained by the 3D substrate or can be removed by fluid flow. In an example, a 3D matrix may comprise programmable cell attachment factors or cell migration factors, which are cleaved, blocked, competitively displaced, or otherwise neutralized by a stimulus, thereby causing a reduction in cell retention properties and facilitating the harvest of cells, such as by fluid flow. In another example, a 3D substrate may be programmably functionalized with factors that promote cellular egress from the matrix environment to facilitate harvest of cells, such as by cell migration or by application of fluid flow. In some embodiments using a stimulus-responsive 3D substrate to facilitate cell harvest, the chemical or biological mechanisms facilitating cell harvest may be directed at a subset of a plurality of cells. For example, in an embodiment utilizing the modulation of 3D substrate cell attachment factors to facilitate cell harvest, one or more of a plurality of cell attachmentWSGR Docket No.67566-702601 factors may be specifically modulated, such that the cell adhesion properties of the 3D substrate are modulated for a specific population of a plurality of cells to permit the harvest of a subset of a plurality of cells. A solid-state bioreactor may be configured with a 3D substrate comprising two or more cell attachment factor(s) directed at two or more populations of cells, such as feeder cell type, which may support the growth of one or more distinct cell type(s) in culture by contributing an undefined and complex mixture of soluble and extracellular matrix (ECM) components. Upon application of a stimulus which modulates the attachment of one of these classes of cells (e.g., the feeder cells or the other cell type(s)), a specific subset of cells may be harvested from the 3D substrate while the other subset(s) remain substantially retained within the 3D substrate. Such a configuration may provide benefits of streamlining cell harvest and formulation workflows or increasing the purity of harvested cells in co-culture applications.

[0268] In some embodiments, cells may be harvested by collecting the cell-laden 3Dsubstrate. In some embodiments, cells may be subsequently isolated from the 3D substrate, outside of the solid-state bioreactor. In other embodiments, one or more types of cells may be cultured in a 3D substrate in a manner and composition suitable for downstream use. In an example, one or more types of cells present in a natural tissue may be cultured in a solid-state bioreactor using a 3D substrate which resembles a natural tissue extracellular matrix, and which is otherwise biocompatible. Upon the harvest of the cultured tissue (e.g., the resulting cell-laden 3D substrate), such a composition may be used for a purpose of tissue replacement or regeneration. Examples of such applications include the growth of tissue grafts, such as skin, muscle, cartilage, brain tissue, lymph nodes, or other types of tissues and cell-matrix biological structures. In another example, animal muscle cells may be cultured in a 3D substrate suitable for human consumption using a solid-state bioreactor. Upon the harvest of the cultured tissue, such a composition may be used as a source of food.

[0269] In an example, a solid-state bioreactor may be configured to harvest cells duringoperation for the purpose of in-process monitoring outside of or separate from the solid-state bioreactor. In some embodiments, a solid-state bioreactor comprises one or more specific cell compartment(s) or cell processing units separate from and may not be in fluidic communication with one or more other compartments or cell processing units of the bioreactor, from which cells may be harvested without harvesting cells from the other compartment(s) or cell processing units, thereby enabling in-process monitoring without disrupting the manufacturing process. Cells harvested in-process through the use of a solid-WSGR Docket No.67566-702601 state bioreactor in such a configuration may be further analyzed within a bioprocessing system or collected and analyzed separately from the bioreactor and bioprocessing system.

[0270] In some embodiments, cells harvested from a 3D substrate may be subjected toone or more additional cell culture operations (e.g., cell passaging) within the solid-state bioreactor. Passaging may comprise harvesting cells from a culture and transferring the cells or a subset thereof to one or more culture vessel(s) under a different condition, such as at a new (e.g., lower or higher) cell concentration or density, or in fresh growth medium, thereby establishing new culture(s). A solid-state bioreactor apparatus and cell processing system may be configured to harvest cells from a solid-state bioreactor, and subsequently re-seed a new 3D cell culture within the solid-state bioreactor. In some embodiments, a solid-state bioreactor may be configured with a number of cell processing units, some of which are used in a first passage, and others which are used in a second or subsequent passage. In certain embodiments, a solid-state bioreactor may be configured with a cell processing unit that is reused in one or more passages. In some embodiments, a solid-state bioreactor may be configured to re-seed passaged cells in the first 3D substrate, or in a separate 3D substrate, such as by mixing the cells with a fresh liquid formulation of soluble 3D matrix material and subsequently forming a de novo 3D substrate. In some embodiments, the solid-state bioreactor is configured for a re-seeding or passaging procedure designed to extend the duration of 3D cell culture, increase the total capacity for cell expansion and proliferation, or perform multiple cell engineering or other bioprocessing processes in series. In some embodiments, a solid-state bioreactor may be configured to perform a Multiplex Automated Genome Engineering (MAGE) procedure, in which cells are cultured, engineered, harvested, and optionally selected or subsets of cells isolated, over two or more passages, such as for the purpose of executing a series of cell engineering procedures or iterative or multiplex genome engineering. For example, over two or more passages of 3D cell cultures using a solid-state bioreactor, cells may be exposed to multiple rounds of genome engineering, such as by using CRISPR-Cas9, for the purpose of developing or manufacturing highly engineered cell products. In an example, the product harvested from the cell processing system is a cell therapy product. In another example, the product harvested from the cell processing system is usable to generate a cell therapy product. The cell therapy product may be generated at a scale of one human dose. Cell processing systems and methods

[0271] In an aspect, the present disclosure provides systems for manufacturing a cellproduct. A system may include a compartment, or a plurality of compartments, configured toWSGR Docket No.67566-702601 retain or that retains a plurality of cells. The system may include a fluid flow path in fluid communication with the compartment. The fluid flow path may be configured to flow or may flow a fluid along a first direction having an angle with respect to a second direction of flow within the compartment. The angle may be greater than zero degrees. The fluid flow path may be further configured to provide or may provide material from a fluid to the compartment. The material from the fluid may be usable to generate a cell product. The system may include an analysis unit coupled to the compartment and the fluid flow path. The analysis unit may be disposed within the same housing as the compartment, the fluid flow path, or both the compartment and the fluid flow path. Alternatively, the analysis unit may be disposed in a separate housing than the compartment, fluid flow path, or both the compartment and fluid flow path. The analysis unit may be configured to monitor or may monitor the plurality of cells or the fluid as the materials are provided to the compartment during generation of the cell product.

[0272] In another aspect, the present disclosure provides systems for cell processing. Asystem may include a cell processing unit, a preservation unit, an analysis unit, or combinations thereof. The cell processing unit may be configured to retain and process or may retain and process a plurality of cells to generate a cell product. The preservation unit may be coupled to the cell processing unit. The preservation unit may be configured to preserve or may preserve the cell product. The analysis unit may be coupled to the cell processing unit, the preservation unit, or both. The analysis unit may be configured to detect or monitor or may detect or monitor one or more properties of the plurality of cells of the cell product.

[0273] In another aspect, the present disclosure provides methods of manufacturing a cellproduct. A method may include providing a plurality of cells to a system. The system may include a compartment or a plurality of compartments that retain the plurality of cells. The system may include a fluid flow path in fluid communication with the compartment. The system may include an analysis unit coupled to the compartment and the fluid flow path. The analysis unit may be disposed in the same housing as the compartment, the fluid flow path, or both the compartment and the fluid flow path. Alternatively, the analysis unit may be disposed in a different housing than the compartment, the fluid flow path, or both. The method may include providing material from a fluid in the fluid flow path to the compartment to generate the cell product. The fluid flow path may flow the fluid along a direction having an angle with respect to a second direction of flow within the compartment. The angle may be greater than zero degrees. The method may include using the analysis unit to monitor theWSGR Docket No.67566-702601 plurality of cells of the fluid as the materials are provided to the compartment during generation of the cell product.

[0274] In another aspect, the present disclosure provides methods for cell processing. Amethod may include providing a plurality of cells to a system. The system may include a cell processing unit, a preservation unit, an analysis unit, or combinations thereof. The cell processing unit may retain the plurality of cells. The preservation unit may be coupled to the cell processing unit. The analysis unit may be coupled to the cell processing unit, the preservation unit, or both. The method may include processing the cells in the cell processing unit to generate a cell product. The method may include preserving the cell product in the preservation unit. The method may include detecting or monitoring one or more properties of the plurality of cells of the cell product during processing of the cells, preserving the cell product, or both.

[0275] The systems and methods described herein (e.g., integrated cell processingsystems) may permit the use of the 3D matrix and compositions of matter described elsewhere herein for the purpose of processing cells. Processing cells may include automated cell processing, cell culture, cell manipulation, or any combination thereof for production of cellular products. Cellular products may include cell therapies, gene therapies, biologics, biobanked cell stocks, or combinations thereof. The systems and methods described herein may be usable for manufacturing or research applications (e.g., genetic and environmental manipulation of calls and cell line creation). In some embodiments, the systems and methods may include a solid-state bioreactor. The solid-state bioreactor may permit culture or cellular manipulation of adherent, microcarrier, or suspension cell culture. The systems and methods described herein may be usable for reproducing cell products with high cell health and viability. A cell product produced using the systems and methods described herein may have a viability of greater than or equal to about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or higher. In an example, a cell product produced using the systems and methods described herein may have a cell viability of greater than or equal to about 75%. In another example, the cell viability may be greater than or equal to about 90%. In another example, the cell viability may be greater than or equal to about 95%.

[0276] Cell processing systems as described herein may include one or more receivingunits, bioreactors, cell processing units, preservation units, analysis units, control units, thermal control units, fluidic units, or any combination thereof. In an example, a cell processing system comprises a bioreactor coupled to a receiving unit, cell processing unit, preservation unit, or analysis unit. In another example, a cell processing system comprises aWSGR Docket No.67566-702601 bioreactor coupled to a cell processing unit, preservation unit, and analysis unit. In an example, the bioreactor is a solid-state bioreactor. The bioreactor may be configured to subject a plurality of cells to cell culture, membrane transduction, expansion, gene delivery, cell selection, or any combination thereof.

[0277] In an example, the cell processing system comprises a receiving unit. Thereceiving unit may be coupled to the bioreactor, a cell processing unit, preservation unit, an analysis unit, or any combination thereof. The receiving unit may be configured to receive a plurality of cells from an environment external to the cell processing system. The receiving unit may be configured to receive a plurality of cryopreserved (e.g., frozen) cells. The receiving unit may comprise a thermal unit, as described elsewhere herein, and may subject cryopreserved cells to controlled thawing. The receiving unit may be configured to automatically transfer the plurality of cell to the processing unit or to a bioreactor subsequent to thawing. Alternatively, or in addition to, the receiving unit may be configured to automatically transfer the cells to a thermal unit for thawing or a cell processing unit upon receipt of the plurality of cells.

[0278] Cell processing systems may comprise one or more control systems. Controlsystems may include computer systems configured to control or operate the cell processing system. The cell processing system may comprise one or more computer processors operatively coupled to one or more units of the cell processing system. The one or more computer processors may be operatively coupled to a cell processing unit, analysis unit, bioreactor, thermal control unit, receiving unit, preservation unit, fluid flow unit, or any combination thereof. In an example, the one or more computer processors are operatively coupled to the cell processing unit and are configured to direct the cell processing unit to automatically process the plurality of cells within the cell processing unit (e.g., within the 3D matrix), direct the cell processing unit to separate the plurality of processed cells from the 3D matrix, and direct the cell processing unit to collect the plurality of processed cells. In another example, the one or more computer processors are operatively coupled to the receiving unit and may be programmed to automatically direct the receiving unit to provide the plurality or cells to the cell processing unit.

[0279] Cell processing system or a bioreactor may include one or more cell processingunits. In an example, a cell processing system comprises one or more bioreactors comprising one or more cell processing units. In an example, a cell processing system comprises one or more bioreactors comprising one or more cell processing units and additional cell processingWSGR Docket No.67566-702601 units separate from the bioreactor(s). In another example, the cell processing system may comprise one or more cell processing units and may not include a bioreactor.

[0280] A cell processing unit may comprise any components or feature configured to orusable to process cells. A cell processing unit may be configured or otherwise used to generate a cell product. A cell process unit may comprise a compartment configured to retain a plurality of cells (e.g., cell compartment), fluid flow path configured to provide fluid to the cell compartment, one or more interfaces disposed between fluid flow paths, cell compartments, or both, or any combination thereof. In an example, a cell processing unit comprises at least one cell compartment, at least one fluid flow path, and at least one interface. In another example, a cell processing unit comprises at least one cell compartment, at least one fluid flow path, and does not include an interface. In another example, a cell processing unit comprises at least one compartment, at least one interface, and no fluid flow path. In another example, a cell processing unit comprises at least one compartment and no fluid flow path or interface.

[0281] A cell processing unit may comprise at least 1, 2, 3, 4, 5, 6, 8, 10 or more cellcompartments. A cell processing unit may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more fluid flow paths. In an example, the fluid flow path(s) are perfusion compartments. A cell processing unit may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more interfaces. The interfaces may be disposed between one cell compartment and another cell compartment or fluid flow path. Interfaces may be disposed between one fluid flow path and another fluid flow path. The number of interfaces may be greater than, equal to, or less than the number of cell compartments or fluid flow paths. The number of fluid flow paths may be less than, equal to, or greater than the number of cell compartments.

[0282] A bioreactor may comprise one or more cell processing units. A bioreactor maycomprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more cell processing units. A cell processing system may comprise a bioreactor and one or more additional cell processing units. A cell processing system may include at least 1, 2, 3, 4, 5, 6, 8, 10, or more cell processing units separate from a bioreactor (e.g., the cell processing system does not include a bioreactor, or the cell processing units are not a part of the bioreactor).

[0283] A cell processing unit may be configured to process or may process a plurality ofcells. Processing cells may include, but is not limited to, cell enrichment, cell selection, cell isolation, cell expansion or culturing, washing, concentration, buffer exchange, de-clumping, or any combination thereof. A cell processing unit may comprise a compartment (e.g., cell compartment) configured to provide one or more processes to the cells. For example, the cellWSGR Docket No.67566-702601 compartment may be configured to subject the plurality of cells to cell culture, membrane transduction, expansion, gene delivery, cell selection, or any combination thereof. Cell retention

[0284] The systems and methods described herein may provide integrated cell retention.Cell processing may include providing media or other liquids to cells, removal or media or other liquids from cells, or replacement of media or other liquids in the system. Cells may be retained within the system to provide effective separation of cells from spent media and other fluids, molecules, reagents, and cellular byproducts to be removed or harvested from the culture environment. The volume of the cell processing system containing cells may be a subset of the total working volume of the cell processing system. In the cell processing system at least some other volume (e.g., not containing cells) may contain other fluid components, such as growth media and buffers, or gasses. The former cell-containing volume is referred to herein as a cell compartment(s). The cell compartment may be disposed in a cell processing unit, a solid-state bioreactor, or any combination thereof. A solid-state bioreactor may be a cell processing unit. A cell processing unit may be any device, unit, or module configured to process or that processes cells.

[0285] Cell compartment(s) may be provided in a single contiguous configuration, ormultiple discrete configurations. Cell compartment(s) provided in a discrete configuration may comprise a plurality of cell compartments physically separated from one another, for example, by a cell-free volume. The cell compartment, regardless of configuration, may provide for the substantial retention of cells by virtue of its design or its configuration utilizing a 3D substrate. The cell compartment may be in fluidic communication with a connected cell-free or substantially cell-free volume. The cell-free or substantially cell-free volume may be a fluid flow path (e.g., perfusion compartment), gas exchange compartment, or both. The cell compartment may be a vessel or other compartment configured to retain a 3D matrix. Alternatively, the 3D matrix may be the cell compartment. The cell compartment may represent a physical 3D volume within the cell processing unit (e.g., solid-state bioreactor or other cell processing unit) and comprises a physical form defined by the dimensions and shape of the 3D matrix or the bounds of cell occupancy within the cell processing unit.

[0286] In some embodiments, a cell compartment comprises a 3D matrix for cellprocessing (e.g., cell culture, gene delivery, transfection, etc.), as described elsewhere herein. In such a configuration, the 3D substrate, such as a 3D matrix, may provide for the substantial retention of cells. For example, a 3D substrate may retain cells, therebyWSGR Docket No.67566-702601 comprising the cell compartment, by virtue of cellular adhesion to the substrate, by encapsulation, by the porosity of the 3D substrate being unsuitable for cell egress, by a flow configuration or the fluid dynamics within the 3D substrate during perfusion or fluidic flow being unable to substantially displace cells from the 3D substrate, or by other physical and biological interactions among the 3D substrate, cells, and fluids, or combinations of these, as well as the effect of gravity, depending on configuration. In some embodiments, the interface(s) between the cell compartment(s) and the other volume(s) (e.g., fluid flow path) of the cell processing unit (e.g., solid-state bioreactor or other cell processing unit) may be defined by the boundary of the 3D substrate in which the cells reside. Such an interface and configuration may be considered a phase separation, e.g., the cell compartment(s) being defined as a solid or semi-solid phase comprising the 3D substrate, and the other compartment(s) being defined as liquid or gas phases, comprising media, atmosphere, or another aspect of the cell processing environment. In some embodiments, a pre-formed 3D substrate for cell culture is provided within the cell processing unit. Cells may be loaded into the cell processing unit and allowed to settle by gravity, attach by cell adhesion, migrate into, or otherwise become localized to or within the 3D substrate. In some embodiments, cell- signaling or cell-binding factors are provided to promote such cellular localization. In some embodiments, a liquid matrix formulation (e.g., polymer precursor mixture) is loaded into a cell processing unit and allowed to form a 3D substrate, such as by gel-sol transition, in situ polymerization, in situ crosslinking of the matrix, or combinations thereof. In some embodiments, the 3D substrate is configured such as to not occupy the entire volume of cell processing unit, e.g., by loading a suitable volume of liquid matrix such as to form a “head space” which is not occupied by 3D substrate upon matrix formation. The liquid matrix formulation (e.g., polymer precursor mixture) may contain cells to permit formation of a cell- laden 3D substrate upon matrix formation. Alternatively, cells may be added to the 3D substrate subsequent to matrix formation. In some embodiments, cells may be added prior to matrix formation and the generated 3D matrix may subsequently be seeded with additional cells. In some embodiments, the volume and specific spatial configuration of the cell compartment(s) may be further defined by a phase separation, such as gas (e.g., “head space”) or by use of a phase-separated liquid. For example, liquid matrix (e.g., polymer precursors) may be loaded into the cell processing unit with a separate immiscible liquid such as oil (concurrently, prior to, or subsequent to), such that the immiscible liquid phase excludes the matrix from a specific volume of the bioreactor during formation of the 3D substrate. Such phase-separated non-matrix liquid may be removed (e.g., washed out) fromWSGR Docket No.67566-702601 the cell processing unit such as to form a perfusion or gas exchange volume and interface(s) with the 3D substrate within the cell processing unit. In some embodiments, multiphasic liquids are utilized. Multiphasic liquid may comprise at least a liquid matrix (e.g., polymer precursors) and a separate immiscible liquid which form two or more discrete phases or volumes separated by one or more interfaces upon formation of the 3D matrix.

[0287] A cell processing unit may comprise at least one fluid flow path. The fluid flowpath may be configured to provide or may provide reagents to the 3D matrix and to remove waste (e.g., waste metabolites) from the 3D matrix, as shown in FIGs.16A and 16B. In an example, during cell culture, the fluid flow path may provide reagents to the 3D matrix to provide conditions sufficient for cell culture as shown in FIG.16A. The reagents may include, but are not limited to, glucose, oxygen, buffering agents, proteins, amino acids, lipids, inorganic salts, trace elements, vitamins, reducing agents, or any combinations thereof. The fluid flow path may be configured to remove or may remove waste from the 3D matrix to maintain conditions sufficient for cell culture. Removed waste may include, but are not limited to, lactate, carbon dioxide, ammonia, other metabolites, or any combination thereof. In another example, during cell activation, the fluid flow path may be configured to provide or may provide growth factors, signaling factors, or both to the cells encapsulated within the 3D matrix. For example, the fluid flow path may provide interleukins (e.g., IL-2), antibodies, complexes (e.g., CD3 / 28), or any combination thereof to the 3D matrix. In another example, the fluid flow path may be configured to provide viruses, DNA, RNA, reagents, or any combination thereof for gene delivery. Gene delivery reagents may include, but are not limited to, viruses, DNA, RNA, lipid-complexes (LNP), electroporation buffer, adjuvants, or any combination thereof. The fluid flow path may be configured to wash the 3D matrix before, during, or subsequent to activation or gene delivery. The wash-out may include providing media or other wash solution to the 3D matrix to remove growth factors, signaling factors, gene delivery reagents, or any combination thereof. In another example, a cell processing unit may comprise a fluid flow path configured to deliver liquid nutrients and reagents and another fluid flow path configured to deliver gaseous nutrients and reagents, as shown in FIG.16B. The 3D matrix may be disposed between a fluid flow pathway (e.g., first fluid flow pathway) configured to provide fluidic nutrients (e.g., nutrients disposed in a liquid phase, such as media) to the 3D matrix and another fluid flow pathway (e.g., second fluid flow pathway) configured to provide gaseous nutrients and reagents to the 3D matrix. Either the first or the second fluid flow pathway, or both, may be configured to flow a gas- exchange liquid (e.g., an oxygenated media) to provide gaseous nutrients and reagents to theWSGR Docket No.67566-702601 3D matrix. In an example, the second fluid flow pathway may be configured to flow a gas- phase fluid such that gas from the gas-phase fluid can diffuse into the 3D matrix or media compartments via a gas-permeable and liquid-impermeable membrane (e.g., polydimethylsiloxane (PDMS), silicones, etc.). In an example, a liquid delivering and gas delivering fluid flow path may be disposed on the same side of a 3D matrix.

[0288] A cell processing unit may comprise a membrane or interface. The membraneor interface may be disposed between a compartment, such as a cell compartment, and a fluid flow path. The membrane may be disposed between a first cell compartment and another cell compartment. The membrane may be disposed between a first fluid flow path and another fluid flow path. The membrane may be disposed between a cell compartment and another compartment configured to flow fluid (e.g., liquid or gas). The membrane may be configured to permit transport of material through the membrane. For example, the membrane may permit transport of nutrients, reagents, or other components in a liquid or gas phase through the membrane. In some embodiments, the cell processing unit does not comprise a membrane or semi permeable membrane between the compartment and the fluid flow path or other volume. In some embodiments, a cell compartment further comprises, or is coupled to, one or more membranes or semi-permeable substrate(s), such as a semi-permeable membrane. A membrane may have a thickness that permits transport of fluids or fluids comprising nutrients, reagents, or other components from a fluid flow path to a cell compartment (e.g., 3D matrix or 3D matrix in a cell compartment) or vice versa. A membrane may have a thickness of greater than or equal to about 5 micrometers (µm), 10 µm, 15 µm, 20 µm, 30 µm, 40 µm, 50 µm, 75 µm, 100 µm, 200 µm, 300 µm, 400 µm, 500 µm, 600 µm, 700 µm, 800 µm, 900 µm, 1 millimeter (mm), 1.5 mm, 2 mm, 3 mm, 4 mm, 5mm, or thicker. A membrane may have a thickness of less than or equal to about 5 mm, 4 mm, 3 mm, 2 mm, 1.5 mm, 1 mm, 900 µm, 800 µm, 700 µm, 600 µm, 500 µm, 400 µm, 300 µm, 200 µm, 100 µm, 75 µm, 50 µm, 40 µm, 30 µm, 20 µm, 15 µm, 10 µm, 5 µm, or less. A membrane may have a thickness from about 5 µm to 10 µm, 5 µm to 15 µm, 5 µm to 20 µm, 5 µm to 30 µm, 5 µm to 40 µm, 5 µm to 50 µm, 5 µm to 75 µm, 5 µm to 100 µm, 5 µm to 200 µm, 5 µm to 300 µm, 5 µm to 400 µm, 5 µm to 500 µm, 5 µm to 600 µm, 5 µm to 700 µm, 5 µm to 800 µm, 5 µm to 900 µm, 5 µm to 1 mm, 5 µm to 1.5 mm, 5 µm to 2 mm, 5 µm to 3 mm, 5 µm to 4 mm, 5 µm to 5 mm, 10 µm to 15 µm, 10 µm to 20 µm, 10 µm to 30 µm, 10 µm to 40 µm, 10 µm to 50 µm, 10 µm to 75 µm, 10 µm to 100 µm, 10 µm to 200 µm, 10 µm to 300 µm, 10 µm to 400 µm, 10 µm to 500 µm, 10 µm to 600 µm, 10 µm to 700 µm, 10 µm to 800 µm, 10 µm to 900 µm, 10 µm to 1 mm, 10 µm to 1.5 mm, 10 µm to 2 mm, 10 µm to 3 mm, 10 µm toWSGR Docket No.67566-702601 4 mm, 10 µm to 5 mm, 15 µm to 20 µm, 15 µm to 30 µm, 15 µm to 40 µm, 15 µm to 50 µm, 15 µm to 75 µm, 15 µm to 100 µm, 15 µm to 200 µm, 15 µm to 300 µm, 15 µm to 400 µm, 15 µm to 500 µm, 15 µm to 600 µm, 15 µm to 700 µm, 15 µm to 800 µm, 15 µm to 900 µm, 15 µm to 1 mm, 15 µm to 1.5 mm, 15 µm to 2 mm, 15 µm to 3 mm, 15 µm to 4 mm, 15 µm to 5 mm, 20 µm to 30 µm, 20 µm to 40 µm, 20 µm to 50 µm, 20 µm to 75 µm, 20 µm to 100 µm, 20 µm to 200 µm, 20 µm to 300 µm, 20 µm to 400 µm, 20 µm to 500 µm, 20 µm to 600 µm, 20 µm to 700 µm, 20 µm to 800 µm, 20 µm to 900 µm, 20 µm to 1 mm, 20 µm to 1.5 mm, 20 µm to 2 mm, 20 µm to 3 mm, 20 µm to 4 mm, 20 µm to 5 mm, 30 µm to 40 µm, 30 µm to 50 µm, 30 µm to 75 µm, 30 µm to 100 µm, 30 µm to 200 µm, 30 µm to 300 µm, 30 µm to 400 µm, 30 µm to 500 µm, 30 µm to 600 µm, 30 µm to 700 µm, 30 µm to 800 µm, 30 µm to 900 µm, 30 µm to 1 mm, 30 µm to 1.5 mm, 30 µm to 2 mm, 30 µm to 3 mm, 30 µm to 4 mm, 30 µm to 5 mm, 40 µm to 50 µm, 40 µm to 75 µm, 40 µm to 100 µm, 40 µm to 200 µm, 40 µm to 300 µm, 40 µm to 400 µm, 40 µm to 500 µm, 40 µm to 600 µm, 40 µm to 700 µm, 40 µm to 800 µm, 40 µm to 900 µm, 40 µm to 1 mm, 40 µm to 1.5 mm, 40 µm to 2 mm, 40 µm to 3 mm, 40 µm to 4 mm, 40 µm to 5 mm,50 µm to 75 µm, 50 µm to 100 µm, 50 µm to 200 µm, 50 µm to 300 µm, 50 µm to 400 µm, 50 µm to 500 µm, 50 µm to 600 µm, 50 µm to 700 µm, 50 µm to 800 µm, 50 µm to 900 µm, 50 µm to 1 mm, 50 µm to 1.5 mm, 50 µm to 2 mm, 50 µm to 3 mm, 50 µm to 4 mm, 50 µm to 5 mm, 75 µm to 100 µm, 75 µm to 200 µm, 75 µm to 300 µm, 75 µm to 400 µm, 75 µm to 500 µm, 75 µm to 600 µm, 75 µm to 700 µm, 75 µm to 800 µm, 75 µm to 900 µm, 75 µm to 1 mm, 75 µm to 1.5 mm, 75 µm to 2 mm, 75 µm to 3 mm, 75 µm to 4 mm, 75 µm to 5 mm, 100 µm to 200 µm, 100 µm to 300 µm, 100 µm to 400 µm, 100 µm to 500 µm, 100 µm to 600 µm, 100 µm to 700 µm, 100 µm to 800 µm, 100 µm to 900 µm, 100 µm to 1 mm, 100 µm to 1.5 mm, 100 µm to 2 mm, 100 µm to 3 mm, 100 µm to 4 mm, 100 µm to 5 mm, 200 µm to 300 µm, 200 µm to 400 µm, 200 µm to 500 µm, 200 µm to 600 µm, 200 µm to 700 µm, 200 µm to 800 µm, 200 µm to 900 µm, 200 µm to 1 mm, 200 µm to 1.5 mm, 200 µm to 2 mm, 200 µm to 3 mm, 200 µm to 4 mm, 200 µm to 5 mm, 300 µm to 400 µm, 300 µm to 500 µm, 300 µm to 600 µm, 300 µm to 700 µm, 300 µm to 800 µm, 300 µm to 900 µm, 300 µm to 1 mm, 300 µm to 1.5 mm, 300 µm to 2 mm, 300 µm to 3 mm, 300 µm to 4 mm, 300 µm to 5 mm, 400 µm to 500 µm, 400 µm to 600 µm, 400 µm to 700 µm, 400 µm to 800 µm, 400 µm to 900 µm, 400 µm to 1 mm, 400 µm to 1.5 mm, 400 µm to 2 mm, 400 µm to 3 mm, 400 µm to 4 mm, 400 µm to 5 mm, 500 µm to 600 µm, 500 µm to 700 µm, 500 µm to 800 µm, 500 µm to 900 µm, 500 µm to 1 mm, 500 µm to 1.5 mm, 500 µm to 2 mm, 500 µm to 3 mm, 500 µm to 4 mm, 500 µm to 5 mm, 600 µm to 700 µm, 600 µm to 800 µm, 600 µm to 900 µm, 600 µm to 1 mm, 600 µm to 1.5 mm,WSGR Docket No.67566-702601 600 µm to 2 mm, 600 µm to 3 mm, 600 µm to 4 mm, 600 µm to 5 mm, 700 µm to 800 µm, 700 µm to 900 µm, 700 µm to 1 mm, 700 µm to 1.5 mm, 700 µm to 2 mm, 700 µm to 3 mm, 700 µm to 4 mm, 700 µm to 5 mm, 800 µm to 900 µm, 800 µm to 1 mm, 800 µm to 1.5 mm, 800 µm to 2 mm, 800 µm to 3 mm, 800 µm to 4 mm, 800 µm to 5 mm, 900 µm to 1 mm, 900 µm to 1.5 mm, 900 µm to 2 mm, 900 µm to 3 mm, 900 µm to 4 mm, 900 µm to 5 mm, 1 mm to 1.5 mm, 1 mm to 2 mm, 1 mm to 3 mm, 1 mm to 4 mm, 1 mm to 5 mm, 1.5 mm to 2 mm, 1.5 mm to 3 mm, 1.5 mm to 4 mm, 1.5 mm to 5 mm, 2 mm to 3 mm, 2 mm to 4 mm, 2 mm to 5 mm, 3 mm to 4 mm, 3 mm to 5 mm, or 4 mm to 5 mm. In an example, a semipermeable membrane may have a thickness from about 18 to 25 µm or from about 110 to 150 µm.

[0289] Semipermeable membrane materials may include Polysulfone (PS / PSF),Polyethersulfone (PES), Polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (PTFE), Nylon (Polyamide), Polycarbonate (PC, PCTE), Teflon (Polytetrafluoroethylene), Cellulose Nitrate, Cellulose Acetate, Mixed Cellulose (MCE), Glass Fiber, Polypropylene (PP), Polyacrylonitrile (PAN), Polyester (PETE), Polyethylene (PE), Polyether ether ketone (PEEK), thermoplastic, metal (such as silver, aluminum), ceramic, or any combinations thereof. Membranes may be surface treated, such as with a wetting agent. For example, a wetting agent may comprise a few molecular thicknesses of polyvinylpyrrolidone (PVP) deposited on the membrane surfaces of materials such as PCTE. As another example, a wetting agent may comprise a deposition of a polymer such as a protein, including but not limited to collagen, albumin, fibronectin, and laminin. For example, treatment, such as adsorption or coating, of collagen may increase the contact angle and hydrophilicity of a plastic material including membranes and other aqueous-fluid contacting surfaces. Porous or semipermeable membranes may be manufactured by track etching or as a woven mesh, nanofiber mesh, non-woven matrix, or combination thereof. Membranes may be selected based on properties including porosity (percent of total area occupied by pores, for example from less than about 1% to 16% for track-etched PC and PE, from about 40% to 80% for other materials and construction methods), pore size (e.g., pore diameter), hydrophilicity and hydrophobicity, bubble point (minimum amount of pressure required to push air bubbles through the largest pore of a wet membrane), maximum operating temperature, sealing compatibility (e.g., ultrasonic, heat, radiofrequency, insert molding), burst strength, water flow rate (e.g., mL / min / cm2at a given pressure), air flow rate (e.g., L / min / cm2at a given pressure), optical properties (e.g., clarity), solvent resistance, chemical compatibility, sloughing or particle shedding, biocompatibility, extractables and adsorption characteristics,WSGR Docket No.67566-702601 biofouling and protein binding (e.g., certain hydrophilic materials, such as nylon and cellulose nitrate, exhibit high protein binding capacity, while regenerated cellulose and cellulose acetate bind virtually no protein).

[0290] In an example, a membrane may be disposed between the 3D matrix or cellcompartment and the media compartment. The membrane may be a track etched PC or PETE membrane with a thickness from about 18 to 25 µm. The membrane may have a pore size from about 1 to 12 µm pore size and a porosity from about 1 to 16%. In an example, the membrane has a pore size of less than about 1 µm. In another example, the membrane has a pore size of less than about 2 µm. In another example, the membrane has a pore size of less than about 3 µm.

[0291] In another example, the system may comprise a gas-permeable membrane.The gas-permeable membrane may be dense ( e.g., nonporous and devoid of interconnected pore networks such that gas molecules transport through diffusion) or porous (e.g., assisted by hydrophobicity, porous membranes may not permit the pressure-driven permeation of liquid into the gaseous pores of the membrane up to a certain critical pressure). Gas- permeable membranes may be made from materials including PDMS, silicone, Teflon (e.g., Teflon AF2400), poly[1-trimethylsilyl-1-propyne] (PTMSP), polyimide, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polycarbonate (PC), or any combination thereof. Other membrane materials used may include polymers of intrinsic microporosity (PIMs), thermally rearranged (TR) polymers, and heterogeneous membranes, including thin-film composite (TFC) membranes (e.g., an ultrathin dense polymer layer formed or deposited onto a rigid membrane support, permitting the simultaneous integration of the selectivity / permittivity of the dense membrane and the mechanical strength of thesupport layer) and mix...

Claims

WSGR Docket No.67566-702601 CLAIMS WHAT IS CLAIMED IS:

1. A system for manufacturing a cell product, comprising:a compartment configured to retain a plurality of cells; a fluid flow path in fluid communication with said compartment, wherein said fluid flow path is configured to (i) flow a fluid along a first direction having an angle with respect to a second direction of flow within said compartment, wherein said angle is greater than zero degrees, and (ii) provide material from said fluid to said compartment to thereby generate said cell product; and an analysis unit coupled to and within a same housing as said compartment and said fluid flow path, wherein said analysis unit is configured to monitor said plurality of cells or said fluid as said materials are provided to said compartment during generation of said cell product.

2. The system of claim 1, wherein said angle is greater than 45 degrees.

3. The system of any one of claims 1 or 2, wherein said cell product is a cell therapyproduct.

4. The system of claim 3, wherein said system is configured to generate said cell therapyproduct at a scale of at least one human dose equivalent.

5. The system of any one of claims 1-4, wherein said analysis unit is configured tomonitor said plurality of cells in said compartment.

6. The system of any one of claims 1-5, system further comprising a cell processing unitin fluid communication with said compartment.

7. The system of any one of claims 1-6, wherein said compartment is configured toprovide to said plurality of cells one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection.

8. The system of any one of claims 1-7, wherein said housing has a volume of about0.01 cubic meters (m3) to about 2 m3.

9. The system of any one of claims 1-8, wherein said analysis unit comprises one ormore sensors configured to monitor an environment comprising said plurality of cells.

10. The system of any one of claims 1-9, wherein said analysis unit comprises one ormore sensors configured to analyze said cell product.

11. A system for cell processing, comprising:a cell processing unit configured to retain and process a plurality of cells to generate a cell product;WSGR Docket No.67566-702601 a preservation unit coupled to said cell processing unit, wherein said preservation unit is configured to preserve said cell product; and an analysis unit coupled to said cell processing unit or said preservation unit, wherein said analysis unit is configured to detect or monitor one or more properties of said plurality of cells or said cell product.

12. The system of claim 11, wherein said cell product is a cell therapy product.

13. The system of claim 11 or claim 12, further comprising a receiving unit configured toreceive said plurality of cells.

14. The system of claim 13, wherein said plurality of cells is a plurality of cryopreservedcells.

15. The system of claim 14, wherein said receiving unit is configured to subject saidplurality of cryopreserved cells to controlled thawing to generate a plurality of thawed cells.

16. The system of claim 15, wherein said receiving unit is configured to automaticallytransfer said plurality of thawed cells to said cell processing unit.

17. The system of any one of claims 11-16, wherein said preservation unit is configuredto generate a cryopreserved cell product.

18. The system of any one of claims 11-17, further comprising a bioreactor coupled tosaid cell processing unit, said preservation unit, or said analysis unit.

19. The system of claim 18, wherein said bioreactor is a solid-state bioreactor.

20. The system of claim 18, wherein said bioreactor is configured to provide to saidplurality of cells one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection.

21. The system of any one of claims 11-20, wherein said analysis unit is configured toprovide in situ, real-time detection or monitoring of said one or more properties of said plurality of cells or said cell product.

22. The system of any one of claims 11-21, wherein said analysis unit is configured toprovide release testing of said cell product.

23. The system of any one of claims 11-22, wherein said cell processing unit, saidanalysis unit, and said preservation unit are integrated withing a housing.

24. The system of claim 23, wherein said housing has a volume of about 0.01 cubicmeters (m3) to about 2 m3.

25. The system of any one of claims 11-24, wherein said analysis unit comprises one ormore sensors configured to monitor an environment comprising said plurality of cells.WSGR Docket No.67566-70260126. The system of any one of claims 11-25, wherein said analysis unit comprises one ormore sensors configured to analyze said cell product.

27. A method for manufacturing a cell product, comprising:(a) providing a plurality of cells to a system comprising (i) a compartment thatretains said plurality of cells, (ii) a fluid flow path in fluid communication with said compartment, and (iii) an analysis unit coupled to and within a same housing as said compartment and said fluid flow path; (b) providing material from said a fluid in said fluid flow path to saidcompartment to generate said cell product, wherein said fluid flow path flows said fluid along a first direction having an angle with respect to a second direction of flow within said compartment, wherein said angle is greater than zero degrees; and (c) using said analysis unit to monitor said plurality of cells or said fluid as saidmaterials are provided to said compartment during generation of said cell product.

28. The method of claim 27, wherein said angle is greater than 45 degrees.

29. The method of any one of claims 27 or 28, wherein said cell product is a cell therapyproduct.

30. The method of claim 29, wherein said cell therapy product is generated at a scale of atleast one human dose equivalent.

31. The method of any one of claims 27-30, wherein said analysis unit monitors saidplurality of cells in said compartment.

32. The method of any one of claims 27-31, wherein said analysis unit monitors saidplurality of cells in real time.

33. The method of any one of claims 27-32, further comprising using a cell processingunit to generate said cell product.

34. The method of any one of claims 27-33, further comprising subjecting said pluralityof cells to one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection.

35. The method of any one of claims 27-34, wherein said housing has a volume of about0.01 cubic meters (m3) to about 2 m3.

36. A method for cell processing, comprising:(a) providing a plurality of cells to a system comprising (i) a cell processing unitthat retains said plurality of cells, (ii) a preservation unit coupled to said cell processing unit, and (iii) an analysis unit coupled to said cell processing unit or said preservation unit;WSGR Docket No.67566-702601 (b) processing said plurality of cells in said cell processing unit to generate a cellproduct; (c) preserving said cell product in said preservation unit; and(d) detecting or monitoring one or more properties of said plurality of cells or saidcell product during said processing or said preserving.

37. The method of claim 36, wherein said cell product is a cell therapy product.

38. The method of claim 36 or 37, further comprising providing said plurality of cells toreceiving unit coupled to said cell processing unit, said preservation unit, or said preservation unit.

39. The method of claim 38, wherein said plurality of cells provided to said receiving unitis a plurality of cryopreserved cells.

40. The method of claim 39, wherein said receiving unit subjects said plurality ofcryopreserved cells to controlled thawing to generate a plurality of thawed cells.

41. The method of claim 40, wherein said receiving unit automatically transfers saidplurality of thawed cells to said cell processing unit.

42. The method of any one of claims 36-41, wherein said preservation unit generates acryopreserved cell product.

43. The method of any one of claims 36-42, further comprising a bioreactor coupled tosaid cell processing unit, said preservation unit, or said analysis unit.

44. The method of claim 43, wherein said bioreactor is a solid-state bioreactor.

45. The method of claim 43, further comprising using said bioreactor to subject saidplurality of cells to one or more processes selected from the group consisting of cell culture, membrane transduction, expansion, gene delivery, and selection.

46. The method of any one of claims 36-45, wherein said analysis unit provides in situ,real-time detection or monitoring of said one or more properties of said plurality of cells or said cell product.

47. The method of any one of claims 36-46, further comprising using said analysis unitfor release testing of said cell product.

48. A system of cell processing, comprising:a plurality of three-dimensional (3D) matrices, wherein a 3D matrix of said plurality of 3D matrices is configured to contain or encapsulate a population of cells; and a plurality of fluid flow paths, wherein a fluid flow path of said plurality of fluid flow paths is independent from and adjacent to said 3D matrix, and wherein said fluid flow path isWSGR Docket No.67566-702601 configured to deliver a fluidic material to said 3D matrix to process said population of cells into a cell product.

49. The system of claim 48, wherein said cell product is a cell therapy product.

50. The system of claim 48 or claim 49, wherein said 3D matrix and said fluid flow pathare separated by a membrane.

51. The system of any one of claims 48-50, wherein said 3D matrix is configured as aslab comprising a first dimension and a second dimension that are larger than a third dimension.

52. The system of claim 51, wherein said third dimension is less than or equal to about 10millimeters (mm).

53. The system of any one of claims 48-52, wherein said 3D matrix is configured as afiber comprising a diameter that is smaller than said long dimension.

54. The system of claim 53, wherein said diameter is less than or equal to about 10 mm.

55. The system of any one of claims 48-54, wherein said 3D matrix is configured as asphere and wherein said tangential flow of said fluid flows along an outer circumference of said sphere.

56. The system of any one of claims 48-55, wherein said plurality of 3D matrices arefluidically coupled to one another.

57. The system of any one of claims 48-56, wherein said fluid flow path is disposedbetween a first 3D matrix and a second 3D matrix.

58. The system of claim 57, wherein said fluid flow path is configured to deliver saidfluidic material to said first 3D matrix and said second 3D matrix.

59. The system of any one of claims 48-58, wherein said 3D matrix comprises a pore sizeof less than or equal to about 400 micrometers (µm).

60. The system of any one of claims 48-59, wherein said 3D matrix comprises a porosityof greater than or equal to about 80%.

61. The system of any one of claims 48-60, wherein an interfacial area between said fluidflow path and said 3D matrix is greater than or equal to about 5 square centimeters (cm2).

62. The system of any one of claim 48-61, wherein said 3D matrix comprises a porous,solid matrix having interconnected pores with an average pore size sufficient to permit growth of lymphocyte cells within said 3D matrix.

63. The system of any one of claims 48-62, wherein said 3D matrix is a programmable3D matrix.WSGR Docket No.67566-70260164. The system of any one of claims 48-63, wherein said 3D matrix has a surface area tovolume ratio of greater than or equal to about 0.1.

65. The system of any one of claims 48-64, wherein said 3D matrix has a conductivityfrom about 10-5Siemens per m (S / m) to about 10-1S / m.

66. The system of any one of claims 48-65, wherein said 3D matrix is configured toprovide activation of said plurality of cells via membrane signal transduction in said 3D matrix.

67. A method for cell processing, comprising:(a) providing (i) a population of cells, (ii) a plurality of three-dimensional (3D)matrices, and (iii) a plurality of fluid flow paths, wherein said population of cells is contained or encapsulated within a 3D matrix of said plurality of 3D matrices, and wherein a fluid flow path of said plurality of fluid flow paths is independent from and adjacent to said 3D matrix; (b) using said fluid flow path to deliver fluidic material to said 3D matrix; and(c) in a presence of said fluidic material delivered to said 3D matrix, processingsaid population of cells into a cell product.

68. The method of claim 67, wherein said cell product is a cell therapy product.

69. The method of claim 68, wherein said cell therapy product is produced at a scale of atleast one human dose equivalent.

70. The method of claim 67 or claim 68, further comprising functionalizing said 3Dmatrix to generate a functionalized 3D matrix.

71. The method of claim 70, wherein said functionalizing comprises coupling one ormore biomolecules to said 3D matrix.

72. The method of claim 71, wherein said one or more biomolecules are usable formembrane signal transduction.

73. The method of claim 70, further comprising polymerizing pre-functionalized polymerprecursors to generate said 3D matrix.

74. The method of claim 71, further comprising releasing said one or more biomoleculesfrom said 3D matrix or replacing said one or more biomolecules with additional or different biomolecules.

75. The method of any one of claims 67-74, wherein said 3D matrix and said fluid flowpath are separated by a membrane, and wherein said fluidic material is transported through said membrane.WSGR Docket No.67566-70260176. The method of any one of claims 67-75, where said 3D matrix has a dimension lessthan or equal to about 10 millimeters (mm).

77. The method of any one of claims 67-76, wherein said plurality of 3D matrices arefluidically coupled to one another.

78. The method of any one of claims 67-77, wherein said fluid flow path is disposedbetween a first 3D matrix and a second 3D matrix of said plurality of 3D matrices.

79. The method of claim 78, wherein said fluid flow path delivers said fluidic material tosaid first 3D matrix and said second 3D matrix.

80. The method of any one of claims 67-79, wherein said 3D matrix comprises a pore sizeof less than or equal to about 400 micrometers (µm).

81. The method of any one of claims 67-80, wherein said 3D matrix comprises a porosityof greater than or equal to about 80%.

82. The method of any one of claims 67-81, wherein an interfacial area between said fluidflow path and said 3D matrix is greater than or equal to about 5 square centimeters (cm2).

83. The method of any one of claims 67-82 wherein said 3D matrix comprises a porous,solid matrix having interconnected pores with an average pore size sufficient to permit growth of lymphocyte cells within said 3D matrix.

84. The method of any one of claims 67-83, wherein said 3D matrix has a surface area tovolume ratio of greater than or equal to about 0.1.

85. The method of any one of claims 67-84, wherein said 3D matrix is a programmable3D matrix.

86. A method of cell processing, comprising:(a) providing a three-dimensional (3D) matrix containing a plurality of cells in acell processing unit; (b) in said cell processing unit, automatically:(i) processing said plurality of cells within said 3D matrix to produce aplurality of processed cells; and (ii) separating said plurality of processed cells from said 3D matrix; and(c) collecting said plurality of processed cells.

87. The method of claim 86, wherein said plurality of cells are encapsulated in said 3Dmatrix or immobilized to the 3D matrix.

88. The method of claim 86 or 87, wherein said plurality of processed cells is a celltherapy product.WSGR Docket No.67566-70260189. The method of claim 88, wherein said cell therapy product is produced at a scale of atleast one human dose equivalent.

90. The method of any one of claims 86-89, further comprising providing a mixturecomprising a matrix precursor and said plurality of cells to said cell processing system.

91. The method of claim 90, further comprising, in said cell processing unit, applying afirst stimulus to said mixture comprising said matrix precursor and said plurality of cells to generate said 3D matrix encapsulating said plurality of cells.

92. The method of claim 91, wherein said first stimulus is a thermal stimulus.

93. The method of claim 91, wherein said first stimulus is a chemical stimulus.

94. The method of claim 91, wherein said first stimulus is an enzymatic stimulus.

95. The method of claim 91, further comprising functionalizing said 3D matrix with oneor more biomolecules concurrently with said applying said first stimulus to generate said 3D matrix.

96. The method of claim 91, wherein (ii) comprises applying a second stimulus to said 3Dmatrix to release said cell product from said 3D matrix.

97. The method of claim 96, wherein said second stimulus is a thermal stimulus.

98. The method of claim 96, wherein said second stimulus is a chemical stimulus.

99. The method of claim 96, wherein said second stimulus is an enzymatic stimulus.

100. The method of any one of claims 86-99, further comprising, prior to (a), generatingsaid 3D matrix in presence of said plurality of cells such that said 3D matrix encapsulates said plurality of cells.

101. The method of claim 100, wherein (a) comprises loading said 3D matrixencapsulating said plurality of cells is loaded into a compartment in said cell processing unit.

102. The method of any one of claims 86-101, wherein (a) comprises separately providingsaid plurality of cells and said 3D matrix to said cell processing unit.

103. The method of claim 102, wherein said 3D matrix is generated within a compartmentin said cell processing unit.

104. The method of claim 102, wherein said 3D matrix is generated external to said cellprocessing system and subsequently provided to said cell processing unit.

105. The method of any one of claims 86-104, further comprising functionalizing said 3Dmatrix to generate a functionalized 3D matrix.

106. The method of claim 105, wherein said functionalizing comprises coupling one ormore biomolecules to said 3D matrix.WSGR Docket No.67566-702601107. The method of claim 106, wherein said one or more biomolecules are usable formembrane signal transduction.

108. The method of claim 105, further comprising releasing said one or more biomoleculesfrom said 3D matrix or replacing said one or more biomolecules with additional biomolecules.

109. The method of any one of claims 86-108, wherein (i) comprises activating saidplurality of cells within said 3D matrix to obtain said cell product.

110. The method of any one of claims 86-108, wherein (i) comprises transducing saidplurality of cells within said 3D matrix to obtain said cell product.

111. The method of any one of claims 86-108, wherein (i) comprises transfecting saidplurality of cells within said 3D matrix to obtain said cell product.

112. The method of any one of claims 86-111, wherein said 3D matrix has a conductivityfrom about 10-5Siemens per m (S / m) to about 10-1S / m.

113. The method of any one of claims 86-112, further comprising activating said pluralityof cells via membrane signal transduction in said 3D matrix.

114. A cell processing system, comprising:a cell processing unit configured to retain a three-dimensional (3D) matrix, wherein said 3D matrix is configured to contain or encapsulate a plurality of cells; and one or more computer processors operatively coupled to said cell processing unit, wherein said one or more computer processors are individually or collectively programmed to automatically (i) process said plurality of cells within said 3D matrix to produce a plurality of processed cells, (ii) direct said cell processing unit to separate said plurality of processed cells from said 3D matrix, and (iii) direct said cell processing unit to collect said plurality of processed cells.

115. The system of claim 114, further comprising a receiving unit configured to receivesaid plurality of cells from a user.

116. The system of claim 115, wherein said one or more computer processors isoperatively coupled to said receiving unit and individually or collectively programmed to automatically direct said receiving unit to provide said plurality of cells to said cell processing unit.

117. The system of claim 115, wherein said receiving unit is configured to receive saidplurality of cells in a cryopreserved state, and wherein said receiving unit is configured to provide controlled thawing to said plurality of cells in said cryopreserved state.WSGR Docket No.67566-702601118. The system of any one of claims 114-117, further comprising an analysis unitconfigured to monitor said plurality of cells during processing of said plurality of cells.

119. The system of claim 118, wherein said analysis unit comprises one or more sensorsconfigured to monitor an environment comprising said plurality of cells.

120. The system of claim 118, wherein said analysis unit comprises one or more sensorsconfigured to analyze said cell product.

121. The system of any one of claims 114-120, further comprising a housing configured toretain said cell processing unit and said one or more computer processors.

122. The system of claim 121, wherein said housing has a footprint of about 0.01 cubicmeters (m3) to about 2 m3.

123. The system of any one of claims 114-122, wherein said cell processing unit isconfigured to generate said 3D matrix in presence of said plurality of cells.

124. The system of any one of claims 114-123, wherein said cell processing unit isconfigured to receive a pre-formed 3D matrix.

125. The system of claim 124, wherein said cell processing unit is configured to receivesaid pre-formed 3D matrix comprising said plurality of cells.

126. The system of claim 125, wherein said cell processing unit is configured toencapsulate said plurality of cells in said pre-formed 3D matrix.

127. The system of any one of claims 114-126, wherein said 3D matrix has a conductivityfrom about 10-5Siemens per m (S / m) to about 10-1S / m.

128. The system of any one of claims 114-127, wherein said 3D matrix is configured toprovide activation of said plurality of cells via membrane signal transduction in said 3D matrix.

129. A method for cell processing, comprising:(a) providing a plurality of cells ex vivo to a cell processing unit;(b) in said cell processing unit, contacting said plurality of cells with a selectionreagent, wherein, upon said contacting, said selection reagent (i) couples with cells of a sub-population of said plurality of cells and (ii) perturbs a state of one or more cells of said sub-population to product (A) a population of perturbed cells and (B) a population of non-perturbed cells; and (c) processing either said population of perturbed cells or said population of non-perturbed cells into a cell therapy product.WSGR Docket No.67566-702601130. The method of claim 129, wherein said cell therapy product is produced at a scale ofat least one human dose equivalent.

131. The method of claim 129 or claim 130, wherein said population of perturbed cellshave an altered growth rate or viability as compared to said population of non-perturbed cells.

132. The method of any one of claims 129-131, wherein said cell processing unitcomprises a three-dimensional (3D) matrix encapsulating said plurality of cells.

133. The method of claim 132, further comprising contacting said plurality of cells withsaid selection reagent within said 3D matrix.

134. The method any one of claims 129-133, wherein said cell processing unit comprises avessel comprising said plurality of cells.

135. The method of claim 134, further comprising contacting said plurality of cells withsaid selection reagent within said vessel.

136. The method of any one of claims 129-135, wherein said selection reagent comprises abinding agent and a selection agent.

137. The method of claim 136, wherein said selection agent is a cytotoxic agent.

138. The method of claim 136, wherein said binding agent is coupled to said selectionagent via a linker.

139. The method of claim 138, wherein said linker is a cleavable linker.

140. The method of claim 136, wherein said binding agent is an antibody.

141. The method of claim 136, wherein said binding agent is an aptamer.

142. The method of any one of claims 129-141, wherein said population of perturbed cellscomprises dead cells or cell degradation products.

143. The method of any one of claims 129-142, wherein said population of perturbed cellshave an altered cell morphology as compared to said population of non-perturbed cells.

144. A system for manufacturing a cell therapy product, comprising:a three-dimensional (3D) matrix configured to contain or encapsulate a population of cells at a density of greater than about 80 million cells per cubic centimeter (cell / cm3), wherein said system is capable of processing cells of said population of cells, with aid of said 3D matrix, into said cell therapy product.

145. The system of claim 144, wherein said density is at least 100 million cells / cm3.

146. The system of claim 144 or claim 145, wherein said 3D matrix has a surface area tovolume ratio greater than or equal to 0.1.

147. The system of any one of claim 144-146, wherein said 3D matrix has a porosity of atleast about 80%.WSGR Docket No.67566-702601148. The system of any one of claim 144-147, further comprising a plurality of 3Dmatrices comprising said 3D matrix.

149. The system of any one of claim 144-148, further comprising a fluid flow path in fluidcommunication with said 3D matrix, wherein said fluid flow path is adjacent to and independent from said 3D matrix.

150. The system of claim 149, further comprising a membrane disposed between said fluidflow path and said 3D matrix.

151. The system of any one of claim 144-150 wherein said 3D matrix is programmable.

152. A method for manufacturing a cell therapy product, comprising (i) providing apopulation of cells at a density of greater than about 80 million cells per centimeters (cells / cm3) retained within a three-dimensional (3D) matrix and (ii) with aid of said 3D matrix, processing cells of said population of cells into said cell therapy product.

153. The method of claim 152, wherein said processing of said cells comprises culturingsaid population of cells.

154. The method of claim 152 or 153, wherein said processing of said cells comprises insitu electroporation of said cells.

155. The method of claim 154, wherein said 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m.

156. The method of any one of claims 152-155, wherein said processing of said cellscomprises activation of said cells in said 3D matrix.

157. A bioreactor for processing a population of cells comprising a three-dimensional (3D)matrix configured to retain said population of cells, wherein said bioreactor has a volume of at most about 2 cubic meters (m3) and is configured to, with aid of said 3D matrix and at high reproducibility, processes said population of cells into a cell product.

158. The bioreactor of claim 157, wherein said cell product has a viability of greater thanor equal to about 75%.

159. The bioreactor of claim 157 or 158, wherein said volume is of at most 1 m3.

160. A method for processing a population of cells comprising (i) providing a populationof cells retained within three-dimensional (3D) matrix of a bioreactor, wherein said bioreactor has a volume of at most 2 cubic meters (m3) and (ii) processing, with aid of said 3D matrix and at high reproducibility, said population of cells into a cell product.

161. The method of claim 160, wherein said cell product has a viability of greater than orequal to about 75%.WSGR Docket No.67566-702601162. The method of claim 160 or 161, further comprising, prior to (i), generating said 3Dmatrix in presence of said population of cells.

163. The method of any one of claims 160-162, further comprising, subsequent to (ii),dissolving said 3D matrix and collecting said population of cells to generate said cell product.

164. A system for cell therapy manufacturing, comprising:a three-dimensional (3D) matrix configured to retain a population of cells at a density of at least about 5 million cells per cubic centimeters (cells / cm3), wherein said system is capable of processing cells of said population within said 3D matrix for a time period of at most about 48 hours to generate a cell product, and wherein said cell product is usable to generate a cell therapy.

165. The system of claim 164, wherein said density is at least 10 million cells / cm3.

166. The system of any one of claims 164 or 165, wherein said 3D matrix has a surfacearea to volume ratio greater than or equal to 0.1.

167. The system of any one of claims 164-166, wherein said 3D matrix has a porosity of atleast about 80%.

168. The system of any one of claims 164-167, wherein said 3D matrix has a dimension ofless than about 200 micrometers (µm).

169. The system of any one of claims 164-168, wherein said 3D matrix comprises aporous, solid matrix having interconnected pores with an average pore size sufficient to permit growth of lymphocyte cells within said 3D matrix.

170. The system of any one of claims 164-169, further comprising a plurality of 3Dmatrices comprising said 3D matrix.

171. The system of any one of claims 164-170, further comprising a fluid flow path in fluidcommunication with said 3D matrix, wherein said fluid flow path is adjacent to and independent from said 3D matrix.

172. The system of claim 171, further comprising a membrane disposed between said fluidflow path and said 3D matrix.

173. A method for manufacturing a cell therapy, comprising (i) providing a population ofcells at a density of at least about 5 million cells per centimeters (cells / cm3) retained within a three-dimensional (3D) matrix; (ii) processing said cells of said population of cells within said 3D matrix for a time period of at most about 48 hours to generate a cell product; and (iii) using said cell product to generate said cell therapy.

174. The method of claim 173, wherein said processing of said cells comprises culturingsaid population of cells.WSGR Docket No.67566-702601175. The method of claim 173 or 174, wherein said processing of said cells comprises insitu electroporation of said population of cells.

176. The method of claim 174, wherein said 3D matrix has a conductivity from about 10-5Siemens per m (S / m) to about 10-1S / m.

177. The method of any one of claims 173-176, wherein said processing of said cellscomprises activation via membrane signal transduction in said 3D matrix.

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