Systems and methods for processing cells

Abstract

The present disclosure provides systems and methods for processing cells. Systems of the present disclosure can include a compartment, a three-dimensional (3D) matrix, a fluidic flow path, an analysis unit, a preservation unit, or any combination thereof. Methods of the present disclosure can process cells via, for example, expansion, culture, gene delivery, activation, or any combination thereof. The systems and methods described herein can be used to produce cell products, including, for example, cell therapy products.

Description

[0001] Cross-references This application claims the 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 incorporated herein by reference in its entirety. Background Technology

[0002] Cell processing technologies have applications in a variety of fields, such as life science research, as well as industrial and therapeutic manufacturing. Bioreactor technologies and bioprocessing systems are useful tools for facilitating the culture, processing, and manufacture of cells and valuable cell byproducts. Summary of the Invention

[0003] This disclosure provides systems and methods for processing cells. Cell processing systems can allow for fully automated cell processing. Automated cell processing can include cell expansion, gene delivery, development of cell therapies, biopharmaceutical manufacturing, or any combination thereof. Compared to other cell processing systems, the systems and methods described herein can include simpler fluid dynamics, which allows for cell processing in smaller and less expensive instruments. The systems and methods described herein allow for cell processing with reduced cell manipulation and in an environment that replicates cells in their natural environment, which can maintain or produce healthier cells compared to other cell processing systems and methods. Therefore, the systems and methods described herein can provide improved cell manipulation and processing, resulting in cells or cell products with improved reproducibility, viability, and overall health compared to cells produced using other systems and methods.

[0004] In one aspect, this disclosure provides a system for manufacturing a cell product, the system 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) allow fluid to flow in a first direction, the first direction being angled relative to a second flow direction within the compartment, wherein the angle is greater than zero degrees, and (ii) supply material from the fluid to the compartment thereby generating the cell product; and an analysis unit coupled to the compartment and the fluid flow path and located within the same housing as the compartment and the fluid flow path, wherein the analysis unit is configured to monitor the plurality of cells or fluid during cell product generation when material is supplied to the compartment.

[0005] In some embodiments, the material includes reagents or nutrients. In some embodiments, 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 a cell therapy product at a scale equivalent to at least one human dose. In some embodiments, the compartment is configured to contain multiple cells. In some embodiments, multiple cells or portions thereof are fixed within the compartment. In some embodiments, multiple cells are encapsulated within the compartment.

[0006] In some embodiments, the analytical unit is configured to monitor multiple cells in a compartment. In some embodiments, the analytical unit is configured to monitor multiple cells in real time. In some embodiments, the analytical unit monitors or detects one or more properties selected from: biomarker expression, phenotype, viability, concentration, potency, sterility, endotoxin, and safety. In some embodiments, the system further includes 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: cell enrichment, cell selection, cell separation, washing, concentration, buffer exchange, and depolymerization.

[0007] In some embodiments, the compartment is configured to provide multiple cells with one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection. In some embodiments, the selection is negative selection. In some embodiments, the housing has a volume of approximately 0.01 cubic meters (m³). 3 ) to about 2 m 3 The volume. In some implementations, the volume is at most 1m³. 3 In some implementations, the volume is at most 0.5 m³. 3 In some implementations, the system is a sterile, closed system.

[0008] In some embodiments, the analytical unit includes one or more sensors configured to monitor an environment containing multiple cells. In some embodiments, the one or more sensors are selected from metabolite sensors, dissolved gas sensors, pH sensors, temperature sensors, viscosity sensors, and pressure sensors. In some embodiments, the analytical unit includes one or more sensors configured to analyze cell products. In some embodiments, the one or more sensors are selected from 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, conductivity sensors, radio frequency impedance sensors, dielectric spectroscopy, permittivity sensors, fluorescence imaging, mass spectrometry, optical density, and magnetic resonance angiography. In some embodiments, the system further includes one or more temperature sensors and a temperature control unit. In some embodiments, the system further includes multiple compartments containing chambers and multiple fluid paths containing fluid flow paths.

[0009] In another aspect, this disclosure provides a system for cell processing, the system 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 some embodiments, the system further includes a receiving unit configured to receive a plurality of cells. In some embodiments, the plurality of cells are 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 a cell processing unit. In some embodiments, the system is a sterile, 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: cell enrichment, cell selection, cell separation, washing, concentration, buffer exchange, and depolymerization. In some embodiments, the cell processing unit is configured to contain a plurality of cells. In some embodiments, a plurality of cells or a portion thereof are immobilized within the cell processing unit. In some embodiments, a plurality of cells are encapsulated within the cell processing unit.

[0011] In some embodiments, the system further includes a bioreactor coupled to a cell processing unit, a preservation unit, or an analysis unit. In some embodiments, the bioreactor is a solid-state bioreactor. In some embodiments, the bioreactor is configured to provide a plurality of cells with one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection.

[0012] In some embodiments, the analytical unit is configured to provide in-situ real-time detection or monitoring of one or more properties of multiple cells or cell products. In some embodiments, one or more properties of the multiple cells or cell products are selected from cell viability, density, concentration, cytometry, pH, dissolved oxygen, and metabolites. In some embodiments, the analytical unit is configured to provide release testing of the cell product. In some embodiments, the release test includes one or more assay methods selected from: imaging cytometry, quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR), enzyme-linked immunosorbent assay (ELISA), cell-based assays, cell counting, viable cell counting, and imaging bead counting.

[0013] In some embodiments, the cell processing unit, analysis unit, and storage unit are integrated within the housing. In some embodiments, the housing has a volume of approximately 0.01 cubic meters (m³). 3 ) to about 2 m 3 The volume. In some implementations, the volume is at most 1m³. 3 In some implementations, the volume is at most 0.5 m³. 3 In some embodiments, the analytical unit includes one or more sensors configured to monitor an environment containing multiple cells. In some embodiments, the one or more sensors are selected from metabolite sensors, dissolved gas sensors, pH sensors, temperature sensors, viscosity sensors, and pressure sensors. In some embodiments, the analytical unit includes one or more sensors configured to analyze cell products. In some embodiments, the one or more sensors are selected from 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, conductivity sensors, radio frequency impedance sensors, dielectric spectroscopy, permittivity sensors, fluorescence imaging, mass spectrometry, optical density, and magnetic resonance angiography.

[0014] In another aspect, this disclosure provides a method for manufacturing a cell product, the method comprising: providing a plurality of cells to a system including (i) a compartment for retaining the plurality of cells, (ii) a fluid flow path in fluid communication with the compartment, and (iii) an analytical unit coupled to the compartment and the fluid flow path and located within the same housing as the compartment and the fluid flow path; providing material from fluid in the fluid flow path to the compartment to generate the cell product, wherein the fluid flow path causes the fluid to flow in a first direction, the first direction being angled relative to a second flow direction within the compartment, wherein the angle is greater than zero degrees; and using the analytical unit to monitor the plurality of cells or fluid during cell product generation when material is provided to the compartment.

[0015] In some embodiments, the material includes reagents or nutrients. In some embodiments, 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 produced at a scale of at least one human dose equivalent. In some embodiments, the compartment is configured to contain multiple cells. In some embodiments, multiple cells or portions thereof are fixed within the compartment. In some embodiments, multiple cells are encapsulated within the compartment.

[0016] In some embodiments, the analytical unit monitors multiple cells in a compartment. In some embodiments, the analytical unit monitors multiple cells in real time. In some embodiments, the analytical unit monitors or detects one or more properties selected from: biomarker expression, phenotype, viability, concentration, potency, sterility, endotoxin, and safety. In some embodiments, the method further includes using a cell processing unit to generate a cell product. In some embodiments, the cell processing unit provides one or more processes selected from: cell enrichment, cell selection, cell separation, washing, concentration, buffer exchange, and depolymerization. In some embodiments, the method further includes subjecting multiple cells to one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection. In some embodiments, the selection is negative selection. In some embodiments, the housing has a volume of approximately 0.01 cubic meters (m³). 3 ) to about 2 m 3 The volume.

[0017] In another aspect, this disclosure provides a method for cell processing, the method comprising: providing a plurality of cells to a system including (i) a cell processing unit holding 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 processing or preservation.

[0018] In some embodiments, the cell product is a cell therapy product. In some embodiments, the method further includes providing a plurality of cells to a receiving unit coupled to a cell processing unit, a storage unit, or a storage unit. In some embodiments, the plurality of cells provided to the receiving unit are a plurality of cryopreserved cells. In some embodiments, the receiving unit subjectes 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 a cell processing unit. In some embodiments, the storage unit generates a cryopreserved cell product.

[0019] In some embodiments, the method further includes subjecting multiple cells to one or more processes selected from: cell enrichment, cell selection, cell separation, washing, concentration, buffer exchange, and depolymerization. In some embodiments, the method further includes a bioreactor coupled to a cell processing unit, a preservation unit, or an analysis unit. In some embodiments, the bioreactor is a solid-state bioreactor. In some embodiments, the method further includes using the bioreactor to subject multiple cells to one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection.

[0020] In some embodiments, the analytical unit provides in-situ real-time detection or monitoring of one or more properties of multiple cells or cell products. In some embodiments, one or more properties of the multiple cells or cell products are selected from cell viability, density, concentration, cytometry, pH, dissolved oxygen, and metabolites. In some embodiments, the method further includes using the analytical unit to perform release testing of the cell product. In some embodiments, the release testing includes one or more testing methods selected from: imaging cytometry, qPCR, dPCR, ELISA, cell-based assays, cell counting, viable cell counting, and imaging bead counting.

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

[0022] In some embodiments, the cell product is a cell therapy product. In some embodiments, the 3D matrix and the fluid flow path are separated by a membrane. In some embodiments, the 3D matrix is ​​configured as a plate, the plate comprising a first dimension and a second dimension greater 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, the fiber comprising a diameter 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 a tangential flow of fluid flows along the outer circumference of the sphere. In some embodiments, multiple 3D matrices are fluidly coupled to each other. In some embodiments, the 3D matrix is ​​configured to contain multiple cells. In some embodiments, the 3D matrix is ​​configured to immobilize multiple cells. In some embodiments, the 3D matrix is ​​configured to encapsulate multiple cells.

[0023] In some embodiments, a fluid flow path is disposed between a first 3D matrix and a second 3D matrix. In some embodiments, the fluid flow path is configured to deliver fluid material to both the first and second 3D matrices. 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 of about 0.5 µm to about 100 μm. In some embodiments, the 3D matrix comprises a porosity greater than or equal to about 80%. In some embodiments, the interfacial area between the fluid flow path and the 3D matrix is ​​greater than or equal to about 5 square centimeters (cm²). 2 In some embodiments, the 3D matrix comprises a porous solid matrix having interconnected pores with an average pore size sufficient to allow lymphocytes to grow 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 greater than or equal to about 0.1. In some embodiments, the 3D matrix has a surface area to volume ratio of about 10. -5 Siemens / m (S / m) to approximately 10 -1The conductivity is measured in S / m. In some embodiments, the 3D matrix is ​​configured to provide activation of multiple cells via membrane signal transduction within the 3D matrix.

[0024] In another aspect, this disclosure provides a method for cell processing, the method comprising: providing (i) a cell population, (ii) a plurality of three-dimensional (3D) matrices, and (iii) a plurality of fluid flow paths, wherein the cell population is retained within the 3D matrices of the plurality of 3D matrices, and wherein the fluid flow paths of the plurality of fluid flow paths are independent of and adjacent to the 3D matrices; using the fluid flow paths to deliver fluid material to the 3D matrices; and processing the cell population into a cell product in the presence of the fluid material delivered to the 3D matrices.

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

[0026] In some embodiments, the method further includes functionalizing a 3D matrix to generate a functionalized 3D matrix. In some embodiments, functionalization includes conjugating one or more biomolecules to the 3D matrix. In some embodiments, the one or more biomolecules are selected from proteins, peptides, cytokines, antibodies, and nucleic acid molecules. In some embodiments, the one or more biomolecules may be used for membrane signal transduction. In some embodiments, the method further includes polymerizing a prefunctionalized polymer precursor to generate a 3D matrix. In some embodiments, functionalization occurs simultaneously with the provision of (i) a cell population, (ii) multiple three-dimensional (3D) matrices, and (iii) multiple fluid flow paths. In some embodiments, functionalization occurs after the provision of (i) a cell population, (ii) multiple three-dimensional (3D) matrices, and (iii) multiple fluid flow paths.

[0027] In some embodiments, the method further includes releasing one or more biomolecules from the 3D matrix, or replacing 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 fluid material is transported through the membrane. In some embodiments, the 3D matrix has a size of less than or equal to about 10 millimeters (mm). In some embodiments, the size is less than or equal to about 1 mm. In some embodiments, the size is less than or equal to about 500 micrometers (μm). In some embodiments, the size is less than or equal to about 200 μm. In some embodiments, multiple 3D matrices are fluidly coupled to each other. In some embodiments, a fluid flow path is provided between a first 3D matrix and a second 3D matrix among the multiple 3D matrices. In some embodiments, the fluid flow path delivers fluid 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 to about 400 micrometers (μm). In some embodiments, the 3D matrix comprises a pore size of 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, the interfacial area between the fluid flow path and the 3D matrix is ​​greater than or equal to about 5 square centimeters (cm²). 2 In some embodiments, the 3D matrix comprises a porous solid matrix having interconnected pores with an average pore size sufficient to allow lymphocytes to grow within the 3D matrix. In some embodiments, the 3D matrix has a surface area to volume ratio greater than or equal to about 0.1. In some embodiments, the 3D matrix is ​​a programmable 3D matrix.

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

[0030] In some embodiments, multiple cells are encapsulated in a 3D matrix. In some embodiments, the 3D matrix is ​​configured to retain multiple cells. In some embodiments, the 3D matrix is ​​configured to immobilize multiple cells. In some embodiments, the multiple treated cells are cell therapy products. 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 includes providing a mixture comprising a matrix precursor and multiple cells to a cell processing system.

[0031] In some embodiments, the method further includes, in a cell processing unit, applying a first stimulus to a mixture comprising a matrix precursor and a plurality of cells to generate a 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 includes, while applying the first stimulus to generate the 3D matrix, functionalizing the 3D matrix with one or more biomolecules. In some embodiments, (ii) includes applying a second stimulus to the 3D matrix to release cell products 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 includes generating a 3D matrix in the presence of multiple cells before providing the 3D matrix, such that the 3D matrix encapsulates the multiple cells. In some embodiments, providing the 3D matrix includes loading the 3D matrix encapsulating the multiple cells into a compartment in a cell processing unit. In some embodiments, providing the 3D matrix includes providing the multiple cells and the 3D matrix separately 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 outside the cell processing system and subsequently provided to the cell processing unit.

[0033] In some embodiments, the method further includes functionalizing the 3D matrix to generate a functionalized 3D matrix. In some embodiments, functionalization includes conjugating one or more biomolecules to the 3D matrix. In some embodiments, the one or more biomolecules are selected from proteins, peptides, cytokines, antibodies, and nucleic acid molecules. In some embodiments, the one or more biomolecules can be used for membrane signal transduction. In some embodiments, the method further includes functionalizing the 3D matrix with one or more biomolecules prior to (i). In some embodiments, the method further includes functionalizing the 3D matrix with one or more biomolecules concurrently with (i). In some embodiments, the method further includes releasing one or more biomolecules from the 3D matrix, or replacing one or more biomolecules with additional biomolecules. In some embodiments, (i) includes activating multiple cells within the 3D matrix to obtain a cell product. In some embodiments, (i) includes transducing multiple cells within the 3D matrix to obtain a cell product. In some embodiments, (i) includes transfecting multiple cells within the 3D matrix to obtain a cell product. In some embodiments, the 3D matrix has about 10 -5 Siemens / m (S / m) to approximately 10 -1The conductivity is measured in S / m. In some embodiments, the method further includes activating multiple cells via membrane signal transduction in a 3D matrix.

[0034] In another aspect, this 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) guide the cell processing unit to separate the plurality of processed cells from the 3D matrix, and (iii) guide the cell processing unit to collect the plurality of processed cells.

[0035] In some embodiments, the method further includes a receiving unit configured to receive a plurality of cells from a user. In some embodiments, one or more computer processors are operatively coupled to the receiving unit and are individually or collectively programmed to automatically direct the receiving unit to provide a plurality of cells to a cell processing unit. In some embodiments, the receiving unit is configured to receive a plurality of cells in a cryopreserved state, and wherein the receiving unit is configured to provide controlled thawing of the plurality of cells in the cryopreserved state. In some embodiments, the method further includes an analytical unit configured to monitor the plurality of cells during processing. In some embodiments, the analytical unit is configured to monitor the plurality of cells in real time. In some embodiments, the analytical unit monitors or detects one or more properties selected from: biomarker expression, phenotype, viability, concentration, potency, sterility, endotoxin, and safety. In some embodiments, the analytical unit includes one or more sensors configured to monitor an environment containing the plurality of cells. In some embodiments, the one or more sensors are selected from metabolite sensors, dissolved gas sensors, pH sensors, temperature sensors, viscosity sensors, and pressure sensors. In some embodiments, the analytical unit includes one or more sensors configured to analyze cell products. In some embodiments, one or more sensors are selected from 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, conductivity sensors, radio frequency impedance sensors, dielectric spectroscopy, permittivity sensors, fluorescence imaging, mass spectrometry, optical density, and magnetic resonance angiography. In some embodiments, the cell processing unit is further configured to provide one or more processes selected from: cell enrichment, cell selection, cell separation, washing, concentration, buffer exchange, and depolymerization. In some embodiments, cell selection is negative selection. In some embodiments, the method further includes a housing configured to retain the cell processing unit and one or more computer processors. In some embodiments, the housing has a volume of approximately 0.01 cubic meters (m³). 3 ) to about 2 m 3 The space it occupies.

[0036] In some embodiments, the 3D matrix is ​​configured to contain multiple cells. In some embodiments, the 3D matrix is ​​configured to immobilize multiple cells. In some embodiments, the 3D matrix is ​​configured to encapsulate multiple cells. In some embodiments, the cell processing unit is configured to generate the 3D matrix in the presence of multiple cells. In some embodiments, the cell processing unit is configured to receive a pre-formed 3D matrix. In some embodiments, the cell processing unit is configured to receive a pre-formed 3D matrix containing multiple cells. In some embodiments, the cell processing unit is configured to encapsulate multiple cells in the pre-formed 3D matrix. In some embodiments, the 3D matrix has approximately 10 -5 Siemens / m (S / m) to approximately 10 -1 The conductivity is measured in S / m. In some embodiments, the 3D matrix is ​​configured to provide activation of multiple cells via membrane signal transduction within the 3D matrix.

[0037] In another aspect, this disclosure provides a method for cell processing, the method comprising: providing a plurality of cells ex vivo to a cell processing unit; in the cell processing unit, contacting the plurality of cells with a selection reagent, wherein upon contact, the selection reagent (i) is cell-coupled to a subpopulation of the plurality of cells, and (ii) perturbing the state of one or more cells in the subpopulation to produce (A) a perturbed cell population and (B) an undisturbed cell population; and processing the perturbed cell population or the undisturbed cell population into a cell therapy product.

[0038] In some embodiments, the cell therapy product is produced at a scale of at least one human dose equivalent. In some embodiments, the perturbed cell population has an altered growth rate or viability compared to an undisturbed cell population. In some embodiments, the method further includes contacting a plurality of cells with a selection agent and simultaneously processing either the perturbed or undisturbed cell population into a cell therapy product. In some embodiments, the method further includes processing the perturbed or undisturbed cell population after contacting the plurality of cells with the selection agent.

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

[0040] In some embodiments, the selection agent comprises a binder and a selector. In some embodiments, the selector is a cytotoxic agent. In some embodiments, the binder is coupled to the selector via a adapter. In some embodiments, the adapter is a cleavable adapter. In some embodiments, the binder is an antibody. In some embodiments, the binder is an aptamer. In some embodiments, the perturbed cell population comprises dead cells or cell degradation products. In some embodiments, the perturbed cell population has altered cell morphology compared to an undisturbed cell population. In some embodiments, the treatment comprises one or more processes selected from: cell culture, transduction, transfection, transformation, amplification, in situ electroporation, and gene delivery.

[0041] In another aspect, this disclosure provides a system for manufacturing cell therapy products, the system comprising: a three-dimensional (3D) matrix configured to have a density greater than about 80 million cells per cubic centimeter (cells / cm³). 3 The system preserves cell populations at high density, and can process the cells of the cell population into cell therapy products using a 3D matrix.

[0042] In some implementations, the density is at least 100 million cells / cm³. 3 In some embodiments, the 3D matrix is ​​configured to contain a cell population. In some embodiments, the 3D matrix is ​​configured to immobilize a cell population. In some embodiments, the 3D matrix is ​​configured to encapsulate a cell population. 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 includes a plurality of 3D matrices comprising the 3D matrix. In some embodiments, the method further includes a fluid flow path in fluid communication with the 3D matrix, wherein the fluid flow path is adjacent to and independent of the 3D matrix. In some embodiments, the method further includes a membrane disposed between the fluid flow path and the 3D matrix. In some embodiments, the 3D matrix is ​​programmable.

[0043] In another aspect, this disclosure provides a method for manufacturing a cell therapy product, the method comprising (i) providing a concentration of more than about 80 million cells / cm³ (cells / cm²). 3 (ii) The density of the cell population is preserved within a three-dimensional (3D) matrix, and (iii) the cells of the cell population are processed into cell therapy products using the 3D matrix.

[0044] In some embodiments, cell treatment includes culturing cell populations. In some embodiments, cell treatment includes in-situ electroporation of cells. In some embodiments, the 3D matrix has approximately 10 -5 Siemens / m (S / m) to approximately 10 -1Electrical conductivity (S / m). In some embodiments, cell treatment includes activating cells in a 3D matrix.

[0045] In another aspect, this disclosure provides a bioreactor for processing cell populations, the bioreactor comprising a three-dimensional (3D) matrix configured to retain the cell population, wherein the bioreactor has a maximum of about 2 cubic meters (m³). 3 The volume of the cell population is configured to process the cell population into cell products with high reproducibility using a 3D matrix.

[0046] In some embodiments, the cell product has a viability of ≥75%. In some embodiments, the volume is at most 1 m³. 3 In some implementations, the volume is at most 0.5 m³. 3 .

[0047] In another aspect, this disclosure provides a method for processing a cell population, the method comprising (i) providing a cell population retained within a three-dimensional (3D) matrix of a bioreactor, wherein the bioreactor has a mass of up to 2 cubic meters (m³). 3 (ii) The volume of the cell population and the processing of the cell population into cell products with high reproducibility using a 3D matrix.

[0048] In some embodiments, the cell product has a viability of about 75%. In some embodiments, the method further includes generating a 3D matrix in the presence of a cell population before step (i). In some embodiments, the method further includes dissolving the 3D matrix and collecting the cell population after step (ii) to generate the cell product.

[0049] In another aspect, this disclosure provides a system for manufacturing cell therapies, the system comprising: a three-dimensional (3D) matrix configured to contain at least about 5 million cells per cubic centimeter (cells / cm³). 3 The system preserves a high density of cell populations, wherein the system is capable of treating the population of cells within a 3D matrix for a period of up to approximately 48 hours to generate cell products, and wherein the cell products can be used to generate cell therapies.

[0050] In some implementations, the density is at least 10 million cells / cm². 3 In some implementations, the density is at least 20 million cells / cm³. 3In 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 size of less than about 200 micrometers (μm). In some embodiments, the size is less than about 100 μm. In some embodiments, the size is less than about 10 μm. In some embodiments, the size 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 allow lymphocytes to grow within the 3D matrix. In some embodiments, the system further comprises a plurality of 3D matrices containing 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 to and independent of 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, this disclosure provides a method for manufacturing cell therapy, the method comprising (i) providing at least about 5 million cells / cm³ (cells / cm²) 3 (ii) Preserving the density of cell populations within a three-dimensional (3D) matrix; (iii) Treating the cell populations within a 3D matrix for a period of up to approximately 48 hours to generate cell products; and (iv) Using the cell products to generate cell therapies.

[0052] In some embodiments, cell treatment includes culturing a cell population. In some embodiments, cell treatment includes in-situ electroporation of the cell population. In some embodiments, the 3D matrix has approximately 10 -5 Siemens / m (S / m) to approximately 10 -1 The conductivity in S / m. In some embodiments, cell treatment includes activation via membrane signal transduction in the 3D matrix.

[0053] Other aspects and advantages of this disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only illustrative embodiments thereof. As will be appreciated, this disclosure is capable of other and different embodiments, and several details thereof can be modified in various obvious respects without departing from this disclosure. Therefore, the drawings and descriptions are to be considered illustrative rather than limiting in nature.

[0054] Incorporation All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and independently indicated to be incorporated by reference. Where a publication, patent, or patent application incorporated by reference contradicts the disclosure contained in this specification, this specification is intended to supersede or give precedence to any such contradictory material. Attached Figure Description

[0055] The novel features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments in which the principles of the invention are utilized, along with the accompanying drawings (also referred to herein as “figure” and “FIG.”), in which: Figure 1 Example cell products that can be generated by the systems and methods described herein are shown; Figure 2 An example of chimeric antigen receptor (CAR)-T cell therapy manufacturing is shown; Figure 3 An example integrated cell processing system and consumables are schematically shown; Figure 4 An example cell processing unit and a solid-state bioreactor are schematically shown; Figure 5A and 5B An example process provided by the example integrated cell processing system is shown; Figure 5A An example process provided by an example integrated cell processing system is illustrated schematically; and Figure 5B An example cell processing workflow is illustrated schematically; Figure 6 The glucose level as a function of time is schematically shown for an example bioreactor configuration; Figure 7 An example workflow for producing cell products from an example cell processing system is illustrated schematically. Figure 8 An example unit integrated into an example cell processing system is schematically shown; Figure 9A The transfer of fluids, nutrients, or reagents in example large-volume and microfluidic bioreactors is illustrated schematically. Figure 9B The transfer of fluids, nutrients, or reagents in an example tangential or perfusion bioreactor is illustrated schematically. Figure 10A and 10B An example 3D matrix configuration and fluid interface are schematically illustrated; Figure 11A and 11BThe functional physical properties of an example 3D matrix are illustrated schematically. Figure 12A and 12B An example of 3D matrix functionalization is illustrated schematically; Figure 13 Example interfaces between the 3D matrix and the fluid flow path, and between the 3D matrix and the cell substrate, are schematically shown; Figure 14 An example cell loading method is illustrated schematically; Figure 15 The illustration schematically shows the loading of an example 3D matrix into a cell compartment; Figure 16A and 16B An example of reagent delivery and removal from a 3D matrix is ​​illustrated schematically; Figure 17 The sample reagent delivery size and corresponding size cutoff value are shown; Figure 18 An example cell processing unit including a 3D matrix and flow paths is shown; Figure 19 The single-fluid flow path and dual-fluid flow path configurations are schematically shown; Figure 20 An example cell processing unit configured for tangential flow is schematically shown; Figures 21A-21C An example plate-like 3D matrix configuration and fluid flow path are schematically illustrated; Figure 22A and 22B An example fibrous 3D matrix configuration and fluid flow path are schematically illustrated; Figure 23 An example cell processing unit including a 3D matrix and fluid flow paths is schematically shown; Figure 24 Images of an example cell processing unit, including a 3D matrix and fluid flow paths, are shown. Figure 25 An exploded view of an example cell processing unit is shown; Figure 26 An example box comprising multiple cell processing units is shown; Figure 27 An example of cell processing consumables is illustrated schematically; Figure 28 An alternative container configuration is schematically shown; Figure 29 Example 3D matrix configurations and bioreactor vessel types are schematically illustrated; Figure 30 An example upstream module for cell processing is illustrated schematically; Figure 31An additional example upstream module for cell processing is illustrated schematically; Figure 32 An example downstream module for cell processing is illustrated schematically; Figure 33 Additional example upstream and downstream modules for cell processing are schematically shown; Figure 34 An example sterile fluid interface is schematically shown; Figure 35 The workflow for generating sterile fluid coupling is illustrated schematically; Figure 36 This schematically illustrates a comparison between non-sterile connectors and sterile fluid connectors; Figure 37 This schematically illustrates another workflow for generating sterile fluid coupling using an in-situ sterile connector; Figure 38 An example hybrid interconnect is schematically shown; Figure 39 The diagram schematically illustrates the automation of aseptic interconnection; Figure 40 The workflows for negative selection and magnetic cell selection are illustrated schematically; Figure 41 Examples of in situ stimulation of cells within a 3D matrix and in suspension are shown; Figure 42 An example of negative cell selection of CD19 cells from peripheral blood mononuclear cells (PBMCs) is shown; Figure 43 Example dose-response curves for negative cell selection using example antibody-drug conjugates are shown; Figure 44 An example in-situ process for generating cell products is shown; Figure 45 Example cell transduction is shown as a function of cell viability in cells transduced in suspension and 3D matrix; Figure 46 Examples of lentiviral transduction are shown in suspended cells and cells encapsulated in a 3D matrix; Figure 47 An example of electroporation within a 3D matrix is ​​shown; Figure 48 Example optical density measurements are shown in 3D matrices and suspension cultures; Figure 49 Example optical density measurements were shown in 3D matrix and liquid culture medium; Figure 50 Examples of monitoring of cell expansion in 3D matrix and suspension culture are shown; Figure 51 Examples of monitoring of cell viability in 3D matrix and suspension are shown; Figure 52 Example flow cytometry measurements of cultures expanded in 3D matrix and suspension culture are shown; Figure 53 Example release test is shown; Figure 54 This demonstrates an example workflow for manufacturing cell and gene therapies; Figure 55 An example process executed during the workflow is illustrated schematically; Figure 56 This shows an example workflow for CAR-T manufacturing; Figure 57 This schematically illustrates another example of a CAR-T cell manufacturing workflow; Figure 58 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein; Figure 59A and 59B An example configuration for scaling up cell processing is illustrated schematically; Figure 60A-60C An example electroporation configuration is illustrated schematically; Figures 61A-61B Other example electroporation configurations are illustrated schematically; Figure 62A and 62B An example optical density measurement is illustrated schematically; Figure 63 Another example optical density measurement configuration is illustrated schematically; Figure 64 Example consumables are shown schematically; Figure 65 The internal components of the example consumable are shown schematically; Figure 66 An example diagram of electronic devices and components used in an example cell processing system is shown schematically. Figure 67 An example irrigation module is illustrated schematically; Figure 68 Another example irrigation module is illustrated schematically; Figure 69 Another example irrigation module is illustrated schematically; Figure 70 The workflow of the example irrigation module is illustrated schematically; Figure 71 An example filtering module is illustrated schematically; Figure 72Another example filtering module is illustrated schematically; Figure 73 Another example filtering module is illustrated schematically; Figure 74 Another example filtering module is illustrated schematically; Figure 75 Example cell expansion of donor cells in 3D cell cultures and suspensions is shown; Figure 76 The transduced sample cells show expansion and viability; Figure 77 Example images of the 3D matrix prior to cell harvesting are shown; Figure 78 An example 3D bioreactor is shown during cell expansion; Figure 79 An example sensor that can be used for 3D cell culture is shown; Figure 80 Examples of phenotypic quality of cells subjected to 3D culture versus suspension culture are shown; Figure 81 Example T cell activation and expansion using a functionalized matrix are shown; Figure 82 Examples of antibody conjugation with functionalized 3D matrix are shown; Figure 83 Example cell separation using size selection is shown; Figure 84 A sample filtering module is shown; Figure 85 Another example filtering module is shown; Figure 86 Example cell filtering is shown; and Figure 87 Another example of cell filtration is shown. Detailed Implementation

[0056] Although various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will 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.

[0057] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first value in a series of two or more values, the terms "at least," "greater than," or "greater than or equal to" apply to each value in the series. 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.

[0058] Whenever the terms "not exceeding," "less than," or "less than or equal to" precede the first value in a series of two or more values, the terms "not exceeding," "less than," or "less than or equal to" apply to each value in the series. 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.

[0059] As used herein, the terms “automatic” or “automatically” generally refer to a device, system, or process that operates without or substantially without external instruction or intervention. For example, a system or device may be configured to operate a process from start to finish, or to operate a process from start to finish, without or substantially without interference from a human operator. Alternatively or supplementarily, a system or device may be configured to operate a part of a process, or to operate a part of a process, without or substantially without interference from a human operator.

[0060] The terms "cell processing system" or "bioprocessing system" (which are used interchangeably) generally refer to a multi-component system configured for processing cells. A cell processing system may include a bioreactor, cell processing units, analytical systems, control systems, preservation systems, other components useful for processing cells, or any combination thereof. In one example, a cell processing system includes a bioreactor containing cell processing units and additional cell processing units separate from the bioreactor. In another example, a cell processing system includes cell processing units and does not include a bioreactor. In yet another example, a cell processing system may be a bioreactor including cell processing units integrated with one or more other components, such as analytical systems, control systems, or other systems.

[0061] As used herein, the term "cell processing unit" generally refers to a device, module, or unit configured to process cells. A cell processing unit may include compartments configured to retain cells, or compartments that retain cells (e.g., cell compartments). As described elsewhere herein, a cell compartment may include or contain a three-dimensional substrate providing cell retention. A cell processing unit may include one or more other components that facilitate or assist in cell processing. One or more other components may include one or more of the following: fluid flow paths, interfaces, sensors, or other components useful for cell processing. A cell processing unit may be configured to process cells without amplifying or culturing them. Alternatively or supplementally, a cell processing unit may be configured to amplify or culture cells. A cell processing system or bioreactor may include one or more cell processing units.

[0062] As used herein, the term "bioreactor" generally refers to an apparatus or system that supports a biologically active environment for the growth or expansion of cell populations. A bioreactor may include a container, tank, compartment, or other environment in which whole cells are grown or cultured, or in which cells are processed to generate cell products or cell expression products. A bioreactor may include a single container, tank, compartment, or other environment. Alternatively or supplementally, a bioreactor may include multiple containers, tanks, compartments, or other environments. A bioreactor may provide a controlled environment for biological reactions, chemical reactions, or both. A bioreactor may include one or more cell processing units, as described elsewhere herein, that provide an environment for cell expansion or culture. A bioreactor may include one or more cell processing units.

[0063] As used herein, the term "cell therapy product" generally refers to a therapeutic product intended for implantation, transplantation, infusion, or transfer to cells or tissues of a subject, such as a patient. A cell therapy product can be a cellular product sufficient to treat the subject or patient. A cell therapy product can be a generic product or a personalized cell therapy product. Personalized cell therapy products can be manufactured from the subject's own cells or from cell lines or other cell sources.

[0064] As used herein, the term "automated system" generally refers to a system configured to process cells with little or no operator intervention. For example, an automated system may not have, or may not allow, the transfer of substances into or out of the system by an operator during operation. For instance, an automated system may allow the transfer of samples, cells, or consumables into the system by an operator when the system is not running (e.g., before operation), but may not allow intervention during cell processing. In another example, an automated system may allow the input of samples, cells, consumables, reagents, etc., into the system before, during, or after operation, but may not allow intervention in cell processing. An automated system may be coupled to one or more other modules or devices (e.g., a sequencer) such that substances (e.g., samples or cells) can be transferred to the coupled unit, module, or device with little or no human intervention.

[0065] As used herein, the term "closed system" generally refers to a system configured to process cells with minimal or no environmental contamination. A closed system can be a sterile closed system. A sterile closed system can be a system that prevents microorganisms from entering or leaving the system to protect cell products, personnel, or facilities from contamination.

[0066] As used herein, the term "integrated system" generally refers to a system comprising multiple components, devices, apparatuses, or combinations thereof coupled together such that the components, devices, apparatuses, or combinations thereof function as a single system. The individual components of the system may be housed in a single housing. Alternatively or complementarily, the individual components of the system may be housed in separate housings and coupled electronically, fluidly, or pneumatically to function as a single system. In one example, the integrated system may include the robotic or mechanical transfer of objects (e.g., consumables, reagents, samples, cells, etc.) between separate housings. In one example, some components, devices, or apparatuses may be housed in a single housing, while others may be housed in another housing or their respective housings. Various components, devices, or apparatuses may include their own controllers. For example, an individual component, device, or apparatus may include a controller associated with that component, device, or apparatus. The system may also include a master controller that allows the individual components, devices, or apparatuses to be controlled as a functional system. Components, devices, or apparatuses may be coupled together via electronic, fluid, pneumatic, or other connections. Fluid or pneumatic connections can provide sterile coupling between the various components of the integrated system.

[0067] As used interchangeably herein, the terms “three-dimensional (3D) matrix” or “3D substrate” generally refer to a matrix or substrate configured for 3D cell processing. A 3D matrix or substrate can be configured to allow cell proliferation, migration, differentiation, or any combination thereof. As described elsewhere herein, a 3D matrix can include polymeric matrices, such as hydrogel matrices, 3D scaffolds, or combinations thereof.

[0068] As used herein, the term "microfluidics" generally refers to fluid handling components at the micrometer (μm) scale. For example, microfluidic fluid flow paths (e.g., channels), chambers, or other fluid handling components 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 can be configured or otherwise used to handle fluid volumes on the order of milliliters (mL) to picoliters (pL). For example, microfluidic devices can handle 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 larger.

[0069] As used in this article, the term "real-time" typically refers to a response time of less than about one second, one-tenth of a second, one-hundredth of a second, one millisecond, or less. In one example, real-time can refer to simultaneous or substantially simultaneous processing, detection, monitoring, analysis, or any combination thereof.

[0070] As used herein, the term "object" generally refers to a human or other mammal, bird, or other organism (e.g., a plant). For example, an object can be a vertebrate, mammal, rodent (e.g., a mouse), primate, ape, or human. Animals can include, but are not limited to, farm animals, sporting animals, and pets. An object can be a healthy or asymptomatic individual, an individual who has or is suspected of having a disease (e.g., cancer) or is susceptible to a disease, or an individual who is a candidate for a therapy (e.g., cell therapy). An object can be a patient.

[0071] As used herein, the term "sample" generally refers to a biological sample of an object. A biological sample may include a cell population, such as a heterogeneous or homogeneous cell population. A biological sample may provide a cell line or cell sample for cell processing. A sample may be a cell line or cell culture sample. A sample may include one or more cells. A sample may include one or more microorganisms. A biological sample may be derived from another sample. A sample may be a tissue sample, such as a biopsy, core biopsy, needle aspiration, or fine needle aspiration. A sample may be a fluid sample, such as a blood sample, urine sample, saliva sample, single sample, or single sample of white blood cells. A sample may be a skin sample. A sample may be a buccal swab. A sample may be a plasma or serum sample.

[0072] Cell treatment applications Cells are a vital material resource for life science research and development in both basic and applied scientific practices. Primary and secondary cells (including cell lines) are frequently propagated, expanded, and library-built for research purposes. Cells are also frequently modified, for example, through genome editing, for research applications. Furthermore, cell therapies (such as adoptive cell therapy and in vitro cell-based gene therapy) represent an emerging therapeutic modality with the potential to treat many types of human diseases, such as cancer, autoimmune diseases, neurological disorders, and age-related diseases.

[0073] The methods and systems described herein can be used for the production of cell therapies. Cell therapies, including adoptive cell therapy (ACT) and gene therapy based on in vitro cells, represent an emerging therapeutic modality with the potential to treat many types of human diseases, including, for example, cancer, autoimmune diseases, neurological diseases, and age-related diseases. In autologous therapies, cells can be collected from a patient, modified ex vivo, and reintroduced into the patient. In allogeneic therapies, cell sources different from those of the patient can 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 (iPSCs). Many emerging cell therapies utilize genome editing technologies to engineer cells to possess 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 can consist of immune cells or cells of immune lineages, such as T cells and hematopoietic stem cells and progenitor cells (HSPCs). Several advanced cell therapies delivering other cell types may have been approved or are in various stages of development, including therapies using a variety of 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., naturally occurring stem cells, such as those in tissues, bone marrow, and umbilical cord blood, as well as induced stem cells, such as iPSCs), and cell products derived from stem cells through directed differentiation or genetic programming to achieve targeted therapeutic cell states. Despite a large number of approved products and ongoing clinical trials, many challenges remain regarding reproducible and cost-effective manufacturing processes for cell therapy products with therapeutic and industrial value, gene therapy products based on in vitro cells, and other cell expression products.

[0074] The systems and methods described herein can be used to develop a variety of cell products. For example, and as... Figure 1 As shown, the systems and methods described herein can be used to culture or otherwise produce cell products from adherent cell types, stem cells and stem cell-derived cells, rare cell types, or any combination thereof. The systems and methods described herein can be used for personalized medicine, point-of-care therapy, or both.

[0075] Cell manufacturing can be a bottleneck, limiting patient accessibility, increasing costs, and reducing the commercial viability of cell therapy products. For example, each clinical dose of a cell therapy product may require 10 [cells / units / etc.]. 6 -10 9Each therapeutic cell. Improving the efficiency and rate of cell expansion while maintaining key properties can be challenging. Performing complex bioprocessing procedures at dose scale can also be challenging, and multiple instrument platforms can be used to execute various processes. Various processes constituting complex or multi-step cell processing workflows can be performed in series, increasing manufacturing time. Manufacturing challenges can be reflected in the high costs of researching and developing novel cell therapies, as well as the high costs of approved cell therapy products. Reproducible manufacturing can also be a challenge due to the functional heterogeneity of cells and the resulting variations in quality properties and clinical outcomes. Sources of product variability can include, but are not limited to: inter-patient variability and intra-population heterogeneity in the source material, or variability introduced during the manufacturing process, including variability during cell isolation, expansion, or genome engineering.

[0076] In the United States, approximately ten advanced cell therapies have been approved for human use. Although thousands of patients may be eligible for cell therapy, only tens of thousands have received it to date. Currently, hundreds of cell therapies are under development or being tested in clinical trials. Dozens more cell therapies are expected to be approved for human use in the next decade. New advances in clinical cell manufacturing can be used to alleviate the burden of researching and developing novel cell therapies and to make these effective treatments available to more patients. Furthermore, cell therapies can be manufactured in centralized facilities, which increases the complexity of the logistics of transporting and manufacturing fresh or cryopreserved cell products. Advances in clinical cell therapy manufacturing that enable alternative manufacturing paradigms, such as distributed manufacturing and point-of-care manufacturing, can reduce costs and otherwise improve patient accessibility. Advances in cell manufacturing that reduce the cost, labor, logistical complexity, biological variability, and turnaround time of cell therapy production can increase patient access to powerful new therapeutic modalities.

[0077] The systems and methods described herein can be used for the production of gene therapy, valuable cellular byproducts, and biotherapies. Cellular processes, whether natural or genetically engineered, are capable of producing a wide variety of chemicals and biocompounds that are essential in the pharmaceutical, chemical, food, energy, and agricultural industries. The bioproduction of clinically and commercially important bioproducts and chemicals from living cells can be complex and time-consuming, requiring sophisticated infrastructure, manufacturing equipment, and workflows. Bioreactors are key tools in bioproduction, enabling the controlled and large-scale production of valuable cellular byproducts. These specialized systems are designed to provide optimal environments for cellular growth and metabolic activity, allowing for the efficient production of biopharmaceuticals such as enzymes, therapeutic proteins, vaccines, viral particles, and other biopharmaceuticals. By precisely controlling parameters such as temperature, pH, oxygen levels, and nutrient supply, bioreactors ensure that cells are maintained under conditions that maximize their productivity.

[0078] One application of bioreactors is the production of biopharmaceuticals using recombinant DNA technology. Cells (e.g., natural or genetically engineered cells) can be cultured in bioreactors to produce therapeutic proteins such as insulin, monoclonal antibodies, and hormones. These proteins can be used to treat a variety of diseases, including diabetes, cancer, and autoimmune disorders. For example, monoclonal antibodies produced in bioreactors can be used for targeted cancer therapies that specifically bind to cancer cells and mediate their destruction without harming normal cells. Bioreactors can also be used to produce viral vectors that can deliver therapeutic genes to patients in the form of in vivo gene therapies. For example, onasemnogeneabeparvovec-xioi (Zolgensma) uses an adeno-associated virus vector (AAV9) to deliver a fully functional copy of the human surviving motor neuron (SMN) gene directly into patient cells. The SMN gene is essential for the survival of motor neurons, and mutations in this gene can cause spinal muscular atrophy (SMA). By providing a novel, normally functioning SMN gene, Zolgensma addresses the genetic root cause of the disease, allowing motor neuron cells to produce proteins that improve muscle movement and function. This gene therapy could be used to treat pediatric patients under two years old with SMA, a rare and often fatal genetic disease that causes muscle weakness and progressive loss of movement. Zolgensma can be administered via a single intravenous infusion, highlighting the practical advantages of in vivo gene therapy. However, the production of viral vectors, such as gene therapies, can be tightly controlled to ensure their safety and efficacy. Bioreactors can provide a controlled environment that supports the growth of virus-producing cells to high densities, thereby facilitating the production of sufficient quantities of vectors for clinical applications. The use of bioreactors in bioproduction provides a reliable, efficient, and scalable method for generating high-value biological products. As biotechnology continues to advance, the capabilities of bioreactors can be expanded, leading to greater innovation and applications in medicine and other fields.

[0079] Cells serve as a material resource for life science research and development in both basic and applied scientific practices. Therefore, cell culture and cell engineering workflows are an integral part of modern life science practice. Furthermore, many research and development applications utilize large quantities of cells or cells from reproducible sources over time. Examples of research and development workflows and experimental paradigms utilizing cells include, but are not limited to, high-throughput screening, cell-based assays, and stem cell research. Primary and secondary cells (including cell lines) can frequently be propagated, expanded, and library-built for research. Cells can also frequently be modified for research, including through genome editing, via a variety of rather complex biological processing workflows. In all these biological processing workflows, manual and semi-automated approaches require significant time, labor, and expense, ultimately increasing the cost and reducing the flexibility of cell-based research. Therefore, new advances in cell manufacturing that shorten turnaround times, labor, or costs associated with cell biological processing workflows can accelerate cell-based life science research.

[0080] The systems and processes described herein can be used to produce a variety of cell products. Cell products can include products containing whole cells or products generated from whole cells. In one example, the systems and methods described herein can be used to produce chimeric antigen receptor (CAR)-T cell therapy. Figure 2 As shown, CAR-T cell therapy manufacturing can include rapid and non-rapid manufacturing methods. Manufacturing methods can include point-of-care manufacturing, distributed manufacturing, or centralized manufacturing. The systems and methods described herein can provide cell therapy manufacturing using smaller production volumes and higher cell densities than other cell therapy manufacturing systems and methods.

[0081] The system described herein can be a cell processing system, an integrated cell processing system, an automated cell processing system, or any combination thereof. The cell processing system can be an integrated cell processing system, such as... Figure 3As shown. The integrated cell processing system 301 can be configured to allow flexible cell input 302. The integrated cell processing system may include a graphical user interface (e.g., a touchscreen interface) that provides the user with real-time information related to cell processing. Cell input 302 may include whole blood, Leukopak, cryopreserved cells, or any combination thereof. The integrated cell processing system 301 can be configured to allow the use of flexible sample carriers 303. The system may include a radio frequency identification (RFID) reader for carrier identification and a cloud-based electronic document system. Sample carriers may include, but are not limited to, cryopreservation bags, tubes, containers, boxes, or any combination thereof. The integrated cell processing system may also include an integrated preservation unit 304. Preservation unit 304 may be a cryopreservation unit. Preservation unit 304 can be configured to freeze or thaw cells, cell samples, or other biological products. Preservation unit 304 can be configured for rapid cryopreservation.

[0082] The cell processing system may include a processing unit and an analysis unit. The cell processing unit 401 and the analysis unit 402 may be configured separately. Alternatively or supplementarily, the cell processing unit and the analysis unit may be integrated into a single cell processing and assay unit 403, such as... Figure 4 As shown. The cell processing and assay unit 403 can be configured to provide various functions, including but not limited to cell enrichment, cell separation, washing, concentration, buffer exchange, depolymerization, or any combination thereof. The cell processing and assay unit 403 can 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, replicative viral load, etc.) or any combination thereof. The output from the cell processing and assay unit 403 (e.g., cells, culture medium, etc.) can be provided to a bioreactor (e.g., a solid-state bioreactor) 404. The bioreactor 404 can be configured to provide various cell processing functions, including but not limited to cell culture, membrane transduction, amplification, gene delivery, cell selection (e.g., negative or positive selection), or any combination thereof.

[0083] A cell processing system can be a benchtop system configured to perform various cell processing procedures, such as Figure 5AAs shown. Cell processing system 501 can provide cell separation 502, activation 503, gene transfer 504, amplification 505, formulation 506, harvesting 507, or any combination thereof. Integrated cell processing system 501 can be used for research, preclinical cell therapy development, clinical research, or any combination thereof. Cell processing system 501 can provide several non-limiting benefits, including reduced costs and labor, increased process reproducibility, improved and reduced processing time to accelerate breakthroughs, improved outcomes, or any combination thereof. Cell processing system 501 can provide fully automated or partially automated functions performed in any sequence to enable hands-free or contact-reduced operation.

[0084] The cell processing system described in this article can be used to run various cell processing operations and workflows. For example... Figure 5B As shown, an example cell processing workflow may include cell thawing 510, cell culture 511, cell engineering 512, assay 513, cell selection 514, freezing 515, or any combination thereof. Various processes may include automated and non-automated processes. Processes may be performed in any order, and some processes may be performed more than once while processing a cell population. Thawing 510 may include using a thermal unit to alter the state of the provided cells. The thermal unit may be integrated with a preservation unit. Cell culture 511 may be performed in a bioreactor (e.g., a 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), viruses), or any combination thereof. Assay 513 may include, but is not limited to, cytology, imaging, molecular assays, cell-based assays, or any combination thereof. Cell selection 514 may include, but is not limited to, in situ selection (e.g., with or without beads), non-in situ selection, upstream selection, downstream selection, or any combination thereof. The produced cells or bioproducts may be subjected to freezing 515 via a thermal unit.

[0085] The systems and methods described herein can provide continuous nutrient delivery before, during, or after cell treatment. Figure 6 The diagram schematically illustrates glucose levels (which can be an indicator of the nutrients provided) as a time function for example bioreactor configurations. In static or batch configurations, glucose levels may start high and decrease over time until depletion or near depletion occurs. In fed-batch configurations, glucose levels may start high and decrease exponentially until a single glucose bolus is added, restoring the level to or near the initial level. This cycle of decrease and single bolus injection can be repeated multiple times, e.g., until cell products are formed. In continuous flow configurations, glucose levels may be maintained constant or otherwise tightly controlled at target levels.

[0086] In one example, the cell processing system can be configured to provide a feed-in batching workflow. Figure 7 An example workflow for producing cell products from an example cell processing system is schematically illustrated. The workflow may include providing an isolated cell population 701. The isolated cells may be mixed with a gel precursor material 702. The mixture containing the cells and the gel precursor may be loaded into a compartment (e.g., a compartment of a cassette) and polymerized 703 to encapsulate the cells in a 3D polymer matrix. An activating agent 704 may be provided to the cells encapsulated within the 3D matrix. The cells may be cultured or expanded within the 3D matrix 705 for at least 1, 2, 3, 4, 5, 6, 8, 10 days or more. In one example, the cells may be cultured or expanded within the 3D matrix 705 for two to three days. Genes may be delivered to the expanded cells 706, for example, via providing the cells with a mixture of virus and culture medium. During or after gene delivery, the cells may be cultured 707 to a target cell concentration or number. Once the target cell concentration or number is reached, the 3D matrix may be liquefied and recovered from the compartment 708. The recovered cells may be harvested 709 as a cell product or for downstream processing.

[0087] The cell processing system described herein may include multiple units and can be configured to provide multiple processes, such as Figure 8 As shown. The cell processing system can be configured, for example, to receive liquid-phase cell material 801 via sterile interconnects. The system may include a preservation unit comprising a thermal unit configured to provide controlled-rate thawing. The preservation unit may be configured to receive frozen cell material and thaw the cell material. Thawed cells or liquid-phase cells may be provided to cell processing module 802 for depolymerization, enrichment, concentration adjustment, mixing with liquid gel precursors, or any combination thereof. A mixture of cells and liquid gel precursors may be provided to a compartment of bioreactor 803 for polymerization. Bioreactor 803 may be configured for culture medium perfusion, in situ cell selection, cell stimulation, gene delivery, or any combination thereof. Cells processed in the bioreactor may be provided to cell processing unit 804. Cell processing unit 804 may be the same cell processing unit as 802 or a different cell processing unit. Cell processing unit 804 may be integrated with analysis units 805 and 806. Cell processing unit 704 and analysis unit 706 can be configured for depolymerization, washing, concentration adjustment, material capture for release testing, release testing, buffer exchange for preservation, or any combination thereof. Cells can be provided to a cryopreservation bag 807 or other container within the preservation unit for preservation (e.g., cryopreservation).

[0088] Cell processing platform In one aspect, this disclosure provides cell processing systems, apparatus, and methods for use in cell processing. The described systems may include or provide various cell processing, cell culture, or cell expansion applications. The described systems and methods may provide two-dimensional or three-dimensional cell culture. In one example, the described systems and methods provide two-dimensional (2D) cell culture. In another example, the described systems and methods provide three-dimensional (3D) cell culture.

[0089] Two-dimensional and three-dimensional cell culture In cell culture, cells can be classified based on their growth characteristics and requirements, particularly whether they adhere to a surface. Adherent cells can attach to solid or semi-solid surfaces (such as tissue culture plastics or microcarrier beads) to grow and proliferate. These cells can originate from organs where cell adhesion is naturally dependent. Adherent cells can grow as a monolayer, attached to the bottom of a culture dish, flask, or bead, or otherwise grow into three-dimensional (3D) structures, such as cell clusters or organoids. During or before subculture, passage, or harvesting, adherent cells can be detached from the solid substrate using enzymatic or mechanical methods. Suspension cells can grow without attaching to any surface. Suspension cells can grow freely floating in the culture medium. Suspension cells can include blood-derived cells, such as lymphocytes or hematopoietic stem cells.

[0090] Both adherent and suspension cells can be cultured in two-dimensional (2D) systems, where adherent cells survive and grow while adhering to a solid substrate (e.g., Petri dishes, flasks, or plates). For example, adherent cells can grow in a monolayer, while suspension cells can settle to the bottom surface where they survive and grow due to gravity (e.g., without mixing). For adherent cell types, the 2D culture approach can lack scalability due to the linear relationship between the maximum potential viable mass and the available surface area of ​​the cultured cells. Furthermore, surface attachment during subculture, passage, or harvest can involve more intricate handling and may require specific types of coated culture vessels for efficient growth. Strategies to scale up production by increasing available surface area can utilize multilayer plates or flasks comprising multiple 2D surfaces. However, these types of 2D cell culture devices can present several challenges. For example, uniformly distributing cells within or between layers can be challenging. To improve the process, procedures that alter the device orientation (e.g., rotation) during cell seeding can provide a more uniform distribution. Uniform perfusion of culture medium into multilayer containers to support long-term culture can be challenging. Providing uniform gas exchange (e.g., supplying adequate oxygen) using impermeable, permeable membranes can also be challenging, as each layer can include a gas exchange membrane to locally oxygenate the cells. For suspension cell types, 2D culture pathways can also lack scalability and exhibit other biological challenges. Without methods to mix or keep cells in suspension, cells can settle to the bottom due to gravity. If cell density is too high, the formation of a dense layer of settling cells can lead to uneven delivery of nutrients and oxygen (whether by providing nutrients or nutrients and gases through excess culture medium on top of the settling cells, or by providing gases through a permeable, impermeable membrane below the cell layer), resulting in increased variability or poor quality. Table 1 shows examples of differences between 2D and 3D cultures. Solid-state cell processing can offer broad applicability to various cell types in therapeutic applications. For example, the systems and methods described herein can allow for the generation of cell products from low-yield cell sources, such as umbilical cord blood containing small amounts of hematopoietic stem cells and progenitor cells (HSPCs). Generating cell products from low-yield cell sources in 2D can be challenging due to low amplification capacity, loss of stemness traits, reduced migration and implantation, decreased long-term reproducibility, or any combination thereof. The solid-state cell processing systems and methods described herein can provide: an 880-fold increase in HSPCs, a 10- to 100-fold increase in stemness marker genes, a 10-fold increase in homing genes, a 73-fold increase in long-term HSC frequency, and an extended duration of in vivo hematopoietic remodeling.

[0091] Table 1. Examples of differences between 2D and 3D cell cultures. Stirred or mixed suspension cultures (which may utilize horizontal impellers, paddles, and oscillating wave mixing mechanisms) can generate unfavorable hydrodynamic environments and stresses (e.g., shear stress), negatively impacting cell growth and quality maintenance. In perfusion suspension cultures, cells can be lost in waste culture during recirculation, culture removal, or both. Using cell retention devices in fluid perfusion systems can reduce or prevent cell loss during perfusion, which can separate cells from waste culture or retain cells in the bioreactor, such as through an alternating tangential flow (ATF) filtration process, where cells are retained in an ATF filter while waste culture is removed. Alternative cell retention techniques include other filtration approaches, including but not limited to: passive flow-based retention (e.g., approaches using inertial or hydrodynamic forces, such as using helical flow or deterministic displacement configurations); and active flow-based retention (e.g., approaches using acoustic or magnetic forces, such as using acoustic transducers to separate cells from waste culture via acoustic particle focusing). These cell retention methods for suspension perfusion can cause reliability issues or degrade 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 implementations), and active cell retention strategies (e.g., acoustic focusing) can subject cells to non-physiological and intense forces. Furthermore, neither 2D nor suspension culture pathways offer the benefits of the 3D cell culture environment described herein.

[0092] Suspension bioreactors can utilize carrier particles, such as microcarriers. In cell culture applications, microcarriers can comprise nonporous beads or granular substrates having a 2D surface on which cells are positioned or adhered. Granular substrates can comprise glass, polystyrene, microporous materials (e.g., cell-impermeable polymeric 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 the 3D culture environment described herein. Other carrier particle approaches may utilize porous or macroporous 2.5D / 3D particles, which can provide a 3D culture environment suitable for culturing adherent cells in suspension bioreactors (e.g., forming cell-carrying matrices, such particles being composed of materials including, but not limited to, cellulose, gelatin, dextran, collagen, acrylamide, and PEG). Such substrates may not be suitable for many types of cells, including, but not limited to, migrating cells, cells capable of mixed suspension-adherent cultures, and non-adherent / suspension cells (such as T cells) that may not be able to remain positioned on the porous carrier during culture and may migrate to the suspension culture phase. Regardless of the carrier particle type (porous or non-porous), the carrier particle approach can have significant limitations. Loading cells onto or into carrier particles can be inefficient, depending on optimized seeding density and providing an environment conducive to cell attachment through collisions between cells and carrier particles. Furthermore, random or stochastic cell seeding procedures can result in highly variable initial cell numbers seeded into the carriers, as little or no communication can exist between individual carrier particles. Carrier particles seeded with a large initial cell number can reach confluence, while other carrier particles, unseeded or seeded with a smaller initial cell number, may remain empty or fail to reach confluence. Frequent passages can overcome these localized spatial constraints and heterogeneity. Moreover, empty carrier particles may not contribute to the overall usable surface area or volume of the culture system, thus reducing the effective manufacturing scale or yield of such systems below their theoretical maximum.

[0093] While suspension bioreactors (which can be static or hybrid, such as stirred and impeller bioreactors) are suitable for carrier particle culture pathways, they can be configured to create an environment of excessive suspension culture medium in which the carrier particles constitute a small fraction of the total bioreactor volume, for example, 16 grams (g) of carrier per liter (L) of culture. Furthermore, hybrid or stirred bioreactors can expose cells, carrier particles, or both to shear stress and other physical stresses, which can damage or detach cells, or otherwise degrade the carrier particles. Bioreactors configured for carrier particle cell culture can use batch, fed-batch, or perfusion feeding. For fed-batch or perfusion feeding, methods are used to separate and retain the carrier particles from the waste culture medium. Such methods can withstand similar challenges as retaining suspended cells in such bioreactors. Carrier particle culture pathways also suffer from carrier particle clumping and aggregation, which can adversely affect the supply of nutrients, oxygen, and other supporting factors to cells within clumps or aggregates. Clumps and aggregates can further clog, impede, or reduce the efficiency of retention, feeding, and harvesting procedures. When using insoluble or non-biodegradable carrier particles, cells can be harvested and separated from the carrier particles. However, inefficient cell detachment methods can reduce harvesting efficiency or cell yield, while harsh cell detachment methods can reduce the viability or quality of the resulting cell product. For human use, carrier particle contamination is a significant risk and can rely on additional procedures and quality controls to ensure efficient removal of carrier particles and minimal to no residue.

[0094] Packed-bed and fixed-bed bioreactors can be used for carrier particle culture pathways. These types of bioreactors can face numerous limitations and challenges (beyond clumping and aggregation, cell harvesting from the carrier, and others mentioned above) that restrict scalability, increase product variability, or reduce product quality. For example, achieving uniform distribution within the bed can be so challenging that cell distribution, fluid flow, or fluid connectivity across any subset or cross-section of the bed, and between any subset or cross-section of the bed, can lead to, for example, nutrient gradients or aeration limitations within the bed. Maintaining uniform cell distribution or fluid circulation over time within and between batches in both fixed-bed and packed-bed bioreactors can also be challenging. Variations in bioreactor packing and preparation can also introduce variability in the process and product. Because cell distribution and fluid circulation directly affect bioreactor performance (e.g., aeration, nutrient supply, shear stress on cells), this can lead to challenges in manufacturing consistency and performance over time. When scaling up, variations in packed-bed size or volume can rely on extensive, time-consuming empirical optimization of other system properties, such as perfusion flow rates.

[0095] Three-dimensional (3D) culture systems enable cell growth in a 3D matrix environment. Manual 3D culture processes performed in a laboratory setting via static culture (including the use of spheres, droplets, gels, or other 3D substrates in multiwell plates and flasks) may not be suitable for scalable manufacturing and bioprocessing. Instead, automated 3D cell culture systems can offer significant benefits.

[0096] Cells cultured in a 3D microenvironment can exhibit significantly different and generally superior properties compared to 2D cultures. For example, 2D cultures can be limited to a single layer in depth, while 3D cultures can be distributed across 3D space. Cells cultured in 2D can be flatter, more elongated, or both, while cells cultured in 3D can have more natural and complex cell shapes. Cells in 2D cultures can migrate in two dimensions and can come into contact from a single dimension, while cells in 3D cultures can migrate in 3D and can come into contact from multiple dimensions. Cell connections and cell-cell interactions can be less common and less physiologically consistent in 2D cultures compared to 3D cultures. Gene expression can be disrupted in 2D cell cultures, but more closely resembles in vivo expression patterns in 3D cultures. In 2D cultures, the effects of gravity and physical microforces can be minimal or nonexistent, while 3D cultures expose cells to more natural, in vivo-like gravity and physical microforces. Furthermore, 3D culture allows for increased control over the microphysical environment (e.g., stiffness, elasticity, and cell-presenting factors), while 2D culture offers little or no control over these parameters. This additional control over the cellular microenvironment provided by the 3D culture approach can benefit from reduced product variability, which arises both from variability in the source material and by reducing variability introduced during production. 3D matrices that further provide cell retention during bioprocessing can offer process advantages (biological and process / automation aspects), including but not limited to: providing a low-shear environment; reducing or eliminating the use of cell retention equipment during feed or culture perfusion; reducing sensitivity to contamination; and providing instrumentation platforms with simplified designs or fewer components, thereby increasing reliability and reducing costs. For equivalent production yields, 3D culture can utilize smaller working and total volumes than 2D culture due to increased cell concentration (e.g., cell volume per reactor volume, number of cells per reactor volume, or both).

[0097] Implementing three-dimensional cell culture processes using existing bioreactor designs, including static, hollow fiber, packed bed, and other bioreactor configurations, can be challenging. For example, culturing cells in 3D while maintaining cell anchorage on the 3D substrate and ensuring an adequate supply of culture components supporting cell metabolism, such as culture medium, nutrients, and dissolved oxygen, is challenging. In packed bed reactor designs, the culture medium can be essentially perfused through the cell-containing compartment (e.g., the packed bed) and its surroundings. However, establishing a suitable rate of medium flow through the 3D substrate to support cell growth can rely on unsuitably high fluid pressures or subjecting the cells or substrate to unsuitable forces, negatively impacting cell quality or the quality and durability of the 3D substrate. Providing medium flow around the 3D substrate may not be a scalable delivery mechanism because the limited diffusion rate within the 3D substrate can restrict the effective perfusion distance, leading to non-uniform perfusion or necrotic zones within the 3D substrate. Using a 3D substrate with sufficient porosity to enable fluid perfusion at a suitable scale (e.g., using woven fiber beds or aligned fiber beds, given the physical size of the 3D substrate) may fail to provide a suitable 3D microenvironment to obtain the benefits of 3D cell culture (e.g., it may be too microporous, such that cells are effectively in 2D culture attached to a scaffold, or in suspension culture within interstitial spaces). In hollow fiber perfusion bioreactors, a portion of the intracapillary (IC) compartment (which includes the space inside the hollow fiber and the headspace at each end of the outer shell and the dead volume of the IC fluid loop) is exposed to culture medium perfusion by the capillary extracapillary (EC) space. The remaining volume may not be fluidly connected to the extracapillary (EC) space. This can increase the challenges of cell seeding, gas exchange, and nutrient delivery to cells. Because the 3D substrate essentially retains cells, loading the cell matrix into the IC can result in a significant portion of cells being retained in the non-perfused IC space. Similarly, a portion of the EC (which may include the headspace at each end of the outer shell and the dead volume of the EC fluid loop) may be fluidly connected to the IC. Loading cell-carrying matrix into ECs can similarly result in a significant portion of cells being retained in the non-perfused EC space. Furthermore, because hollow fibers can be randomly distributed within the shell, loading ECs with cell-carrying matrix can lead to uneven perfusion caused by the flow of culture medium in the IC.

[0098] Cell processing system The manufacture of cell therapies and other valuable chemical and biological cell products can utilize 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 cell biomass between different workflows and instrumentation platforms. Biomass transfer and processing between workflows and instruments can increase human interaction, thereby increasing the workforce required for production and thus increasing product costs. In an “open” configuration, cell biomass can be exposed to the environment during processing, leading to contamination, manufacturing failures, batch-to-batch variability, difficulty in meeting regulatory standards (including for sample traceability), and the use of a larger footprint or more highly trained personnel in a highly controlled environment (e.g., a cleanroom). In a “closed” configuration, the system can be designed to avoid exposure of cell biomass to the environment (e.g., through the use of sterile barriers and connectors, by incorporating single-use technologies, or both), which can reduce the risk of contamination and increase flexibility in facility design (e.g., fewer controls) and staffing (e.g., less trained personnel). Closed configurations can utilize manual or automated methods forming numerous sterile interconnects to aseptically transfer biomass between process workflows, which can increase the amount of human labor involved or the complexity of automated systems. Non-integrated manufacturing systems, using multiple instrument systems and platforms, present challenges in scheduling and instrument availability, as well as documentation and accurate record keeping, when manufacturing many products simultaneously. Transferring cells between platforms and processes can damage cells when cell concentrations or buffers change, or when cells are held under suboptimal conditions (e.g., in transfer containers) between processes. Within closed processes, cell transfer via the establishment and termination of closed sterile interconnects (e.g., aseptic welding of inputs and outputs at each operation point or on each individual instrument platform during consumable manufacturing, system setup, or system operation) can employ complex fluid or automated architectures, or labor-intensive, complex procedures. Automated cell transfer between processes, such as physically transporting cell biomass between instruments using one or more robotic arms or robotic systems (or alternatively, transporting instruments between cell biomass), can be challenging to scale. Such robotic systems, due to their size and complexity, cannot be well scaled and can exhibit further fundamental limitations in scaling multiple parallel processes (e.g., the number of simultaneous operations a single robotic arm can support can be limited by the time required to perform each operation and the physical range of the robotic arm or motion system). For these reasons, closed-loop integrated systems can improve consistency, purity, and safety, while helping to reduce overall manufacturing costs by reducing labor and facility requirements. Closed-loop integrated systems that perform more, essentially all or entirely cellular processes can be scaled up simply by increasing the number of systems used in manufacturing.

[0099] Another challenge of cell processing platforms and technologies is the integration with cryopreserved input materials and the generation of cryopreserved output materials. When bioprocessing from cryopreserved source materials, frozen samples can be immersed in a 37°C water bath until the ice is visually confirmed to have melted (e.g., wet thawing). This method can be simple and low-cost, but it can rely on specialized technicians to execute it successfully and consistently. Variations in user behavior and laboratory settings can lead to batch-to-batch variability. Untrained operators may choose slightly different and often subjective freezing bag angles, shaking speeds, and thawing termination criteria, resulting in a range of inconsistent results. While sufficient in research settings, water baths present challenges in clinical and cleanroom environments. In addition to the risks of process and product variability, the presence of warm water can introduce unacceptable contamination risks in controlled environments. Integration of automated thawing modules functionally connected to the bioprocessing system can address these challenges.

[0100] When handling cryopreserved products with cells, cell samples or other materials can be suspended in specialized cryopreservation buffers, and the temperature can be lowered to the freezing point using optimized procedures to minimize damage to the cells or product. Cell cryopreservation buffers can be at least slightly cytotoxic, meaning that prolonged exposure of cells to liquid buffers can lead to loss of viability and other quality properties upon thawing. The integration of automated cryopreservation modules functionally connected to cell handling systems can minimize the time cells are exposed to cryopreservation buffers before freezing, while also reducing manufacturing time and complexity, as the product is stable once frozen and can be collected from the system at convenient times.

[0101] As described herein, integrated biological processing systems (e.g., integrated cell processing systems) can offer a variety of advantages over other biological or cell processing systems. For example, the systems described can offer simpler fluid dynamics, such as fewer valves, tubing, and interconnects, which can provide smaller and lower-cost instrumentation, simpler consumables, and ease of sterilization. Compared to other biological or cell processing systems, the systems and methods described herein can provide reduced pathways for cells between processes. Compared to other biological processing systems, the systems and methods described herein can enhance cell phenotypes and provide expanded applications. For example, the systems and methods described herein can provide healthier cells due to reduced handling and manipulation. In some embodiments, a three-dimensional (3D) matrix can be used to replicate the extracellular matrix of natural tissue to provide a superior phenotype. The systems and methods described can be used to process adherent or suspension cells.

[0102] The cell processing systems described herein can be used for immunoassays and target discovery, cell research and cell and gene therapy (CGT) development, clinical cell therapy, induced pluripotent stem cell (iPSC) and adherent cell bioprocessing, continuous biopharmaceutical manufacturing, or any combination thereof. Immunoassay and target discovery applications may include, but are not limited to: large-scale multiplex RNA-based artificial antigen-presenting cells (APCs) for antigen screening and validation of immunoreactivity, 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 are not limited to: multiplex RNA cell engineering to understand the phenotypic and functional consequences of gene expression heterogeneity, development of phenotypic enhancements for cell and gene therapy, cell therapy process development, or any combination thereof. Clinical cell therapy applications may include, but are not limited to: multiplex RNA cell phenotypic engineering and CAR delivery, safer properties due to transient kinetics and reduced or no risk of oncogenic integration, focused on the fields of oncology and autoimmune diseases, long-term delivery of synthetic immune systems, or combinations thereof. Applications of IPSC and adherent cell bioprocessing can include, but are not limited to: scalable iPSC amplification and developmentally programmed bioreactor (e.g., solid-state) methods, scalable production of adherent cell types (such as pancreatic islet cells and cardiomyocytes) bioreactor (e.g., solid-state) methods, research and clinical uses, or combinations thereof. The systems and methods described herein can provide continuous bioproduct manufacturing for high-density cell line culture in a three-dimensional (3D) matrix, featuring continuous culture medium perfusion and secreted protein collection for purification.

[0103] 3D matrix and its application In one aspect, this disclosure provides a system for cell processing. The system for cell processing may include multiple three-dimensional (3D) matrices and multiple fluid flow paths. The 3D matrices may be configured to retain cell populations, or may retain cell populations. The fluid flow paths may be independent of and adjacent to the 3D matrices. The fluid flow paths may be configured to deliver fluid material to the 3D matrices, or may deliver fluid material to the 3D matrices, to process cell populations into cell products.

[0104] In another aspect, this disclosure provides a method for cell processing. The method for cell processing may include providing a cell population, a plurality of 3D matrices, and a plurality of fluid flow paths. The cell population may be contained within the 3D matrices. The fluid flow paths may be independent of and adjacent to the 3D matrices. The method may include using the fluid flow paths to deliver a fluid material to the 3D matrices. The method may include processing the cell population into a cell product in the presence of the fluid material delivered to the 3D matrices.

[0105] In another aspect, this disclosure provides a method for cell treatment. The method for cell treatment may include providing a 3D matrix in a cell treatment unit for retaining, containing, encapsulating, fixing, or any combination thereof of multiple cells. Within the cell treatment unit, multiple cells may be automatically treated within the 3D matrix to produce multiple treated cells. Within the cell treatment unit, the treated cells may be automatically separated from the 3D matrix. The method may also include collecting the multiple treated cells.

[0106] In another aspect, this disclosure provides a system for cell processing. The system for cell processing may include a cell processing unit and one or more computer processors. The cell processing unit may be configured to retain a 3D matrix, or may retain a 3D matrix. The 3D matrix may be configured to encapsulate a plurality of cells, or may encapsulate a plurality of cells. 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 a plurality of cells within the 3D matrix to produce a plurality of processed cells, (ii) guide the cell processing unit to separate the plurality of processed cells from the 3D matrix, (iii) guide the cell processing unit to collect the plurality of processed cells, or any combination thereof.

[0107] In another aspect, this disclosure provides a system for manufacturing cell products. The system may include a 3D matrix configured to produce cells at a density greater than 80 million cells per cubic centimeter (cells / cm³). 3 The density of the 3D matrix is ​​used to retain, contain, encapsulate, or immobilize cell populations, or the 3D matrix contains more than 80 million cells / cm³. 3 The system can preserve, contain, encapsulate, or immobilize cell populations at high density. It can also utilize a 3D matrix to process cell populations into cell therapy products.

[0108] In another aspect, this disclosure provides methods for manufacturing cell therapy products. The methods for manufacturing cell therapy products may include providing a concentration of more than 80 million cells / cm³. 3 The density of cell populations is preserved, contained, encapsulated, or fixed within a 3D matrix, and the cells of the cell populations are processed into cell therapy products using the 3D matrix.

[0109] In another aspect, this disclosure provides a bioreactor for processing cell populations. The bioreactor for processing cell populations may include a 3D matrix configured to retain, contain, encapsulate, or immobilize the cell population. The bioreactor may have a volume of up to about two cubic meters (m³). 3The volume of the bioreactor. The bioreactor can be configured to process cell populations into cell products with high reproducibility using a 3D matrix, or the bioreactor can process cell populations into cell products using a 3D matrix.

[0110] In another aspect, this disclosure provides a method for treating cell populations within a bioreactor. The method for treating cell populations may include providing cell populations that are retained, contained, encapsulated, or immobilized within a 3D matrix of the bioreactor. The bioreactor may have a maximum of about 2 m³. 3 The volume. Methods may include processing cell populations into cell products with high reproducibility using a 3D matrix.

[0111] In another aspect, this disclosure provides a system for the manufacture of cell therapies. The system for the manufacture of cell therapies may include a 3D matrix configured to hold at least about 5 million cells / cm³. 3 The density of the cell population is retained, contained, encapsulated, or fixed, or the 3D matrix contains at least approximately 5 million cells / cm³. 3 The system can preserve, contain, encapsulate, or immobilize cell populations at high density. It can process cells or cell populations within a 3D matrix for up to 48 hours to generate cell products. These cell products can be used to generate cell therapies.

[0112] In another aspect, this disclosure provides methods for manufacturing cell therapies. Methods for manufacturing cell therapies may include providing at least about 5 million cells / cm³. 3 This involves preserving, containing, encapsulating, or immobilizing cell populations within a 3D matrix. Methods may include treating the cell populations within the 3D matrix for a period of up to approximately 48 hours to generate a cell product. This cell product can be used to generate cell therapies.

[0113] Compared to solution-phase cell treatment, 3D matrices can offer several advantages for cell treatment. For example... Figure 9A As shown, the transfer of fluids, nutrients, or reagents in large-volume and microfluidic bioreactors can be challenging. Large-volume and microfluidic bioreactors can transfer cells and fluids from one process to another, increasing cell manipulation and the likelihood of cell damage. Furthermore, large-volume and microfluidic bioreactors can utilize multiple washing and separation processes, subjecting cells to shear stresses that can damage them. In alternative configurations, cells can be processed within a 3D matrix, such as… Figure 9BAs shown. Cell processing within a 3D matrix can reduce shear stress on cells by allowing the transfer of fluids, nutrients, or reagents via tangential flow or perfusion. The 3D matrix and the compartments holding the 3D matrix can be configured such that all or substantially all fluid flows along the surface of the 3D matrix, allowing nutrients and reagents within the fluid to diffuse into the 3D matrix to contact the cells encapsulated therein. Cells can undergo multiple processes within the 3D matrix, thereby reducing cell manipulation. Tangential flow or perfusion of nutrients, reagents, or both can allow for highly controlled levels of those nutrients or reagents within the 3D matrix, enabling processing at high cell densities (e.g., approximately 10 million to 100 million cells / cm²). 3 ) Treat cells.

[0114] 3D substrates used in cell-matrix formulations may comprise porous, permeable solids or mixtures thereof suspended in an interstitial fluid primarily composed of water; and may also comprise suspended cells in an interstitial aqueous liquid, cells adhered to the 3D substrate and in contact with the interstitial aqueous liquid, or both. 3D substrates may comprise three-dimensional structures with defined or indeterminate structures, described as, but not limited to, matrices, meshes, networks, or otherwise described as physical interconnection systems of molecules such as polymers. The structure of the 3D substrate can support the structural integrity of the 3D cell culture environment, providing interstitial spaces in which cells can reside, grow, proliferate, or any combination thereof, and facilitating the transport of nutrients and other essential dissolved substances, as well as materials, reagents, and constructs for genetic engineering. The configuration of the interstitial spaces (characterized by the tissue structure and porosity of the 3D substrate) and the physical properties of the substrate, such as hydrophilicity, cell adhesion properties, and biofouling properties, can allow the diffusion of water-soluble substances, thus making it an ideal medium for cell activity. The configuration of the interstitial space and the structural properties of the 3D substrate can further allow for active exchange of interstitial fluids, such as by using a fluid flow that can partially, substantially, or completely displace the first interstitial fluid with a second interstitial fluid. In some embodiments, the 3D substrate additionally provides for cell retention within the 3D substrate during cell culture or passive or active exchange of interstitial fluids. The three-dimensional substrate can include, but is not limited to, hydrogel matrices, porous solid matrices, inverse opal matrices, and composite matrices (e.g., interpenetrating matrices).

[0115] Hydrogel matrices, such as those used in aqueous environments for cell culture, may contain hydrophilic polymers. Hydrogel matrices may contain all or substantially all of the hydrophilic polymers. Hydrogel matrices may contain greater than or equal to about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more of the hydrophilic polymers. The hydrogel matrix may contain approximately 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% Hydrophilic polymers of 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%.

[0116] Hydrophilic polymers, while being hydrophilic or water-absorbing, can form 3D matrices that are insoluble in water themselves. The polymer matrix can swell without dissolving in the presence of water. This swelling occurs because the hydrophilic (water-absorbing) nature of the polymer chains allows them to absorb and retain large amounts of water, which can be many times their dry weight. Despite their affinity for water, these polymers can be chemically or physically crosslinked, preventing them from dissolving in water. Crosslinking between and within the polymers that make up a hydrogel can confer stability to the hydrogel matrix. Crosslinking can involve forming connections between and within the individual polymer chains, thereby forming a three-dimensional network structure. This network can also include other structural materials (such as in the case of mixed gel materials and matrices formed by interpenetrating networks), functional molecules (e.g., biomolecules), water molecules, and other particles (such as cells) within its structure or volume. Crosslinking can be formed through chemical bonds (e.g., covalent crosslinking) or through physical interactions. Physical interactions can include physical crosslinking, such as ionic bonds, hydrogen bonds, hydrophobic interactions, polyelectrolyte complexes, electrostatic interactions, aggregation, or any combination thereof. The ratio of chemical to physical crosslinking can alter the mechanical strength, degradation rate, biological interactions, or any combination thereof of the hydrogel matrix.

[0117] As described elsewhere in this document, crosslinking can be stable or reversible under given conditions (e.g., stimulation). The insolubility and crosslinking properties of hydrogel matrices in aqueous environments can provide cells with a stable and supportive scaffold. Such 3D substrates can mimic the extracellular matrix where cells naturally reside. 3D substrates can also provide structural support and serve as a medium or substrate through which nutrients and other soluble factors can diffuse or be presented to cells, or otherwise be made available for contact with cells. Furthermore, the physical structure and properties of hydrogels, including porosity (e.g., its pore size and distribution), stiffness, and elasticity, can be customized or engineered as described herein, including for the purpose of regulating cell migration, proliferation, differentiation, phenotype, gene expression, or any combination thereof.

[0118] Hydrogels can be formed from natural polymers, modified polymers, synthetic polymers, or any combination thereof. Hydrogel materials can be selected based on biocompatibility, biodegradability, mechanical strength, ease of modification, or any combination thereof. Materials used as 3D matrices can be modified or altered to be compatible with a given type of cell. Natural polymers can include, but are not limited to, fibrinogen, hyaluronic acid, collagen, matrigol, gelatin, chitosan, alginate, cellulose, heparin, dextran, keratin, agarose, silk fibroin, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide polymers, or any combination thereof. The synthetic polymers may include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylic acid (PAA), poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polyvinyl acetate (PVA), polyethyleneimine (PEI), polyvinylpyrrolidone (PVP), poly(N-isopropylacrylamide) (PNIPAM), polysiloxanes, polyurethanes, poly(ethylene glycol) diacrylate, poly(ethylene glycol) dimethacrylate, their derivatives, or any combination thereof. The 3D matrix may contain a single polymer or may contain a mixture of polymers configured for nutrient diffusion, cellular mechanical support, biocompatibility, or any combination thereof.

[0119] The polymer (e.g., a hydrogel polymer) may or may not be chemically modified. In one example, the polymer (e.g., a hydrogel polymer) is chemically modified. For example, collagen can be provided in a chemically modified form of natural collagen, such as methacrylamide collagen (often abbreviated as collagen-MA or methacrylamide collagen), where the collagen can be functionalized with methacrylate groups. The methacrylamide process can involve, for example, introducing methacrylate groups into the amino acid residues of the collagen molecule using methacrylic anhydride. This chemical modification can occur primarily at the lysine residues and N-terminal groups of the collagen. Using methacrylamide collagen can be advantageous because it enables rapid thermogel-sol transitions (e.g., exhibiting reversible transitions from liquid to gel with increasing temperature) and photocrosslinking upon exposure to light (e.g., ultraviolet light) in the presence of a photoinitiator. The methacrylate groups can form covalent bonds with each other, thereby establishing a crosslinked network. Chemical modification can preserve the biocompatibility and biodegradability of natural collagen, making it an excellent scaffold material for supporting cell adhesion, proliferation, and differentiation. Hydrogels formed from methacrylamide collagen can possess tunable mechanical properties that can be modulated to match the properties of the natural tissue environment. The degree of substitution of the methacrylate groups can be varied, thereby altering the crosslinking density and the physical properties of the resulting hydrogels, such as their thermal responsiveness, stiffness, degradation rate, and pore size. In another example, PEG polymers can be prepared as block copolymers with poly(N-isopropylacrylamide) (PNIPAM), also known as thermogel-type PEG-PNIPAM block copolymers. PEG-PNIPAM block copolymers exhibit a thermally responsive gel-sol transition (e.g., exhibiting a reversible transition from liquid to gel with increasing temperature). Each polymer segment can provide specific properties that contribute to the overall behavior of the copolymer. PEG is a hydrophilic polymer that can be biocompatible and non-immunogenic. PEG can be used in biomedical applications. In copolymers, PEG can act as a segment that stabilizes the copolymer and increases its solubility in aqueous environments. PNIPAM can exhibit thermally responsive behavior. Below its lower critical solution temperature (LCST) (which can be around 32°C), PNIPAM is hydrophilic and soluble in water. Above this temperature, PNIPAM can become hydrophobic and insoluble, leading to polymer precipitation or gel formation. In these copolymers, PEG and PNIPAM can be linked in a block copolymer architecture, where each polymer forms different blocks within the copolymer chain. The proportions of each polymer component can be controlled, thus affecting the properties of the copolymer. The copolymer can contain PNIPAM and secondary polymers. The PNIPAM:PEG ratio (e.g., PNIPAM:PEG) can be from about 1000:1 to about 1:1000.The ratio of PNIPAM to secondary polymer can be selected based on target mechanical properties, chemical properties, responsiveness, synthetic route, copolymer composition, or any combination thereof. The block copolymer structure allows PEG-PNIPAM materials to utilize the water solubility and biocompatibility of PEG and the temperature-sensitive gelation properties of PNIPAM. Other LCST polymers that can be incorporated into stimulus-responsive copolymers include, but are not limited to, poly((meth)acrylamide):poly(… N,N- Diethylacrylamide), poly(2-carboxyisopropylacrylamide), poly( N -(L)-(1-hydroxymethyl)propylmethacrylamide, poly( N -Acryloyl-N'-propylpiperazine), poly( N -Ethylacrylamide, poly( N -methyl- N -Ethylacrylamide, poly( N -n-propylacrylamide), poly( N -ethylmethylacrylamide), poly( N -methyl- N -Isopropylacrylamide), poly( N -Isopropylmethylacrylamide), poly( N -n-propylmethacrylamide), poly( N -methyl- N -Isopropylacrylamide), poly( N -Cyclopropylacrylamide), poly( N -Cyclopropylmethacrylamide, 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 a stimulus-responsive polymer (e.g., chemical, physical, thermal, or any combination thereof). 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-ethylacrylic acid), and poly(... N,N Dimethylaminoethyl methacrylate), poly(vinylimidazolium), polyaspartic acid, polylysine, and polyhistidine. Other photoresponsive polymers include, but are not limited to, poly(ethylene oxide-methacrylate) with pyrene side groups, nitrobenzyl and dithiodipropionic acid modified polyetherimides, and poly-N -Isopropylacrylamide-spiropyran, poly[ N -Isopropylacrylamide- b -2-(acrylamido)-2-methylpropanesulfonate sodium spiropyran. Other glucose-responsive polymers include, but are not limited to, poly[(2-dimethylamino)ethyl methacrylate-] common -3-Acrylamide phenylboronic acid]. Stimulus-responsive polymers, including LCST polymers such as PNIPAM and other polymers listed above, can be synthesized into 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), polyethyleneimine (PEI), and 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 propyl acrylic acid (PAA). Examples of such copolymers include, but are not limited to, PEG-PNIPAM, alginate / N - Isopropylacrylamide (NIPAM), PNIPAM / poly(sodium acrylate), and methacryloxychitosan / PNIPAM. Copolymers can contain two or more components or block components, such as diblock copolymers, triblock copolymers, or blends. Polymers and copolymers can be designed and synthesized to have a wide range of topologies / morphologies, such as linear polymers (homopolymers, di / tri / multiblock copolymers, and organic / inorganic hybrid polymers) and nonlinear polymers (star polymers, (hyper)branched polymers). Polymers and copolymers can be synthesized using a variety of methods, including but not limited to free radical polymerization (FRP), atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), and graft copolymerization.

[0120] As an alternative or supplement, 3D substrates for cell processing can comprise porous solids. These matrices can be made from a variety of materials, including but not limited to plastics (e.g., polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polycarbonate, acrylics, cyclic olefin copolymers, cyclic olefin polymers, or any combination thereof), ceramics, glass, metals, natural fibers, or any combination thereof. Unlike hydrogels, these materials can have defined microscale porosity, allowing for precise control over pore size and distribution. This porosity can be produced by methods such as sintering, foaming, leaching, weaving, 3D printing, or combinations thereof. Solid porous matrices can be more rigid and stable than hydrogels and may not necessarily expand or change shape in the presence of water or biofluids, which can be advantageous for certain applications requiring dimensional stability. Solid porous materials can also be modified to alter their physical, chemical, or biological properties. Functionalization and modification of porous solid 3D scaffold materials can include all methods for modifying these materials for 2D cell culture applications, including but not limited to plasma surface modification, self-assembled monolayer deposition, covalent grafting, surface coating or absorption, or any combination thereof. For example, plastics and other solid scaffold materials can be treated to alter surface energy, surface tension, or wettability, such as by forming carbonyl (-C=O-), carboxyl (HOOC-), hydrogen peroxide (HOO-), and hydroxyl (HO-) groups through plasma treatment or oxidation reactions. As another example, materials can be treated to influence protein binding, protein deposition, biofouling, and cell adhesion, such as surface hydrolysis of polyester scaffolds to promote protein adsorption, thereby improving cell adhesion. Surface functionalization can be performed by exposing the surface to RF plasma. Many gases can be excited and used to functionalize surfaces for a wide variety of applications. Surface functionalization techniques can include the use of air plasma, oxygen plasma, and ammonia plasma, as well as other specialty gases. Each gas can have different effects on the substrate. For example, ammonia plasma and n-heptylamine vapor treatment can be used to attach amine functional groups. Surfaces can be treated with thin polysaccharide films or coatings, including through covalent attachment, such as by using oxidized polysaccharides that form Schiff base bonds with amine-functionalized materials.

[0121] As an alternative or supplement, the 3D substrate can contain an inverse opal (IOPAL) material, which mimics the ordered porous architecture of opal but has an inverted morphology. To form an IOPAL 3D substrate, a first sacrificial material (e.g., an opal template) can be deposited, stacked, or otherwise formed into a 3D spatial configuration (e.g., by using a mold). For example, the opal template can be formed from monodisperse microspheres (e.g., made of polystyrene, silica, polymethyl methacrylate, etc.), which are stacked into a dense structure, such as by sedimentation, vertical deposition, or centrifugation, allowing the spheres to naturally align into a 3D configuration. The 3D structure can include a substantially regular structure (e.g., a face-centered cubic (FCC) lattice). The 3D substrate precursor material can permeate into the gaps in the opal template to fill the voids without disturbing the 3D structure. The 3D substrate material can then be formed into the 3D substrate, such as through a sol-gel method or polymerization. The sacrificial template can then be removed, such as by dissolving it 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 liquefy the template, thus facilitating removal from the IOPAL structure). Removal of the opal template can leave a porous structure with an ordered array of voids, which can mirror the original arrangement of the template. In some examples, removal of the sacrificial porogen creates a network of interstitial spaces within the precise geometric arrangement of the original template, resulting in interconnected voids in the inverted opal structure that can be uniform and highly ordered. Due to its ability to form 3D structures with highly controlled porosity and structural regularity, IOPAL materials can provide uniform diffusion and other desired material properties. IOPAL substrates can contain a variety of materials, including but not limited to hydrogels, porous solids, or any combination thereof.

[0122] In one example, the 3D matrix may include a composite 3D substrate. The composite 3D substrate may be formed from a mixture of 3D substrate materials or an interpenetrating 3D substrate. The composite 3D substrate may include 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 can support cell culture. To support cell culture, the 3D substrate may provide cells with physical 3D space, a liquid culture medium supply, dissolving gases, or other reagents and compositions that support cell culture. The various components or portions thereof constituting the composite 3D substrate may be physically crosslinked, physically associated, or not physically associated. The 3D substrate may include one or more interpenetrating networks, such as an irreversible network of first mechanical strength interpenetrating with a second network characterized by reversible crosslinking. Using a composite 3D substrate or a mixture of 3D substrate materials may offer advantages such as including individual materials to serve a selected function. In another example, the 3D substrate may include a first material providing cell retention and a second material providing a compatible cell growth microenvironment, such individual materials being configured separately and used together to form a 3D substrate suitable for cell retention and growth. Other examples include, but are not limited to, interpenetrating hydrogel matrices, such as those formed from combinations of thermoresponsive hydrogel materials (e.g., methacrylamide collagen and PEG-PNIPAM), and individual porous solid matrices, such as polystyrene, characterized by large pore sizes (e.g., greater than 100 micrometers (μm)) and filled with individual hydrogel matrices such as collagen gels.

[0123] 3D substrate properties The physical properties of 3D substrates used for cell culture may include, but are not limited to, stiffness, flexibility, rigidity, swelling, porosity, stability, optical properties, dimensional properties, or any combination thereof. The physical properties of 3D substrates can be modified or otherwise engineered by altering the composition of the matrix, the method of matrix formation, the soluble environment of the matrix, or any combination thereof. Methods for modifying or adjusting the physical properties of 3D substrates may include: altering the crosslinking density (e.g., by changing the crosslinking potential of one or more base components); altering the component ratios (e.g., by changing the percentage of crosslinked components); altering the molecular weight of the polymer or substrate; adding components (e.g., by adding nanomaterials, including degradable, removable, or sacrificial porogens that may not be components of the formed matrix but otherwise alter the distribution and network structure of the matrix); altering the soluble environment (e.g., such as pH, ionic strength, etc.); or any combination thereof.

[0124] 3D substrates can provide a high surface area to volume ratio to efficiently deliver nutrients, gas exchange, and other reagents, such as viral particles, to 3D cell culture substrates. A 3D matrix or substrate with a high surface area to volume ratio can have a high interfacial area compared to its volume. A 3D substrate with a high surface area to volume ratio can provide a more homogeneous cellular environment compared to a 3D substrate with a low surface area to volume ratio. For example, compared to similar substrates with a lower surface area to volume ratio, a 3D matrix or substrate with a higher surface area to volume ratio can diffuse or transport fluid materials (such as nutrients and reagents) over a shorter distance into the 3D matrix. Shorter diffusion or transport distances can provide a smaller concentration gradient throughout the 3D substrate and a more homogeneous cellular environment throughout the 3D matrix or substrate. 3D matrices or substrates can have a surface area to volume ratio greater than or equal to about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, or greater. The 3D matrix or substrate can have a particle size of 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 of the surface area to volume ratio.

[0125] 3D matrix or substrate can include any shape. For example, a 3D matrix can be a plate, a sphere, a fiber, or any other shape as described elsewhere herein. In one example, the 3D matrix is ​​a plate. In another example, the 3D matrix includes a fiber shape. In yet another example, the 3D matrix includes a spherical shape. The 3D matrix or substrate can have a dimension substantially smaller than any other dimension. For example, the 3D matrix can be a plate, and the thickness of the plate can be substantially smaller than the length or width of the plate. In another example, the 3D matrix can be a sphere, and the diameter can be substantially smaller than the circumference. In yet another example, the 3D matrix can be a fiber, and the diameter of the fiber can be substantially smaller than the length of the fiber. During cell treatment, fluid can flow in a direction perpendicular or substantially perpendicular to the smaller dimension (e.g., fluid flow can be tangential to the longer dimension of the 3D matrix). For example, fluid flow can be parallel to the longer dimension of the plate, or along the surface of the plate arranged at approximately 90, 80, 70, or 60 degrees to the shorter dimension (e.g., fluid flows along the length or width of the plate). In one example, the 3D matrix can include fibers, and fluid can flow along the length of the fibers. In another example, the 3D matrix may comprise a sphere, and fluid may flow along the outer circumference of the sphere. The 3D matrix may have a small size of less than or equal to about 20 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 smaller. In one example, the 3D matrix may have a small size of less than or equal to about 10 mm. In one example, the 3D matrix may have a small size of less than or equal to about 1 mm. In one example, the 3D matrix may have a small size of less than or equal to about 500 μm. In one example, the 3D matrix may have a small size of less than or equal to about 200 μm. In one example, the 3D matrix may have a small size of less than or equal to about 100 μm. In one example, the 3D matrix may have a small size of less than or equal to about 10 μm. In one example, the 3D matrix may include a plate configuration with a small size of less than or equal to about 10 mm. In one example, the 3D matrix may include a plate configuration with a small size of less than or equal to about 1 mm. In one example, the 3D matrix may include a plate configuration with a small size of less than or equal to about 500 μm. In one example, the 3D matrix may include a plate configuration with a small size of less than or equal to about 200 μm. In one example, the 3D matrix includes a fiber configuration, and the fibers include a small size (e.g., diameter) of less than or equal to about 10 mm. The small size can be from about 10 μm to 25 μm, or from 10 μm to 50 μm.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 2mm, 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 200 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 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 5 mm 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 600μm, 200μm to 700 μm, 200μm to 800 μm, 200μm to 900 μm, 200μm to 1 mm, 200μm to 2 mm, 200μm to 3mm, 200μm to 4 mm, 200μm to 5 mm, 200μm to 6 mm, 200μm to 8 mm, 200μm to 10 mm, 200μm to 20 mm, 300μm to 400 μm, 300μm to 500 μm, 300μm to 600 μm, 300μm to 700 μm, 300μm to 800μm, 300μm to 900 μm, 300μm to 1 mm, 300μm to 2 mm, 300μm to 3 mm, 300μm to 4 mm, 300μm 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, 800μm 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 20 mm, 8 mm to 10 mm, 8 mm to 20 mm, or 10 mm to 20 mm. In one example, the smallest dimension is approximately 10 μm to 100 μm.

[0126] The 3D matrix can be configured such that fluid can flow along its surface. In one example, the 3D matrix may include fibrous or plate-like configurations, such as... Figure 10A As shown. Alternatively or supplemented, the 3D matrix may include a spherical configuration. The 3D matrix may include a surface area significantly larger than its volume to allow for the diffusion and transport of nutrients and reagents into the 3D matrix. In one example, the thickness or diameter of the 3D matrix may be substantially smaller than other dimensions of the 3D matrix. Figure 10BAs shown, depending on the cell product being produced, a 3D matrix with a large surface area to volume ratio can allow for the use of small or large culture medium volumes. In one example, the system can be configured for low fluid volumes. Low fluid volumes allow for high flow rates and more frequent batch feeding. Lower fluid volume operation can be used for short process runs, such as zero to three days or zero to five days. The low fluid volume configuration can minimize the amount of activating reagents and genetic payload reagents used, thereby saving costs and improving reagent efficiency. Alternatively, the 3D matrix and system can be configured for large or larger culture medium volumes. Larger culture medium volumes can provide lower fluid flow rates and less frequent batch feeding. Configurations with larger fluid volumes can be used for longer process runs, such as those longer than five days.

[0127] The amount of cell product produced can depend on the volume available for cell processing. The volume available for cell processing can be the volume of a single 3D matrix or a combined volume of multiple 3D matrices. In one 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 multiple 3D matrices. In yet another example, the cell processing system can have multiple 3D matrices, and each 3D matrix can perform a separate cell process, such that the volume available for generating cell product from each process is the volume of a single 3D matrix. In yet another example, the cell processing system can have multiple 3D matrices, each performing the same cell processing operation to generate a single cell product from the multiple 3D matrices. In yet another example, a subset of the multiple 3D matrices can perform a first operation or a series of operations to generate a first cell product, and another subset of the multiple 3D matrices can perform a second operation or a series of operations to generate another cell product. The volume of the 3D matrix can be at least about 1 mL, 2 mL, 3 mL, 5 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 larger. The volume of the 3D matrix can 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 smaller. The volume of the 3D matrix can be approximately 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. mL, 2 mL to 800 mL, 2mL 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 800mL, 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 to 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 The volumes range from 5 mL to 100 mL. In one example, the 3D matrix has a volume of approximately 5 mL to 100 mL.

[0128] The combined volume of multiple 3D matrices can be at least approximately 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, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 8 L, 10 L or larger. The combined volume of 3D matrices can be less than or equal to approximately 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 smaller. The combined volume of the 3D matrix can be approximately 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 40 ...200 mL, 25 mL to 400 mL, 25 mL to 200 mL, 2 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 10L, 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 1 100 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 900 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, 400400 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 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 ...1 L, 900 mL to 2 L, 900 mL to 3 L, 900 mL to 1 L, 900 mL to 1 L, The volumes range from 100 mL to 4 L, 900 mL to 5 L, 900 mL to 6 L, 900 mL to 8 L, 900 mL to 10 L, 1 L 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 one example, the combined volume of multiple 3D matrices is approximately 100 mL to 10 L.

[0129] The efficiency of mass transport and diffusion of nutrients and reagents from fluids flowing through or adjacent to a 3D matrix can depend on the interfacial area between the fluid or flowing fluid and the 3D matrix. The interfacial area between the fluid (e.g., a fluid path or fluid compartment) and the 3D matrix can be greater than or equal to approximately 1 square centimeter (cm²). 2 ), 2 cm 2 3 cm 2 4 cm 2 5 cm 2 6 cm 2 8cm 2 10 cm 2 15 cm 2 20 cm 2 30 cm 2 50 cm 2 Or larger. The interfacial area between the fluid (e.g., a fluid flow path or fluid compartment) and the 3D matrix can be approximately 1 cm². 2 Up to 2 cm 2 1 cm 2 Up to 3 cm 2 1 cm 2 Up to 4 cm 2 1 cm 2 Up to 5 cm 2 1cm 2 Up to 6 cm 2 1 cm 2 Up to 8 cm 2 1 cm 2 Up to 10 cm 2 1 cm 2 Up to 15 cm 2 1 cm 2 Up to 20 cm 2 1 cm 2 Up to 30 cm 2 1cm 2 Up to 50 cm 2 2 cm 2 Up to 3 cm 2 2 cm 2 Up to 4 cm 2 2 cm 2 Up to 5 cm 2 2 cm 2 Up to 6 cm 2 2 cm 2 Up to 8 cm 2 2cm 2 Up to 10 cm 2 2 cm 2Up to 15 cm 2 2 cm 2 Up to 20 cm 2 2 cm 2 Up to 30 cm 2 2 cm 2 Up to 50 cm 2 3 cm 2 Up to 4 cm 2 3 cm 2 Up to 5 cm 2 3 cm 2 Up to 6 cm 2 3 cm 2 Up to 8 cm 2 3 cm 2 Up to 10 cm 2 3 cm 2 Up to 15 cm 2 3 cm 2 Up to 20 cm 2 3 cm 2 Up to 30 cm 2 3 cm 2 Up to 50 cm 2 4 cm 2 Up to 5 cm 2 4 cm 2 Up to 6 cm 2 4 cm 2 Up to 8 cm 2 4 cm 2 Up to 10 cm 2 4 cm 2 Up to 15 cm 2 4 cm 2 Up to 20 cm 2 4 cm 2 Up to 30 cm 2 4 cm 2 Up to 50 cm 2 5 cm 2 Up to 6 cm 2 5 cm 2 Up to 8cm 2 5 cm 2 Up to 10 cm 2 5 cm 2 Up to 15 cm 2 5 cm 2 Up to 20 cm 2 5 cm 2 Up to 30 cm 2 5 cm 2 Up to 50 cm2 6 cm 2 Up to 8 cm 2 6 cm 2 Up to 10 cm 2 6 cm 2 Up to 15 cm 2 6 cm 2 Up to 20 cm 2 6 cm 2 Up to 30 cm 2 6 cm 2 Up to 50 cm 2 8 cm 2 Up to 10 cm 2 8 cm 2 Up to 15 cm 2 8 cm 2 Up to 20 cm 2 8 cm 2 Up to 30 cm 2 8 cm 2 Up to 50 cm 2 10 cm 2 Up to 15 cm 2 10 cm 2 Up to 20 cm 2 10 cm 2 Up to 30 cm 2 10 cm 2 Up to 50 cm 2 15 cm 2 Up to 20 cm 2 15 cm 2 Up to 30 cm 2 15 cm 2 Up to 50 cm 2 20 cm 2 Up to 30 cm 2 20 cm 2 Up to 50 cm 2 or 30 cm 2 Up to 50 cm 2 In one example, the interface area is greater than or equal to approximately 5 cm². 2 .

[0130] like Figure 11AAs shown, treating cells within a 3D matrix can provide improved processing or cell culture conditions, which can improve the health of produced cells and the reliability of the cell manufacturing process. 3D matrices can retain cells within cell compartments, create favorable biophysical microenvironments (e.g., matrix stiffness) that mimic the natural tissue extracellular matrix, establish high local concentrations of secreted factors, and provide programmable substrates or any combination thereof for cell transduction and signal transduction. Figure 11B As shown, 3D matrices can provide tunable physical properties. For example, a 3D matrix comprising a dense, soft matrix can deform as cells proliferate within it. A 3D matrix comprising a porous, rigid matrix can allow cells to proliferate within a matrix with minimal deformation. A 3D matrix comprising a rigid, dense matrix may not allow cell proliferation.

[0131] The physical stiffness, softness, or rigidity of a 3D substrate can alter the ability of cells to grow, proliferate, or otherwise be manipulated within the 3D matrix described herein. The physical stiffness, softness, or rigidity of a 3D substrate can be described and measured based on mechanical parameters such as stiffness, elastic modulus, and viscoelasticity. Elastic modulus (e.g., Young's modulus) is a measure of a material's stiffness. It can 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 can be determined using compression or tensile tests, where the sample is deformed under a given load, and the resulting deformation is measured. The modulus can then be calculated from the slope of the stress-strain curve in the elastic region. Shear modulus describes a material's response to shear stress (parallel to the surface) and is particularly relevant to materials that can undergo significant shear deformation, such as hydrogels. Shear modulus can be measured using rheometry, where the hydrogel sample is subjected to oscillatory shear, and the resulting shear stress and strain are used to calculate the modulus. Bulk modulus describes a material's response to (uniform) hydrostatic pressure. For isotropic materials, the shear modulus (G), elastic modulus (E), and bulk modulus (K) can be correlated with each other as functions of the material's Poisson's ratio (u), as shown by the equation: E = 2G(1+u) = 3K(1-2u). In some embodiments, the 3D matrix can be configured to have an elastic modulus or other physical descriptor that is similar to, or within the range of, that exhibited by, the extracellular matrix (ECM) of natural tissue, such as the extracellular matrix of brain tissue (E is about 1.4-1.9 kPa), or the extracellular matrix of another type of tissue. 3D matrices can have elastic moduli greater than or equal to approximately 0.1 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 greater. 3D matrices can have elastic moduli less than or equal to approximately 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 smaller. 3D matrices can have the following elastic moduli: approximately 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, and 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 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 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, 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 9kPa to 10 kPa. .

[0132] The viscoelasticity of 3D matrices can be tuned or altered for selected cell processing applications. Viscoelasticity describes materials that exhibit both viscous and elastic properties upon deformation. Hydrogels can exhibit significant viscoelastic behavior, allowing them to dissipate energy (e.g., like viscous fluids) and recover their original shape (e.g., like elastic solids). Dynamic mechanical analysis (DMA) can be used to measure viscoelasticity, where a 3D matrix, such as a hydrogel, can be subjected to sinusoidal stress, and the resulting strain can be measured. The phase difference between stress and strain can provide insights into the viscoelastic properties of the material. Stiffness-independent viscoelasticity and relaxation time can influence cell behavior. For example, in hydrogels exhibiting stress relaxation, each force or strain applied to the matrix by cells over time can initially be resisted by a specific stiffness defined by the initial elastic modulus, and subsequently, the resistance decreases over time. For example, stress relaxation can be measured by a stress relaxation test (τ1 / 2). Stress relaxation timescales can be less than or equal to about 1 week, 1 day, 1 hour, 1 minute, 1 second, or less. The stress relaxation timescale can be greater than or equal to about 1 second, 1 minute, 1 hour, 1 day, 1 week or longer.

[0133] The swelling properties of 3D matrices can be tailored for selected cell treatment applications or otherwise modified. Swelling properties can be described and measured based on the swelling ratio, such as the amount of water or buffer that a 3D substrate (e.g., a hydrogel or other polymer matrix) can absorb and retain. Swelling can be an indicator of network hydrophilicity and relative crosslinking density. 3D matrices with stiffer networks tend to exhibit lower swelling, while matrices with more flexible networks tend to exhibit greater swelling. The swelling ratio can indirectly affect mechanical softness and flexibility. The swelling ratio can be a measure of how much the matrix swells beyond its initial state, or the ratio of its swollen volume to its dry volume. For example, to determine the swelling ratio, the dry material can be weighed or its volume measured, immersed in water or other suitable aqueous buffer until equilibrium swelling is reached, and then weighed or remeasured to determine the swelling ratio (e.g., Q). 1a = V s / V d Q1 is the volume swelling ratio relative to dryness, V s V is the saturation volume. d Q is the dry volume; 1b = (V s - V d ) / V d );Q 1c = M s / M d Q 1c M is the mass swelling ratio relative to dryness. s For saturated mass, M d For drying quality; Q 1d = (M s - M d ) / M d Alternatively, the swelling ratio can be determined by weighing or determining the volume of the solvated or hydrated matrix in its initial state (e.g., the initial hydrogel is 2% collagen w / v hydrogel), immersing the matrix in water or other suitable aqueous buffer until equilibrium swelling is reached, and then reweighing or remeasuring the volume to determine the swelling ratio (e.g., Q). 2a = V s / Vi; Q2 represents volume swelling relative to the initial state; V s V is the saturation volume; i Q is the initial volume; 2b = (V s - V i ) / Vi); Q 2c = M s / M i Q 1c M is the mass swelling ratio relative to the initial mass. s For saturated mass, Mi Q represents the initial mass. 2d = (M s - M i ) / M i In some implementations, the 3D matrix is ​​essentially non-swelling (e.g., swelling ratio Q). 2d (Less than approximately 50%). The swelling rate of the 3D matrix can be greater than or equal to approximately 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, or greater. The swelling rate of the 3D matrix can be less than or equal to approximately 500%, 400%, 300%, 250%, 200%, 175%, 150%, 125%, 100%, 75%, 50%, 25%, or less. The swelling ratio of the 3D matrix can be approximately 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%, and 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%, 1 00% 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%, 1 50% 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 one example, the swelling ratio of the 3D matrix is ​​less than or equal to approximately 100%.

[0134] In some embodiments, the 3D volume of the matrix may be constrained by the configuration of the container (such as a flow cell or its compartments) to prevent the 3D matrix 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 programmable swelling matrix that undergoes changes in swelling properties in response to external stimuli. External stimuli may be chemical, thermal, electromagnetic, physical, or any combination thereof. In some embodiments, the container holding the 3D matrix may not physically constrain or otherwise limit the swelling of the 3D volume of the 3D matrix's physical dimensions. For example, the container holding the 3D matrix may include a deformable container. The deformable container may be internally configured such that the 3D matrix swells to occupy an internal space that would otherwise be occupied by a fluid or gas when the 3D matrix is ​​in a non-swelling state, and that internal space is displaced by the 3D matrix upon swelling.

[0135] The porosity of a 3D substrate can be tailored for a given cell processing application or otherwise modified. The porosity of a 3D substrate can be determined using a variety of methods, including visualizing and measuring the morphology and size distribution of pores using microscopy (e.g., stimulated emission depletion microscopy, atomic force microscopy (AFM), or cryo-scanning electron microscopy (Cryo-SEM)). Porosity can also be quantified by measuring the pore volume fraction, for example, porosity e = V p / V t × 100; V p = Pore volume = (W w - W d ) / p(W w = wet weight, W d = dry weight, p = fluid density); V t= Total volume). 3D matrix porosity can affect the swelling behavior and other mechanical properties of the 3D matrix. For example, lower swelling and higher modulus can be associated with lower porosity. The network size or molecular porosity of the matrix can range from nanometer to micrometer scales and can affect nutrient flux throughout the matrix, as well as other aspects of cell retention, proliferation, growth, and cell state or phenotype. In some embodiments, the matrix porosity is on average smaller than that of the cells, but large enough to allow macromolecular diffusion to maintain cell viability. In some embodiments, the matrix porosity is on average larger than that of the cells, such as to facilitate cell proliferation and migration within the 3D matrix while still retaining cells. The average pore diameter can be constant throughout the 3D matrix or can vary throughout the 3D matrix. 3D matrices can have porosities greater than or equal to about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater. In one example, the 3D matrix has a porosity greater than or equal to about 80%. 3D matrices can have porosities of less than or equal to approximately 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or even less. The 3D matrix can have a porosity of approximately 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%.

[0136] The 3D matrix or substrate may have an average pore size that allows nutrients, reagents, and other materials to diffuse or otherwise move through the 3D matrix while retaining or substantially retaining cells within it. In some examples, the 3D matrix comprises a porous solid matrix with interconnected pores. The average pore size of the interconnected pores may be large enough to allow cells (e.g., lymphocytes or other target cells) to grow 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 smaller. In one 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 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 larger. 3D matrices or substrates can have the following average pore sizes: approximately 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 1 ... 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 1μm 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 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, 200μm 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, 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 The pore sizes range from approximately 0.5 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, 600 µm to 800 µm, 600 µm to 900 µm, 600 µm to 1000 µm, 700 µm to 800 µm, 700 µm to 900 µm, 700 µm to 1000 µm, 800 µm to 900 µm, 800 µm to 1000 µm, or 900 µm to 1000 µm. In one example, the 3D matrix or substrate has a pore size of approximately 0.5 µm to 400 µm.

[0137] The stability of 3D substrates can be tailored or otherwise modified for selected cell treatment applications. Some 3D substrate materials can be substantially chemically or biologically stable. For example, porous solid matrices formed from materials such as glass, ceramics, or plastics can be considered stable on an indefinite timescale because they do not undergo substantial chemical or biological degradation under cell treatment use cases (e.g., cell culture conditions). In some embodiments, 3D substrates are substantially stable on a macroscopic scale and on relevant timescales (e.g., hours, days, or weeks). For example, 3D matrices comprising natural polymer hydrogels (such as collagen and fibroin) can be degraded 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 crosslinking agents that can be designed to degrade in response to cell-mediated protease activity. Despite these biological processes, 3D substrates comprising degradable portions can be stable on a macroscopic scale. For example, this degradation process can locally remodel the 3D matrix surrounding cells, thereby allowing cell proliferation, cell migration, or both to occur within a local 3D environment. During local remodeling, the macroscopic matrix can remain substantially structurally intact (e.g., gelled). In some embodiments, the 3D matrix can substantially degrade during cell culture. For example, after a given time, the 3D matrix may no longer be substantially structurally intact. In some embodiments, the matrix material can acquire “self-healing” properties or other dynamic remodeling properties over time. “Self-healing” properties can allow the 3D matrix to autonomously recover its original state after damage or degradation. 3D matrices configured for dynamic remodeling can allow polymer untangling, disturbance of weak physical interactions, rearrangement of reversible interactions, degradation processes, or any combination thereof.

[0138] The optical properties of a 3D matrix can be tailored or otherwise modified for selected cell processing applications. For example, the optical properties of a 3D matrix used in 3D cell culture can provide for monitoring and analysis of the process. In one example, the optical properties of a 3D substrate can determine which imaging and spectroscopic techniques can be effectively applied to observe and measure biological and physical processes occurring within the 3D substrate during cell culture. In some implementations, a highly transparent 3D matrix (e.g., a hydrogel matrix) can be used to enable optical techniques such as measurements of optical density and backscattering. To measure optical density, light of one or more specific wavelengths can be emitted into the 3D substrate, and the intensity of the light transmitted through the 3D substrate can be measured by a detector. A 3D substrate containing relatively more or denser cells can transmit less light. Using Lambert-Beer's law, the transmitted light signal can be used to calculate optical density. To measure backscattering, light of one or more specific wavelengths can be emitted into the 3D substrate, and a sensor near the light source can detect the amount or intensity of light backscattered by cells and other particles present in the 3D substrate. A 3D substrate containing relatively more or denser cells can backscatter more light. Measuring optical density (OD) and backscatter can be useful in-situ process analysis tools in 3D cell culture, such as when assessing cell growth, proliferation, or metabolic activity within a 3D matrix. Such measurements can be used to estimate the concentration of cells or other substances in solution by measuring the absorbance of light at a specific wavelength. In some embodiments, 3D substrates that do not significantly absorb or scatter light at the measurement wavelength (which can interfere with accurate readings) are used to achieve optical density and backscatter measurements. In some embodiments, such as those utilizing thicker or less transparent 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 can be used to allow the use of microscopy techniques, including high-resolution methods such as confocal microscopy. Optical transparency provides minimal light scattering and absorption, allowing for clear imaging of cells and structures within the 3D matrix. In some embodiments, 3D matrices, such as hydrogels, can exhibit light-scattering properties, particularly for thicker or more densely cross-linked hydrogels. Adjusting the refractive index of the hydrogel to match the refractive index of the immersion medium or cells can help reduce scattering. Matching the refractive index of a 3D substrate to that of the surrounding medium (typically a buffer or culture medium) reduces refraction at the interface within the imaging field of view, thereby enhancing image sharpness and depth penetration. Matched refractive indices can provide depth imaging, allowing for more efficient use of techniques such as multiphoton microscopy. Certain 3D substrates, such as porous solids and hydrogels derived from natural polymers, can exhibit autofluorescence, which can interfere with fluorescence microscopy.In some implementations, a 3D matrix with low autofluorescence can be used to allow fluorescence imaging techniques, or the imaging modality can be configured using specific excitation and emission wavelengths (e.g., wavelengths that reduce or minimize autofluorescence for a given material) to minimize the negative impact of autofluorescence on optical sensing.

[0139] 3D matrices can be tailored or otherwise modified to address optical homogeneity, allowing for accurate spectroscopic measurements. Inhomogeneities can result in variable optical path lengths and affect the accuracy of spectroscopic data, such as near-infrared (NIR) and Raman spectroscopy. In some implementations, the 3D substrate is selected for compatibility with specific spectroscopic techniques. In one example, the 3D substrate is configured to allow near-infrared (NIR) spectroscopy, which penetrates deeper into the 3D substrate (such as a hydrogel) (compared to visible light), making it suitable for monitoring cellular processes, water content, solvent interactions, and other overall properties of the cell-loaded 3D substrate. In another example, the 3D substrate is configured to allow Raman spectroscopy, a measurement technique sensitive to molecular vibrations that can provide detailed information about the chemical environment within the 3D substrate. The optical properties of the 3D substrate can be selected or designed to enhance the quality of the Raman signal. The 3D substrate can be incorporated into or otherwise coupled to optical sensors that respond to changes in pH, temperature, specific biomolecules, or any combination thereof. These sensors can provide real-time, in-situ monitoring of the microenvironment, which can enable the monitoring of dynamic cellular processes. In some embodiments, the 3D matrix composition can support light propagation with minimal distortion for techniques such as optical coherence tomography (OCT) or photonic crystal-based sensing. Another optical consideration can be the stability of the 3D substrate under light exposure. Some 3D substrates, such as hydrogels, can readily undergo photodegradation, especially under intense or prolonged light exposure used in certain imaging techniques. This can affect the structural integrity of the hydrogel and the viability of encapsulated cells. In some embodiments, 3D substrates that are not easily photodegraded under the relevant exposure conditions can be used. As described herein, in some embodiments, the 3D matrix can be optically transparent, substantially optically transparent, light-scattering, opaque, and possess other photonic properties such as refractive index and light scattering. In some embodiments, the 3D matrix is ​​optically transparent, substantially optically transparent, or otherwise optically adapted for imaging, such as to allow in situ cell imaging, including by microscopy methods such as wide-field microscopy, bright-field microscopy, light-sheet microscopy, confocal microscopy, or any combination thereof. In some implementations, the 3D matrix is ​​optically suitable for spectroscopic methods, such as allowing in-situ process analysis using NIR spectroscopy or Raman spectroscopy.

[0140] Conductive 3D substrate 3D substrates can be configured to be conductive or provide conductivity. Conductivity can be useful for processing cell types exhibiting phenotype-related electrochemistry or electrophysiology. In some embodiments, the conductivity of the 3D substrate can allow in situ electropermeabilization within the 3D substrate, such as for electroporation cell transfection and genetic engineering purposes. In some embodiments, conductive substrates can be used to incorporate electrical signals as biorelevant physical cues. Electrobiorelevant cues can be used to culture and study electrochemically active cell types, such as neurons and cardiomyocytes. These cells can be highly responsive to electrical stimulation that plays a role in their function, development, and communication. Incorporating electrical cues into the cell culture environment can enhance physiological relevance, thereby supporting the formation and maintenance of relevant cell phenotypes. Direct electrical stimulation can be applied by delivering electrical pulses directly to the 3D substrate, the culture medium, or by electrodes embedded within, around, or throughout the cell culture medium. This method can mimic the natural electrical activity experienced in vivo by cells such as neurons and cardiomyocytes. Stimulation can be configured in various ways (e.g., amplitude, frequency, pulse duration, or combinations thereof) to closely match physiological conditions. For neurons, this can promote axonal growth and synapse formation, while for cardiomyocytes, it can enhance maturation, alignment, contractile strength, or any combination thereof.

[0141] In some embodiments, the materials constituting the 3D substrate, and the 3D substrate itself, can be weakly conductive. For example, a collagen hydrogel matrix can have a very low overall conductivity, such as near that of an insulator. In another example, PEG is a nonionic polymer and is non-conductive. A 3D substrate formed from PEG hydrogel can be non-conductive unless modified. In some embodiments, the 3D substrate can be considered a volume conductor, or a material that allows current to flow through its entire volume (e.g., as opposed to a surface conductor in which current flows primarily along the surface). A volume conductor allows current to flow through its three-dimensional mass. The 3D substrate can have a conductivity greater than or equal to about 10. -10 Siemens / meter (S / m), 10 -9 S / m, 10 -8 S / m, 10 -7 S / m, 10 -6 S / m, 10 -5 S / m, 10 -4 S / m, 10 -3 S / m, 10 -2 S / m, 10 -1 S / m, 10 0 S / m, 10 1 S / m or higher conductivity. 3D substrates can have a conductivity of less than or equal to about 10. 1 S / m, 100 S / m, 10 -1 S / m, 10 -2 S / m, 10 -3 S / m, 10 -4 S / m, 10 -5 S / m, 10 -6 S / m, 10 -7 S / m, 10 -8 S / m, 10 -9 S / m, 10 -10 Conductivity of S / m or lower. Depending on the composition, 3D substrates can have the following conductivity: approximately 10. -10 Up to 10 -9 S / m, 10 -10 Up to 10 -8 S / m, 10 -10 Up to 10 -7 S / m, 10 -10 Up to 10 -6 S / m, 10 -10 Up to 10 -5 S / m, 10 -10 Up to 10 -4 S / m, 10 -10 Up to 10 -3 S / m, 10 -10 Up to 10 -2 S / m, 10 -10 Up to 10 -1 S / m, 10 -10 Up to 10 0 S / m, 10 -10 Up to 10 1 S / m, 10 -9 Up to 10 -8 S / m, 10 -9 Up to 10 -7 S / m, 10 -9 Up to 10 -6 S / m, 10 -9 Up to 10 -5 S / m, 10 -9 Up to 10 -4 S / m, 10 -9 Up to 10 -3 S / m, 10 -9 Up to 10 -2 S / m, 10 -9 Up to 10 -1 S / m, 10 -9 Up to 10 0 S / m, 10 -9 Up to 10 1 S / m, 10-8 Up to 10 -7 S / m, 10 -8 Up to 10 -6 S / m, 10 -8 Up to 10 -5 S / m, 10 -8 Up to 10 -4 S / m, 10 -8 Up to 10 -3 S / m, 10 -8 Up to 10 -2 S / m, 10 -8 Up to 10 -1 S / m, 10 -8 Up to 10 0 S / m, 10 -8 Up to 10 1 S / m, 10 -7 Up to 10 -6 S / m, 10 -7 Up to 10 -5 S / m, 10 -7 Up to 10 -4 S / m, 10 -7 Up to 10 -3 S / m, 10 -7 Up to 10 -2 S / m, 10 -7 Up to 10 -1 S / m, 10 -7 Up to 10 0 S / m, 10 -7 Up to 10 1 S / m, 10 -6 Up to 10 -5 S / m, 10 -6 Up to 10 -4 S / m, 10 -6 Up to 10 -3 S / m, 10 -6 Up to 10 -2 S / m, 10 -6 Up to 10 -1 S / m, 10 -6 Up to 10 0 S / m, 10 -6 Up to 10 1 S / m, 10 -5 Up to 10 -4 S / m, 10 -5 Up to 10 -3 S / m, 10 -5 Up to 10 -2 S / m, 10 -5 Up to 10 -1S / m, 10 -5 Up to 10 0 S / m, 10 -5 Up to 10 1 S / m, 10 -4 Up to 10 -3 S / m, 10 -4 Up to 10 -2 S / m, 10 -4 Up to 10 -1 S / m, 10 -4 Up to 10 0 S / m, 10 -4 Up to 10 1 S / m, 10 -3 Up to 10 -2 S / m, 10 -3 Up to 10 -1 S / m, 10 -3 Up to 10 0 S / m, 10 -3 Up to 10 1 S / m, 10 -2 Up to 10 -1 S / m, 10 -2 Up to 10 0 S / m, 10 -2 Up to 10 1 S / m, 10 -1 Up to 10 0 S / m, 10 -1 Up to 10 1 S / m, or 10 0 Up to 10 1 S / m. In one example, the 3D matrix or substrate has approximately 10. -5 Up to 10 -1 Conductivity in S / m.

[0142] Incorporating ionic solutions or 3D matrix components containing ionic or conductive components can increase matrix conductivity. In one example, the 3D matrix contains ionic or conductive components that increase matrix conductivity. The cell-carrying 3D substrate environment can contain conductive or biomaterials characterized by ionic or electronic conductivity, such as through the presence of mobile ionic substances as dissolved electrolytes, which may be present in cell culture media and buffers. For example, cell culture media and buffers can have a conductivity of about 1.5 to 2.0 S / m, similar to extracellular fluid. The conductivity of such media can depend on the concentration of dissolved ions, such as dissolved ions from added salts (e.g., NaCl, KCl, CaCl2), nutrients, and buffers. The conductivity of cell culture media can help maintain osmotic balance and pH, and also allows for various cellular functions dependent on ion transport and elevator mobility. Conductivity parameters can provide for effective interaction of the applied electric field with the cells without causing excessive thermal or electrolytic effects. In some embodiments, the 3D substrate can provide electrical and electrochemical properties by integrating a conductive material into the 3D substrate, or by using a 3D substrate containing a conductive material, including but not limited to gold (e.g., about 4.1 × 10⁻⁶). 7 S / m), carbon nanotubes (e.g., approximately 10 5 S / m), graphene (e.g., about 10 6 (S / m), conductive polymers (e.g., polypyrrole, about 10-100 S / cm; PEDOT:PSS, about 0.1-1 kS / cm), or any combination thereof. These materials can conduct electrical signals, provide local electrical stimulation, facilitate in-situ electroporation, or any combination thereof.

[0143] In some embodiments, the 3D substrate is filled with an ionic solution that acts as a buffer for electroporation or membrane permeation. The conductivity of the electroporation buffer can be varied and can be tailored for specific applications and cell types. Buffers with lower conductivity (around 0.1 to 1.0 S / m) can be used to reduce Joule heating during the electroporation process. Some electroporation processes can use lower or higher conductivity, depending on the field strength and pulse duration used. Electroporation buffers may include phosphate buffers or solutes such as sucrose to maintain osmotic pressure at minimum ionic strength. The composition and conductivity can be modified to maximize pore formation in the cell membrane while minimizing cell death. Electroporation efficiency can depend on the conductivity and capacitance of the culture medium, cell membrane, and cytosol, as well as cell size and shape. In some embodiments, the conductivity of the 3D substrate and the interstitial fluid can be tuned according to the specific system configuration and biological application.

[0144] As described elsewhere in this document, devices and systems for cell treatment may further incorporate electrical elements that may be coupled to or otherwise electrically communicate with a 3D substrate, an ionic solution, or both to provide an electrical interface with the treated cells (e.g., stimulation, sensing) or to provide membrane permeation.

[0145] Programmable 3D substrate 3D matrices can be functionalized, such as Figure 12A As shown. Functionalization of the 3D matrix can provide a structural microenvironment that enhances or otherwise improves signal transduction. For example, the 3D matrix can be functionalized with cytokines, antibodies, or other components useful for cell treatment. Functionalization can be static, so that the functionalization does not change during cell treatment. Alternatively, functionalization can be changed during cell treatment, so that the 3D matrix can be programmable. Programmable matrices (such as...) Figure 12B (As shown) may include a chemically tethering portion. The chemically tethering portion may be configured to allow the tethering of biomolecular factors, such as cytokines and antibodies, to the 3D matrix. The functional biomolecules may be removably coupled to the 3D matrix such that, during cell treatment, the functional biomolecules can be released from the 3D matrix and removed by washing.

[0146] In some embodiments, the 3D matrix is ​​a programmable 3D matrix. A programmable 3D matrix can physically interact with cells by virtue of including a 3D local environment in which living cells reside (e.g., possessing physical properties such as stiffness and porosity). The 3D substrate may further include cell interaction aspects with designed biological properties. Such cell interaction aspects may include, but are not limited to, factors related to cell adhesion and membrane signaling transduction.

[0147] In some embodiments, the 3D substrate includes features that define cell adhesion or attachment. The 3D matrix material can provide complex and mechanistically diverse adhesive interactions with cells. Cell surface proteins and cell membranes can interact with the 3D substrate via steric interactions, electrostatic interactions, hydrophobic interactions, hydrogen bonds, or other interactions. Transmembrane proteins and cell surface receptors (such as integrins and other proteoglycans) can bind to ligands on or in the 3D substrate (e.g., alone or synergistically) (e.g., when homologous adhesion moieties are provided by a certain 3D substrate composition), thereby providing cell adhesion, mechanical interactions with cells, cell signaling, or combinations thereof. The density of various cell adhesion moieties can depend on the composition of the matrix and can be tuned to promote cell phenotype, morphology, motility, or other cellular properties. In some embodiments, a substrate without intrinsic cell interaction moieties can still allow cell adhesion by the cell producing extracellular matrix and other proteins secreted by the cell within the matrix, which can form a pericellular matrix to which cells can interact and to which 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.

[0148] 3D matrices or 3D substrates can be functionalized to generate functionalized 3D matrices or substrates. Functionalizing a 3D matrix allows it to be stimulus-responsive. Alternatively or complementing this, functionalizing a 3D matrix allows it to interact with multiple cells, for example, to activate or otherwise stimulate cells within a multiple cell group. Alternatively or complementing this, functionalizing a 3D matrix allows it to serve as a substrate for multiple cells or otherwise provide a given cellular environment to multiple cells. 3D matrices or substrates can be functionalized using prefunctionalized polymer precursors to generate 3D matrices. For example, prefunctionalized polymer precursors can be provided in tandem with or as a substitute for unfunctionalized polymer precursors to the cell compartment. The prefunctionalized polymer precursors can be incorporated into the 3D substrate during polymer precursor polymerization. In one example, cells and prefunctionalized polymer precursors can be provided simultaneously, such that the formation of the functionalized 3D matrix occurs concurrently with the retention or encapsulation of multiple cells. Alternatively or complementing this, 3D matrices or substrates can be formed, and functionalization can occur after the polymerization of the 3D matrix or substrate. In one example, functionalizing a 3D matrix involves conjugating or immobilizing one or more biomolecules to the 3D matrix. The one or more biomolecules can be proteins, peptides, cytokines, antibodies, nucleic acid molecules, or any combination thereof. In one example, the one or more biomolecules can be used for membrane cell transduction. During cell treatment, the one or more biomolecules can remain conjugated or homologous to the 3D matrix. Alternatively or supplementally, the functionalized biomolecules can be released from the 3D matrix or substrate and, in some examples, replaced by other biomolecules.

[0149] In some implementations, the 3D substrate contains cell-interacting factors. These factors can provide one or more functions, including, for example (but not limited to, other possible functions), mimicking the tissue microenvironment, imitating or providing extracellular matrix composition, providing membrane signal transduction, regulating cellular properties including cell adhesion, proliferation, migration, differentiation (e.g., including immune cell stimulation), and survival, providing stimulatory, co-stimulatory, pro-survival, or pro-phenotype cellular cues, or any combination thereof. Cell-interacting factors can include, but are not limited to, matrix proteins, glycoproteins, glycosaminoglycans, proteoglycans, ECM-segregated growth factors, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and other secreted proteins, or any combination thereof. For example, peptides can be incorporated into the 3D substrate to mimic specific molecules, sequences, or portions found in the natural extracellular matrix that interact with cell receptors and influence cell adhesion, migration, and fate. RGD or peptides with related motifs can be used to allow cell attachment via the arginine-glycine-aspartic acid adhesion sequence naturally present in the extracellular matrix protein fibronectin, which can enhance cell adhesion through interaction with integrin receptors. The IKVAV peptide (Ile-Lys-Val-Ala-Val) can promote neuronal cell adhesion and neurite growth. The YIGSR peptide (Tyr-Ile-Gly-Ser-Arg) can be used to influence cell attachment and proliferation. As another example, growth factors can be incorporated into the 3D matrix to promote cell proliferation, differentiation, tissue regeneration, or any combination thereof. Fibroblast growth factor (FGF) can be used in neural stem cell culture to promote proliferation. Vascular endothelial growth factor (VEGF) can be used to promote angiogenesis. Bone morphogenetic protein (BMP) can be incorporated for bone tissue engineering to stimulate osteogenic differentiation. In some embodiments, the 3D substrate may contain signal transduction factors, including but not limited to antibodies (such as those binding 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 can be used to modulate cellular immune responses or promote specific pathways of differentiation, regeneration, and function of immune lineage cells. In some embodiments, hormones may be used within the 3D substrate to influence hormonal pathways and support the growth and function of hormone-responsive cell types.

[0150] Cell-interacting factors can be coupled to a 3D matrix. Alternatively or supplementally, cell-interacting factors can be non-covalently associated with the 3D matrix via electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, or any combination thereof. Cell-interacting factors can be covalently coupled to the 3D matrix via cross-linking, immobilization, binding, or any combination thereof. Cell-interacting factors can be encapsulated or otherwise physically retained within the 3D matrix. Cell-interacting factors can be irreversibly coupled to the 3D matrix. Alternatively or supplementally, the 3D matrix can be configured for controlled release of cell-interacting factors.

[0151] In some embodiments, one or more cell-interacting factors are delivered via covalent binding or crosslinking to a 3D substrate, which can stabilize the molecules within the 3D substrate or provide a uniform or homogeneous and controlled signal (e.g., presenting such molecules to cells at a specific average concentration within the 3D substrate, such as milligrams per milliliter (mg / mL) equivalent to a solution-phase formulation). The selection of the 3D substrate material, crosslinking density, functionalization strategy, or any combination thereof can be considered to tune the functionalization of the 3D matrix to provide selectable bioactive compositions for cell contact and interaction. Alternatively, the 3D matrix can be an inactivating composition, such as containing an antibody that can crosslink to the 3D substrate in a manner that prevents binding or other targeting of biological interactions. Functionalization chemistry can include, but is not limited to, covalent bonding chemistry, photocrosslinking, enzymatic crosslinking, Michael addition, or any combination thereof. Carbodiimide chemistry can be used to crosslink proteins to various substrates and involves the use of carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). EDC can activate carboxyl groups to react with amino groups to form stable amide bonds. This chemistry can be combined with N-hydroxysuccinimide (NHS) to improve efficiency. Glutaraldehyde crosslinking can effectively crosslink proteins through their amino groups, forming Schiff bases. This chemistry offers the benefits of simplicity and robustness of the resulting crosslinks. Click chemistry, including copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC), can be highly specific, efficient, and work under mild or biocompatible conditions, making it suitable for coupling sensitive biomolecules. Incorporating photocrosslinkable moieties such as benzophenone, aryl azides, or cinnamoyl groups into biomolecules or polymers allows for crosslinking upon exposure to ultraviolet (UV) or visible light. Photocrosslinking can provide spatial and temporal control over the crosslinking process. Thiol-olefin reactions (UV-induced reactions between thiols and olefins) can generate thioether bonds. Enzymatic cross-linking can include examples such as transglutaminase (an enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues in proteins), as well as laccase and peroxidase (which can catalyze the oxidation of phenolic compounds to form cross-links, such as those used for the modification of lignin or other natural polymers). Thiol-Michael addition reactions between thiol groups and electron-deficient double bonds (such as acrylates or maleimides) can have benefits of cytocompatibility and reactivity under physiological conditions. Some conjugation chemistry can involve the chemical modification or conjugation of carbohydrates (sugars) to proteins, or the chemical modification or coupling of carbohydrate-modified proteins. Chemically enzymatic biological conjugation can use enzymes, such as glycosyltransferases, to attach sugars with specific functional groups to proteins.Glycosyltransferases are used to transfer sugar molecules with pre-attached functional groups (e.g., biotin or other conjugation groups) to target sites on proteins to provide highly specific bioconjugation, allowing sensitive proteins (such as antibodies) to retain protein functionality. These sugars, or functionalized sugars, can act as linkers for subsequent conjugation operations. Sugars on proteins can be modified to introduce azides or alkynes, followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC) to attach various functional groups. Glycosylation sites on proteins can be used to attach polyethylene glycol (PEG) molecules that have been functionalized to react with sugars. This can involve using linkers capable of reacting with the hydroxyl groups of sugars to form stable bonds. Carbohydrates on the protein surface can be chemically modified to introduce aldehyde or ketone groups, which can be used to form oxime or hydrazone bonds with aminooxy or hydrazine-functionalized molecules. Other 3D matrix bioconjugation methods can be used to prepare biofunctional 3D substrates.

[0152] In some implementations, protein-protein interactions are used to immobilize bioactive proteins, utilizing specific and high-affinity binding pairs to create functional 3D substrates. For example, the biotin-streptavidin interaction is one of the strongest non-covalent interactions in nature, exhibiting extremely high affinity (Kd ≈ 10⁻⁶). -15 Biotinylated proteins can be immobilized in hydrogels containing streptavidin, and vice versa. In another example, collagen-binding peptides (CBPs) can be used to link peptides via specific affinity for collagen molecules. Protein immobilization can be achieved by incorporating functionalized CBPs into a 3D matrix containing collagen, utilizing this targeted protein interaction. The interaction between His tags and Ni-NTA (nitroglycerotriacetic acid) can be achieved using short sequences of histidine residues added to the protein, which can bind to nickel ions on Ni-NTA groups integrated within the 3D matrix. As described elsewhere herein, this interaction can be further used for reversible immobilization. Incorporating protein A or G into the 3D matrix facilitates antibody immobilization. Proteins A and G have high affinity for the Fc region of IgG type antibodies. In some embodiments, multivalent linkers can be employed to improve functionalization efficiency and control the density of cell-interacting factors within the 3D matrix. For example, four-armed, six-armed, eight-armed, ten-armed, twelve-armed, and other multi-armed PEG molecules (which may be provided with their specific molecular weight or distribution and further contain chemical conjugation groups, such as four-armed PEG succinimide carboxymethyl ester) can be used to generate stable connections between 3D substrates and cell-interacting factors.

[0153] In some embodiments, one or more cell-interaction 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 the factor via electrostatic interactions. Hydrophobic interactions (such as those driven by the self-assembly of amphiphilic molecules, which aggregate in an aqueous environment to minimize the exposure of their hydrophobic segments) can retain the factor within the 3D substrate. In some embodiments, the transient binding of one or more cell-interaction factors to the 3D substrate can be stabilized by additional crosslinking, such as through the use of chemistry described elsewhere herein.

[0154] In some embodiments, the 3D matrix may be configured with a controlled-release mechanism 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 is one or more proteins. In some embodiments, the cell-interacting factor is a viral vector. In some embodiments, the cell-interacting factor includes both proteins and viral vectors. Protein encapsulation can provide controlled release of bioactive factors into a culture environment or biological system (e.g., a 3D matrix). Protein encapsulation can provide local and sustained release of proteins and other factors, which may, for example, guide cellular behavior or regulate immune responses. The controlled-release mechanism may involve trapping proteins or other factors within a carrier material (such as hydrogels, nanoparticles, microspheres, liposomes, or combinations thereof). The carrier material can protect molecules from degradation and allow controlled release via various mechanisms. In diffusion-controlled release systems, proteins or other factors can diffuse out of the encapsulation material over time. Diffusion can be driven by a concentration gradient. The diffusion rate can be modulated by modifying the porosity and crosslinking density of the encapsulation material. In degradation-controlled release systems, the encapsulating material can be biodegradable, and the encapsulated proteins or other cell-interacting factors can be gradually released as it decomposes. The degradation rate can be tuned by selecting materials or cross-linking agents within the encapsulating material.

[0155] In stimulus-responsive release systems, encapsulation materials can be designed to respond to specific stimuli present in the environment, such as pH, temperature, enzyme activity, or any combination thereof. The stimulus can trigger the release of proteins or cell-cell interaction factors. For example, proteins can be encapsulated within alginate beads using ionic crosslinking with calcium ions. The release rate can be controlled by the ionic strength and pH of the culture medium, which alter the stability of the alginate matrix. In another example, temperature-sensitive hydrogels, such as poly(N-isopropylacrylamide) (PNIPAM), can undergo a phase transition at a given temperature. The phase transition can release the encapsulated protein in response to temperature changes. In yet another example, PLGA microspheres formed from poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer that degrades over time, can release the encapsulated protein during hydrolytic degradation and other processes that degrade the polymer matrix. The polymer composition and molecular weight can alter or modify the degradation rate of such encapsulation materials, thereby altering or modifying the release kinetics. In another example, dendritic polymers or other branched synthetic polymers can encapsulate proteins or other cell-interacting factors within their structure, allowing release to be controlled through polymer design, including its size and surface functionality.

[0156] In one example, proteins or other cell-interaction factors can be delivered to a 3D matrix in the form of liposomes. Liposomes can be spherical vesicles with a lipid bilayer that encapsulate hydrophilic proteins or cell-interaction factors within their aqueous core. Liposomes can be introduced into the 3D matrix environment and release the payload over time via liposome instability and leakage. Modifications to the liposome structure, such as including pH-sensitive or thermosensitive lipids, can further allow for the release of proteins from the liposomal formulation in response to environmental changes. In another example, hydroxyapatite (HA) particles can be used for the sustained release of cell-interaction factors, such as viral particles, within a 3D matrix. Due to its high surface area and porous structure, hydroxyapatite can adsorb proteins and other biomolecules onto its surface or, depending on the formulation, encapsulate these molecules within its matrix. Viral particles can bind to HA particles, allowing them to be gradually released as the HA degrades or as the particles desorb from the surface. The release rate can be tuned by modifying the particle size, porosity, and surface properties of the HA. For example, smaller particles or particles with higher porosity can provide a faster release rate than larger particles or particles with lower porosity. Viral particles and other cell-interacting factors can be encapsulated within biodegradable fibers, where the release rate is tailored to the fiber composition and structure. Viral particles can be encapsulated within fibers produced by electrospinning and other methods. These fibers can provide high surface areas and can be engineered to degrade within a targeted timeframe, releasing cell-interacting factors such as viral particles. Combining different polymers or incorporating nanoparticles into the fibers can further refine the release profile and enhance the stability of 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 surfaces or encapsulate them within their porous structures, providing both protection and controlled release mechanisms. Each controlled release mechanism or encapsulation method offers unique advantages and can be selected based on the specific requirements of the application, such as targeted release profiles, factor stability, and the environmental conditions of the cell treatment system. These strategies allow for precise control over the spatial and temporal availability of bioactive factors, thereby enhancing the effectiveness of cell treatment systems.

[0157] In some embodiments, elastin-like peptides (ELPs) can be used for the immobilization, controlled release, or both of cell-interacting factors within a 3D matrix. The ELP may contain a repeating pentapeptide sequence (Val-Pro-Gly-Xaa-Gly, where Xaa can be any amino acid except proline) exhibiting lower critical solution temperature (LCST) behavior. Below the LCST, the ELP is soluble in water. Above the LCST, the ELP undergoes a phase transition and becomes insoluble in water, then aggregates. This property can be utilized to control the solubility and localization of ELP-tagged proteins within the 3D matrix. By adjusting the ambient temperature just above the LCST, ELP aggregates can be induced, effectively capturing and immobilizing the linked proteins within the matrix. The ELP can be chemically modified to include reactive groups (such as acrylates). Through copolymerization or cross-linking reactions, the ELP can be integrated into the 3D matrix structure. This integration provides that the linked proteins remain within the matrix when the ELP undergoes a phase transition, allowing for controlled spatial distribution. Proteins of interest can be genetically fused with the ELP sequence. This fusion allows proteins to inherit the phase transition properties of ELPs, thus providing responsive and reversible immobilization within a 3D matrix. ELPs can be used to encapsulate proteins within microspheres or nanoparticles, which are then embedded in a 3D substrate. Upon temperature changes, these particles undergo a transition that can control the release rate of the encapsulated proteins. ELPs can also contribute to or enhance the mechanical reinforcement of hydrogels. By behaving somewhat like natural elastin, they can impart elasticity and durability to the 3D matrix, which can be beneficial for creating scaffolds that mimic the natural extracellular matrix.

[0158] The connection or spatial co-localization between cell-interacting factors and the 3D matrix can be formed externally to the 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 device. In some embodiments, a 3D matrix precursor solution (containing a polymer functionalized with cell-interacting factors) is provided, optionally mixed with a cell population and allowed to gel, thereby forming a functionalized cell-interacting 3D substrate. In some embodiments, a stock 3D substrate precursor solution containing a polymer with attachment portions can be provided such that, upon mixing with one or more additional reagents (e.g., reagents including cell-interacting factors), a functionalized cell-interacting 3D substrate or a functionalized cell-interacting 3D substrate precursor solution (e.g., which forms a functionalized cell-interacting 3D substrate upon gelation) is formed. In some embodiments, a 3D substrate with attachment portions or a 3D substrate precursor solution containing attachment portions can be provided, followed by the provision and contacting of an aqueous reagent containing at least one cell-interacting factor with the 3D substrate, thereby forming a connection between the 3D substrate and the cell-interacting factors.

[0159] In some embodiments, a 3D matrix precursor solution comprising a controlled-release formulation of a cell-interacting factor is provided. The 3D matrix precursor solution can be mixed with cells and allows gelation to form a functionalized 3D substrate with controlled release of the cell-interacting factor. In some embodiments, a stock 3D matrix precursor solution is provided, and the controlled-release formulation of the cell-interacting factor is provided separately, such that a 3D substrate comprising controlled release of the cell-interacting factor is generated upon mixing and gelation. In some embodiments, a 3D substrate or a 3D matrix precursor solution is provided, followed by the provision and contacting of an aqueous reagent comprising a controlled-release formulation of at least one cell-interacting factor with the 3D substrate to form a 3D substrate comprising controlled release of the cell-interacting factor.

[0160] In some implementations, functional aspects of the 3D substrate (such as the connection between the 3D substrate and cell-interacting factors) can be reversed, removed, cleaved, or any combination thereof. The connection can be generated by any reversible conjugation or 3D deposition method or a reversible controlled-release formulation. These reversible connections can respond to specific stimuli (e.g., chemical, thermal, physical, enzymatic, etc.) or incorporate competitive displacement mechanisms. Cleavable linkers can be stable until exposed to certain internal or external stimuli, after which they cleave and release the conjugated molecule. Stimulus-responsive linkers can include, but are not limited to, pH-sensitive linkers (e.g., hydrazones), enzyme-responsive linkers (e.g., enzyme-cleavable peptide linkers), redox-responsive linkers (e.g., linkers containing disulfide bonds configured to cleave in a reducing environment), and photocleavable linkers (e.g., O-nitrobenzyl derivatives cleaved upon UV or near-UV light exposure). In one example, the linker is a hydrazone, which is stable at physiological pH and cleavable in an acidic environment. In another example, the linker is a peptide linker configured to be cleaved by matrix metalloproteinases (MMPs). Alternatively, a competitive substitution mechanism can be used, in which the binding of the bioconjugated molecule can be reversed by introducing a competing molecule with a higher affinity for the binding partner to replace the first molecule. For example, a cell-loaded 3D matrix functionalized with one or more cell-interaction factors can be provided. Contacting the cell-loaded 3D matrix with an agent or other stimulus that reverses the connection between the cell-interaction factor and the matrix can release the factor from the 3D matrix to remove it from the 3D microenvironment. The released factor can be removed from the 3D microenvironment by washing, diffusion, fluid flow, or any combination thereof.

[0161] In some implementations, a combination of conjugation and cleavage chemistry is used to time-program signaling cascades by delivering cell cues using a 3D matrix at specified times (such as a specified duration). These capabilities can be used to precisely control complex stimuli, co-stimulatory, survival-promoting, and phenotype-promoting cell cues to provide biological control over cellular processes and products. For example, a cell-loaded 3D matrix substrate comprising one or more functionalized portions is provided. An aqueous reagent comprising one or more components can be introduced into and contacted with the 3D matrix, such that the functional component is incorporated into or onto the matrix via the functionalized portion (e.g., through binding, chemical bonding, ionic bonding, electrostatic interaction, etc.). The 3D matrix can be contacted with a second reagent or other stimulus that reverses the connection between the first component and the functionalized portion of the matrix. Reversing the connection can release the first component, allowing it to be removed from the 3D microenvironment via washing, diffusion, fluid flow, or any combination thereof. A third reagent comprising one or more functional components can be introduced into and contacted with the 3D matrix, such that the functional component is incorporated into or onto the 3D matrix via the first or second functionalized portion. Combinatorial or modular functionalization can provide dynamic or iterative temporal control and programming of the 3D microenvironment. Dynamic or iterative temporal control allows for the use of multiplexed or synchronized cell signaling workflows. The use of two or more orthogonal functionalization components or chemicals further allows for time-dynamic and controllable programming of cell membrane signal transduction.

[0162] In some implementations, the 3D substrate may be sterilized prior to use in cell processing. Sterilization of the 3D substrate can reduce or prevent contamination during cell processing and may allow for the clinically regulatory applicability of using 3D matrices in cell processing workflows. The 3D substrate may be sterilized prior to cell processing (e.g., cell culture) using temperature (e.g., autoclaving), gamma or sterilizing UV irradiation, ethylene oxide exposure, ethanol treatment, dense carbon dioxide gas sterilization, other methods, or combinations thereof. For workflows involving gel-sol transition, the precursor solution may be sterilized prior to matrix formation. Sterilization of the precursor may include, but is not limited to, filtration (e.g., filtration of lower viscosity solutions), gamma or sterilizing UV irradiation of the solution or dry polymer, temperature (e.g., autoclaving), or any combination thereof. The sterilization method used may be selected based on compatibility and the cell processing application. For example, sterilization methods that do not degrade, denature, or otherwise alter the physical and biological properties of the 3D substrate material may be used. 3D substrate materials used to manufacture clinical products, such as those for human use, may be subject to stringent safety and performance standards. For example, 3D substrate materials can be selected for their biocompatibility properties or their ability to be easily or substantially completely removed from the formulated product (e.g., within acceptable limits). 3D substrate materials that may be carried into the formulated product can be selected from a group of materials that do not cause harmful immune responses, toxicity, or infection. Alternatively, 3D substrate materials can be selected or designed based on their ability to be easily or substantially completely removed from the formulated product. 3D substrate materials can be selected from a group of materials that do not carry or substantially do not carry into the product formulation. For example, 3D substrate materials can be removed during cell washing. 3D substrate materials can be selected for nonspecific interactions with the cell product. For example, materials with little or no nonspecific interactions with the cell product can be selected to reduce the material's carryover into the product. 3D substrate materials can be selected for their ability to be removed from the formulated product. For example, the material can be easily washed away, degraded, or both to achieve acceptable levels within the cell product. 3D substrates can be selected or manufactured to minimize the presence of endotoxins, which can cause severe inflammatory responses and are subject to regulatory limits in the formulation of clinical products. 3D substrate materials can be selected from a group of materials that can be consistently manufactured to provide homogeneity in product properties or clinical outcomes. Homogeneity may be subject to regulatory approval. The specific details of these properties may vary based on the intended application and regulatory guidance.

[0163] Reversible Gel-Soluble 3D Substrate In some embodiments, a 3D substrate is formed using a matrix composition that undergoes a stimulus-responsive or reversible gel-sol transition. For example, a stimulus-responsive hydrogel that responds to physical or biochemical stimuli can be used. Physical or biochemical stimuli can include, but are not limited to, temperature, light, electromagnetic fields, sound waves, pH, ions, crosslinking agents, other reagents, or any combination thereof.

[0164] Thermally responsive 3D matrices can exhibit changes in their physical state or properties in response to temperature variations. This behavior can be controlled by a critical temperature, which defines the transition in solubility and structure of the 3D matrix. A 3D matrix can exhibit a first phase below its lower critical solution temperature (LCST) and a second phase above its upper critical solution temperature (UCST). The LCST is a temperature below which the polymer is soluble in a given solvent, and above which the polymer becomes insoluble. When the temperature rises above the LCST, the hydrophilic-hydrophobic equilibrium can shift to hydrophobicity due to changes in polymer-solvent interactions. This shift can lead to polymer aggregation and precipitation from solution or gel formation. This behavior is often caused by an increase in intra-polymer hydrophobic interactions, which overcome polymer-solvent interactions as the temperature increases. For polymers like poly(N-isopropylacrylamide) (PNIPAM), the hydrogen bonds with water that maintain solubility at lower temperatures are broken at higher temperatures, leading to phase separation. Such materials can exhibit reversible phase transitions, allowing for the redissolution of the matrix substrate upon temperature reduction. This temperature-controlled gel-sol transition can facilitate the harvesting of cellular products. Polymers capable of undergoing thermoresponsive gel-sol transitions include, but are not limited to, gelatin, collagen, cellulose, chitosan, starch, carrageenan, hyaluronic acid, xanthan gum, xylan, elastin, dextran, or combinations thereof. Additional chemical modifications can alter the thermoresponsiveness of polymeric hydrogel materials. For example, methacrylamide collagen can be capable of undergoing a reversible thermogel-sol transition, where the thermogelation of natural collagen can be thermally irreversible.

[0165] Other classes of stimulus-responsive 3D substrate materials that can be used include pH-responsive, chemical-responsive, enzyme-responsive, electroresponsive, photoresponsive, or combinations thereof. pH-responsive polymer materials may contain polymers with side acidic or basic groups that accept or contribute protons in response to changes in pH, resulting in a gel-sol transition. Chemically responsive polymer materials may use reversible chemical bonds to mediate the gel-sol transition. Examples of materials and mechanisms with reversible chemical bonds include, but are not limited to: those characterized by disulfide bonds, such as in PEGSSDA crosslinking agents; and ionic condensation of alginate in cations such as Ca2+. 2+Cross-linking exists and can be reversed by chelating agents such as EDTA; and reversible chemical bonds can be broken by introducing competitive molecules, such as FLAG-based interactions that can be broken by introducing competitive FLAG peptides. Enzyme-responsive materials, such as those composed of naturally derived materials such as collagen (collagenase), fibrin (nattokinase), and HA (hyaluronidase), can allow gel-sol transitions and cell release by degrading the hydrogel matrix in the presence of enzymes, or upon addition of enzymes, or under enzyme activity, in suitable buffer and reaction conditions. When using enzyme-responsive materials, care should be taken not to damage cell surface receptors through prolonged enzyme treatment. Using specific enzymes, such as collagenase, can provide advantages over non-specific proteases. Electrosensitive hydrogels can respond to electric fields that affect the polymer binding, structure, or both within them. 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. Photosensitive polymer materials can respond to light of specific wavelengths. For example, a gel matrix comprising a deoxycholic acid-modified β-cyclodextrin derivative and an azobenzene-branched poly(-acrylic acid) copolymer can transition from a gel to a solution phase upon exposure to a first light (e.g., light at a wavelength of 355 nanometers (nm)) and from a solution phase to a gel upon exposure to a second light (e.g., light at 450 nm). Other embodiments of 3D matrix substrates, as described herein, do not undergo a gel-sol transition, such as in the case of static or pre-formed matrices (e.g., porous solid matrices).

[0166] In some embodiments, a liquid 3D matrix precursor solution is provided, mixed with cells, and subjected to gelation (e.g., via the application of stimulation) to form a 3D matrix. For example, a precursor solution, such as a precursor solution of a PEG-PNIPAM polymer, or a precursor solution of collagen or a collagen-based polymer, can be provided, which is in the liquid phase at 4°C but undergoes a gel-sol transition upon being raised to 37°C (a temperature suitable for cell treatment). In another example, a precursor solution, such as alginate or a precursor solution of an alginate-based polymer, can be provided, which is in the liquid phase until mixed with a suitable cation source (e.g., Ca). 2+ ,Br 2+ or Sr 2+A salt solution is mixed. The precursor solution can be mixed with cells and loaded into a cell processing compartment (e.g., a compartment of a bioreactor). A salt solution can be added to the compartment, resulting in gelation of the 3D matrix. Subsequently, a stimulus can be provided to reverse the gel-sol transition. For example, the compartment temperature can be lowered to 4°C, resulting in a gel-sol transition of a thermoresponsive hydrogel matrix, or an ion-chelating agent such as EDTA can be introduced into a chemically responsive alginate-based hydrogel matrix, resulting in a gel-sol transition. A liquid containing dissolved gel matrix material and cells can be harvested, such as by removing the liquid mixture from the compartment and washing or otherwise separating the cells for downstream use as a liquid suspension. In some embodiments, a 3D substrate with stimulus-responsive gel-sol transition properties is provided, and cells can be seeded onto or into the 3D matrix. A stimulus can be provided to reverse the gel-sol transition, thereby facilitating the harvesting of cells from the dissolved gel matrix material.

[0167] Alternative 3D substrate configuration In some embodiments, the 3D substrate or cell matrix may not contain a monolithic matrix composition at the macroscale. For example, the 3D substrate may contain other configurations, including liquid-like solids, colloidal solutions, packed beds, and dense suspensions (e.g., colloidal mixture configurations), which may also provide a matrix-like or 3D-like environment and at least partially serve to retain cells. In some embodiments, the 3D substrate resembles a colloidal mixture, wherein the matrix substrate contains suspended, dispersed insoluble particles, including porous particles (such as beads, gel beads, or gels) as well as ribbon-like and fibrous particles. While such configurations do not form a monolithic hydrogel or monolithic 3D matrix, they can 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 the 3D substrate may be suspended within a porous matrix (such as a hydrogel), effectively forming an interpenetrating network of the 3D matrix substrate that supports cell treatment. The material composition, chemistry, and methods described herein regarding 3D matrices, programmable substrates, membrane signal transduction, biological properties, reversible gel-sol, and other features described elsewhere herein can be used in the described non-monolithic matrix configurations.

[0168] In some embodiments, the solid-state bioreactor or cell processing system may be configured with cell compartments that do not contain a monolithic 3D substrate, such as a monolithic porous solid scaffold or a monolithic 3D matrix such as a hydrogel. In some embodiments, the solid-state bioreactor or cell processing system may be configured to allow maintenance of cell compartments and optional perfusion compartments. For example, the 3D substrate may comprise a dense colloidal solution of particles, ribbons, or other substrates that can support cell culture and provide a cell processing environment similar to that described elsewhere herein. For example, systems including non-monolithic substrates may further include integrated sensing, perfusion, in situ genetic engineering, or any combination thereof. This configuration may utilize perfusion configurations that substantially do not disturb or suspend the 3D substrate environment, such as low flow rates or flow configurations that exert minimal destructive fluid forces (e.g., shear forces) on the 3D substrate. Solid-state bioreactors or cell processing systems may have a semi-permeable membrane interface configured between the cells and the perfusion or fluid flow compartments, said semi-permeable membrane interface retaining or substantially retaining both the cells and the 3D substrate within the cell compartment (e.g., with pore sizes or filter sizes that prevent, for example, the microgels or fibers forming the 3D substrate and the cells from passing through). In this configuration, nutrient supply and other reagents may be delivered primarily via diffusion from the perfusion compartment, by guiding a secondary flow through the cell compartment, or both. In this configuration, nutrient supply and other reagents may be provided via other solid-state bioreactor configurations described herein to provide nutrients and other materials to the 3D substrate, such as those using containers including flasks and bags.

[0169] 3D substrates can be in contact with fluid within the fluid flow path, or they can be separated from the fluid flow path, such as... Figure 13As shown. The 3D matrix can be separated from the fluid flow path via a membrane. The membrane can be porous, allowing reagents and waste to diffuse through it. Alternatively, the interface between the 3D matrix and the fluid flow path can be a liquid interface, allowing the fluid flowing in the fluid flow path to directly contact the 3D matrix. In another example, the interface between the fluid flow path and the 3D matrix can be a thin gel layer. The thin gel layer can be a hydrogel. The thin gel interface can be configured to allow reagents and waste to diffuse through or from the fluid via the thin gel. As described herein, the 3D matrix can be a stimulus-responsive 3D matrix. The 3D matrix can be configured to retain a variety of cell substrates. For example, cells can be directly embedded within the 3D matrix. In another example, cells can be disposed in or on two-dimensional (2D) carriers (e.g., strips), 3D carriers (e.g., gel beads), 2D beads (e.g., magnetic beads), microporous scaffolds (e.g., polystyrene mesh), or any combination thereof. The carrier or scaffold containing cells can then be encapsulated or embedded within the 3D matrix. In some examples, the carrier or scaffold containing cells can be disposed in solution and not encapsulated or embedded within a 3D matrix. In such examples, the carrier or scaffold can be retained within the cell compartment by means of a membrane disposed between the cell compartment and the fluid flow path.

[0170] Cell loading Various systems and methods can be used to deliver components into cell compartments or into a 3D matrix within cell compartments. Systems and methods can provide: cell localization or substantially localization within a 3D substrate, cell compartment, or both. Alternatively, cells can be localized or substantially localized within a region or area of ​​a bioreactor or cell processing system, said region or area being in communication with cell culture medium or perfusion fluid. In some embodiments, one or more liquid 3D substrate precursors are provided, mixed with one or more cell sources, introduced into a solid-state bioreactor or its compartments, and allowed or guided to form a 3D substrate that contains, embeds, or encapsulates cells. For example, a solid-state bioreactor may be configured with a mixing vessel (which can serve to continuously or batch-mix cells with the liquid 3D substrate precursor solution and, in some examples, other reagents) or configured to interface with a cell processing system having a mixing vessel. In another example, the solid-state bioreactor can be configured with a fluid-configured mixing module, or configured to interface with a cell processing system having a fluid-configured mixing module, such that the 3D matrix solution, cells, and other reagents, in some examples, aggregate and mix in a continuous or semi-continuous manner as they are transferred to or into the bioreactor, cell processing unit, or its compartments. The method can provide regions or compartments that position or substantially position the cell-loaded 3D matrix within the bioreactor, such as regions or compartments configured to communicate with cell culture media or perfusion fluids. In one example, a sacrificial material (such as a cell-free 3D matrix material, another liquid, or a gas) can be introduced into the fluid system and used to push the cell-loaded matrix volume into a targeted location within the bioreactor. In another example, a negative pressure can be applied to draw the cell-loaded matrix volume into a targeted location within the bioreactor (e.g., as shown in the image). Figure 15 (As shown). As another example, cell-loaded matrix volumes can be placed at targeted locations within a bioreactor using extrusion, deposition, or printing methods. These and other methods can be used in any combination. Solid-state bioreactors can be configured with fluid pathways and systems that minimize the dead volume of the liquid cell-matrix mixture.

[0171] Cells can be loaded into or onto a 3D substrate using any retention, encapsulation, or adhesion method. In one example, multiple cells are encapsulated or trapped within a 3D matrix. In another example, the 3D matrix or substrate is formed in the presence of multiple cells to generate a 3D matrix encapsulating multiple cells. The 3D matrix encapsulating multiple cells can be generated inside a compartment (e.g., a cell compartment) or otherwise within a compartment, as described elsewhere herein. Alternatively, the 3D matrix encapsulating or trapping multiple cells can be generated outside a compartment and subsequently loaded into the cell compartment. In one example, a cell processing unit can be configured to receive a pre-formed 3D matrix having or containing multiple cells. In one example, multiple cells can be mixed with a matrix precursor in solution, and the solution can be provided to a compartment of a cell processing system or bioreactor for in-situ polymerization. In-situ polymerization of the 3D matrix can include applying a stimulus to the matrix or polymer precursor. In one example, a stimulus can be applied to the matrix or polymer precursor in a compartment of a cell processing system or bioreactor to generate a 3D matrix encapsulating multiple cells in situ. As described elsewhere herein, the stimulus can be a thermal stimulus, a chemical stimulus, an enzymatic stimulus, or any combination thereof. In another example, the 3D matrix can be generated inside or outside the compartment without multiple cells, and multiple cells can be added to the pre-formed 3D matrix, such that the cells adhere to the 3D matrix, migrate into the 3D substrate, or both.

[0172] In some embodiments, cells can be loaded or otherwise deposited into a pre-formed 3D cell culture substrate. For example, a suspended cell source can be introduced into a bioreactor containing a 3D substrate, allowing cells to passively deposit or permitting or guiding cells to actively permeate the 3D substrate to become substantially retained in the 3D matrix. In another example, a solid-state bioreactor can be configured with 3D substrate cell compartments and perfusion compartments oriented such that cells introduced into the perfusion compartment (e.g., via a fluid flow) settle onto and permeate the 3D substrate due to gravity. In some embodiments, the 3D substrate component of a solid-state bioreactor or cell processing system can be configured to retain cells within a 3D matrix environment. For example, a 3D substrate can be provided in a solid-state bioreactor device, the solid-state bioreactor being configured with a matrix environment that promotes cell retention or cell migration into the 3D substrate. In some embodiments, the 3D substrate is functionalized (e.g., statically or programmatically) with factors that promote cell-cell interactions, and such factors can promote specific cell retention in the matrix or promote specific cell migration to the matrix where they are substantially retained. In some embodiments, substrate functionalization can be configured to promote the retention or migration of specific cell types or properties. For example, the 3D substrate of a solid-state bioreactor can be configured with a 3D microenvironment that attracts or retains specific cell types due to the properties of the 3D microenvironment. In another example, the 3D substrate of a solid-state bioreactor can be configured with a 3D microenvironment that supports the viable culture of a specific cell type, making it impossible for other cell types to maintain a viable state or proliferate. In some embodiments, the solid-state bioreactor can be configured with a stimulus-responsive 3D substrate that is capable of attracting or retaining cells in response to stimuli that modulate one or more properties of the 3D substrate. For example, the solid-state bioreactor can be configured with a stimulus-responsive 3D matrix that modulates the substrate porosity. After contacting a cell suspension with a highly porous 3D substrate (such as by adding or seeding cells), a stimulus (e.g., a cross-linking agent) can be applied, which causes a reduction in the porosity of the 3D substrate, resulting in the cells being trapped or substantially retained within the substrate.

[0173] Cells can be loaded into a pre-formed matrix, or alternatively, the matrix can be generated in the presence of multiple cells, such as... Figure 14As shown. Cells can be loaded into a pre-formed 3D matrix by allowing cells to flow into a compartment containing a pre-formed 3D matrix. One side of the compartment may include a membrane or other interface configured to retain the 3D matrix. Cells can adhere to the 3D matrix and migrate into it, where they proliferate. Cells can settle into the 3D matrix due to gravity. In another example, cells can be mixed with a polymer or gel precursor. The polymer or gel precursor may be stimulus-responsive, causing the 3D matrix to form or degrade upon application of a stimulus (e.g., thermal, chemical, or enzymatic stimulation). The 3D matrix may be a thermoresponsive matrix, which is liquid or semi-liquid at low temperatures and solid at higher temperatures. Cells can be embedded or encapsulated within the matrix during matrix formation.

[0174] 3D matrix can be generated within cell compartments. Alternatively, such as Figure 15 As shown, the 3D matrix can be pre-formed and loaded into cell compartments. The pre-formed 3D matrix can be loaded via pressure differential or by applying mechanical force. In some examples, the cell compartment includes a sacrificial 3D matrix that is pushed out by a non-sacrificial 3D matrix or pulled out by applying negative pressure.

[0175] In some embodiments, the solid-state bioreactor includes multiple hollow fibers within a shell, and a cell-loaded 3D substrate is substantially positioned in a region of the intracapillary space (IC) that is in fluid communication with the extracapillary space (EC) of the shell for perfusion. In some embodiments, the solid-state bioreactor can be configured with a hollow fiber structure having a significantly reduced headspace and fluid system dead volume compared to existing hollow fiber bioreactors. In some embodiments, the solid-state bioreactor configured with a hollow fiber design can be configured with features that allow the cell matrix to be substantially positioned within the fibers (rather than in the headspace and dead volume), such as those described above. Alternatively, a hollow fiber configuration providing a substantially spatially uniform, defined, or sufficiently large distribution of hollow fibers within the shell can allow loading of the EC with a cell-loaded 3D substrate in substantially uniform fluid communication with the perfusion medium in the IC. This configuration can also utilize improved headspace, dead volume, or loading procedures to maximize the positioning of the cell matrix within the perfusion compartment of the EC.

[0176] Cell culture and expansion In some embodiments, solid-state bioreactors are configured to culture cells to produce cell biomass, cell products, or both. In one example, the cell product is a cell therapy product. Cell biomass growth can be divided into several distinct phases. Biomass growth may include an initial lag phase, characterized by a low growth rate as cells are adapting to the new environment inside the bioreactor. During the exponential (or logarithmic) growth phase, cell division may continue at a constant or near-constant rate. As nutrients and free space are depleted and byproducts accumulate, the rate of cell biomass growth may slow, and the culture may enter a plateau or cell biomass may decline. The duration of each phase can depend on a number of factors, including initial seeding density, expansion or proliferation rate, culture medium, cell type, or other bioreactor and cellular factors. Solid-state bioreactors can be used for a variety of cell culture methods. In some embodiments, a perfusion method may be used, which minimizes environmental changes and provides a targeted cellular environment throughout the biomass growth process. For example, in a continuous perfusion configuration, fresh culture medium is continuously provided and waste culture medium is removed at a specific or variable rate to maintain a constant total volume in the perfusion fluid system. Alternatively, the total volume of the fluid system can be variable, for example, by using a fluid reservoir within the fluid system capable of accommodating a variable volume. In some embodiments, solid-state bioreactors are configured to perform static batch culture processes, such that the bioreactor is loaded with a fixed working volume of culture medium for use throughout the culture period, and the medium is not replaced during cell incubation. While this configuration can provide cell culture for a specified duration, or may produce lower cell densities and production yields compared to fed-batch and perfusion processes, such configurations may be suitable for certain applications, including short-term cultures during other cell bioprocessing operations. For example, solid-state bioreactors can be provided in a batch static configuration for rapid cell therapy production processes (e.g., T-Charge, FasT-CAR), where cells are not substantially expanded or allowed to substantially proliferate during manufacturing, but are maintained in a viable state during cell selection, activation, genetic engineering, and other bioprocessing operations. In this configuration, the perfusion compartment may not exhibit flow (e.g., configured with a static culture medium volume). In this configuration, the bioreactor can be further configured with gas exchange capabilities (e.g., via the use of a permeable membrane as described elsewhere herein) to continuously supply oxygen to the static culture medium. In one example, the solid-state bioreactor can be configured with a perfusion compartment that provides fluid flow, such as for recirculation or gas exchange purposes, without supplying fresh culture medium to the cell culture medium volume in the perfusion compartment or to the fluid system of the perfusion compartment.In some embodiments, solid-state bioreactors are configured to perform fed-batch or fed-batch culture processes, such that fresh culture medium is provided during bioreactor operation, a volume of waste culture medium is replaced discontinuously, or the waste culture medium is diluted with fresh culture medium. For example, in a fed-batch configuration, a volume of waste culture medium equal to the volume of fresh culture medium introduced into the perfusion fluid system can be removed from the perfusion fluid system, such that the perfusion fluid system maintains a constant total volume up to the working volume of the perfusion system (e.g., substantially all culture medium can be replaced during each feeding). In a fed-batch configuration, fresh culture medium can be added discontinuously or continuously over time, but waste culture medium can be added without removal, thereby increasing the total volume of the perfusion fluid system over time. This configuration can be permitted by using reservoirs aligned with the perfusion fluid system, including fixed-volume and deformable reservoir container types, which can be configured to hold variable volumes of culture medium or other fluids. For example, a solid-state bioreactor configured with a fixed-volume fluid reservoir can maintain a variable volume of liquid over time, and increases or decreases in fluid volume can be accommodated by displacement of a gas (e.g., air). In another example, this configuration can be permitted by using deformable containers within a solid bioreactor, such as perfusion compartments that can expand upon the addition of fluid (e.g., in an accordion configuration).

[0177] Washing and buffer exchange In some embodiments, solid-state bioreactors or cell processing systems are configured to perform fluid exchange operations such that a first fluid within a cell processing unit of the bioreactor or its compartment is substantially replaced with a second fluid. For example, a fluid exchange operation can be used to substantially replace a first interstitial fluid within a 3D substrate with a second fluid. This operation can provide buffer exchange or washing of cells retained within the bioreactor. During cell processing, culture medium exchange can be performed frequently to alter or substantially alter culture medium conditions (e.g., to replace the culture medium with an electroporation buffer suitable for in-situ electroporation), or to substantially remove factors such as activating agents or viral particles. Fluid exchange can purge or replace perfusion culture medium or fluid present in the 3D substrate and cell compartments. Fluid exchange can be performed iteratively to substantially exchange the first liquid as the first liquid in the cell compartment is diluted with the second liquid provided in the perfusion compartment through diffusive mixing. In configurations having two or more perfusion compartments in fluid communication with the cell compartments, a pressure or flow rate difference between the two or more compartments can be used to accelerate the flushing and reagent exchange of the cell compartments and the 3D substrate. For example, the flow rate or fluid pressure in one fluid flow path can be less than or significantly less than the 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.

[0178] In some embodiments, solid-state bioreactors are configured to provide one or more methods for retaining cells within the bioreactor to allow for efficient cell washing and buffer exchange in the presence and absence of a 3D substrate. For example, a 3D substrate can provide cell retention. Alternatively or supplementally, a solid-state bioreactor may include one or more membranes, cell-capturing features, or other cell-capturing mechanisms to retain cells in the absence of a 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 that provides cell retention during operation and one or more semi-permeable membrane interfaces between the 3D substrate (cell compartment) and one or more culture media or perfusion compartments. During solid-state cell culture, the 3D substrate can serve to retain cells and provide a 3D environment for cell culture and growth, while the semi-permeable membrane interfaces serve to retain the 3D substrate and, in some cases, provide additional cell retention, such as by using membranes with a filtration size cutoff value unsuitable for cell passage. In some embodiments, the 3D substrate may be configured to provide a reversible gel-sol transition in response to stimuli, such that cells can be washed in situ within a solid-state bioreactor in this configuration. For example, upon gel-sol stimulation (such as temperature changes, the addition of chemical agents or other reagents, or other stimuli of gel-sol transition), a 3D substrate can transition from a substantially solid or semi-solid state to a substantially liquid or dissolved state; following such a gel-sol transition, cells contained within the 3D substrate can become suspended, surrounded and suspended in the liquefied or dissolved matrix. For hybrid 3D substrates, such as those comprising porous solid scaffolds interpenetrating with hydrogel scaffolds (to which cells attach or primarily interact and grow), stimulus-responsive gel-sol transitions can affect subsets of the 3D substrate while providing a cell culture transition, such as from 3D culture to efficient suspension culture within a porous solid. Subsequently, as described herein, buffers or culture media (such as wash buffers or cryopreservation buffers) can be pumped into and permeate the bioreactor or cell processing system, while a semi-permeable membrane acts to retain cells in the same volume, space, or compartment, such that following this gel-sol transition, cells can be suspended in the liquefied 3D matrix or otherwise not attached to the remaining solid 3D substrate. Solid-state bioreactors in this configuration allow for in-situ washing or buffer exchange. Washed or buffer-exchanged cells can then be collected from the cell compartments, such as through dedicated ports connected to the cell compartments, or by subsequently moving, removing, or degrading the membranes or interfaces between the cells and other compartments. In some embodiments, the solid-state bioreactor or cell processing system can be configured to perform in-situ clinical formulation operations using one or more in-situ washing or buffer exchange procedures to suspend cells in a suitable formulation buffer for harvesting and downstream clinical use.

[0179] Cell harvest In some embodiments, the solid-state bioreactor is configured to perform a cell harvesting operation. In some embodiments, cells are harvested from the solid-state bioreactor at fixed intervals, on demand, or once cells reach a target number, concentration, phenotype, or other relevant indicators (such as those provided by one or more sensors and feedback systems within the bioreactor or bioprocessing system as described elsewhere herein). The 3D matrix can be dissolved, and multiple cells or populations thereof can be collected to generate a cell product. In some embodiments, the solid-state bioreactor is configured with a stimulus-responsive gel-sol 3D substrate to facilitate cell harvesting. The stimulus can be thermal, chemical, enzymatic, or any combination thereof. In one example, applying a single stimulus may allow multiple cells to be released from the 3D matrix. In another example, applying at least two stimuli may allow multiple cells to be released from the 3D matrix. Applying a single stimulus may release a selected amount of cells, and applying a second, different stimulus may release another amount of cells from the 3D matrix. Cells can be harvested by collecting suspended cells or cells suspended in a dissolved matrix, such as by applying a stimulus, through inducing a phase transition ( wholly or partially) of the 3D substrate from a solid or semi-solid phase to a liquid phase. For example, a solid-state bioreactor can be configured to culture cells in a thermally responsive, reversible gel-sol material, such as methacrylamide collagen or polyethylene glycol (PEG)-poly(N-isopropylacrylamide) (PNIPAAm) block copolymer. When the temperature of the bioreactor or cell compartment is lowered, the 3D substrate can undergo a phase transition to a dissolved or liquid state, and cells previously attached to the 3D substrate can be transformed into a suspension phase. The liquefied substrate containing the cells can be harvested from the bioreactor, for example, by using fluid flow. In some embodiments, a permeable or semi-permeable interface can be positioned near the cell compartment or perfusion compartment, and the liquid matrix component can be partially or substantially removed from the cells using washing and buffer exchange methods described elsewhere herein, while retaining the cells within the compartment, for example, to facilitate cell harvesting by reducing viscosity, or to facilitate cell exchange from the matrix reagent into buffers suitable for downstream processing (including, but not limited to, cryopreservation buffers and formulation buffers).

[0180] In some embodiments, the solid-state bioreactor or cell processing system is configured to harvest cells directly from a 3D substrate. For example, fluid flow within or through a cell compartment can be configured to remove cells from the 3D substrate, such as by applying fluid forces capable of disrupting the interaction between the cells and the 3D substrate, to wash the cells out of or from the 3D matrix. In some embodiments, the solid-state bioreactor or cell processing system can be configured with a programmable or stimulus-responsive 3D substrate to facilitate cell harvesting. For example, by using a 3D substrate with stimulus-responsive mass transfer property modulation (such as porosity or swelling), stimulation can facilitate the harvesting of cells cultured and retained within and therein, which causes the 3D substrate to swell or increase porosity, such that the cells are no longer substantially retained by the 3D substrate, or can be removed by fluid flow. In one example, by using a stimulus-responsive 3D substrate configured to modulate the cell attachment or migration properties of a 3D matrix, stimulation can facilitate the harvesting of cells cultured and retained within and within the 3D substrate. This stimulation causes the 3D substrate to interact with the cells in different ways, such that cells may no longer be substantially retained by the 3D substrate or may be substantially retained, or may be removed by fluid flow. In one example, the 3D substrate may contain programmable cell attachment or migration factors that, upon stimulation, can be cleaved, blocked, competitively replaced, or otherwise neutralized, resulting in reduced cell retention properties and promoting cell harvesting, such as by fluid flow. In another example, the 3D substrate may be programmably functionalized with factors that promote cell migration from the matrix environment to facilitate cell harvesting, such as by cell migration or by applying fluid flow. In some embodiments using stimulus-responsive 3D substrates to facilitate cell harvesting, the chemical or biological mechanisms facilitating cell harvesting may target a subset of multiple cells. For example, in embodiments utilizing 3D basal cell attachment factors to facilitate cell harvesting, one or more of a plurality of cell attachment factors can be specifically modulated to adjust the cell adhesion properties of the 3D substrate for specific populations of multiple cells, allowing the harvesting of a subset of multiple cells. Solid-state bioreactors can be configured with a 3D substrate containing two or more cell attachment factors targeting two or more cell populations, such as feeder cell types, which can support the growth of one or more different cell types in culture by contributing an ambiguous and complex mixture of soluble components and extracellular matrix (ECM) components. Upon application of a stimulus regulating the attachment of one class of cells (e.g., feeder cells or other cell types), a specific subset of cells can be harvested from the 3D substrate, while other subsets remain substantially within the 3D substrate. This configuration can provide the benefit of streamlined cell harvesting and formulation workflows or increased purity of harvested cells in co-culture applications.

[0181] In some embodiments, cells can be harvested by collecting a cell-loaded 3D substrate. In some embodiments, cells can subsequently be isolated from the 3D substrate outside the solid-state bioreactor. In other embodiments, one or more cell types can be cultured in the 3D substrate in a form and composition suitable for downstream use. In one example, one or more cell types present in natural tissues can be cultured in a solid-state bioreactor using a 3D substrate that is similar to and otherwise biocompatible with the extracellular matrix of natural tissues. Upon harvesting the cultured tissue (e.g., the resulting cell-loaded 3D substrate), this composition can be used for tissue replacement or regeneration purposes. 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 biostructures. In another example, animal muscle cells can be cultured in a 3D substrate suitable for human consumption using a solid-state bioreactor. Upon harvesting the cultured tissue, this composition can be used as a food source.

[0182] In one example, a solid-state bioreactor can be configured to harvest cells during operation for process monitoring at a location outside or separate from the bioreactor. In some embodiments, the solid-state bioreactor includes one or more specific cell compartments or cell processing units that are separate from and may not be in fluid communication with one or more other compartments or cell processing units of the bioreactor. Cells can be harvested from these specific cell compartments or cell processing units without needing to harvest cells from the other compartments or cell processing units, thereby enabling process monitoring without interrupting the manufacturing process. Cells harvested during the process using a solid-state bioreactor of this configuration can be further analyzed within the bioprocessing system or collected and analyzed separately from the bioreactor and bioprocessing system.

[0183] In some embodiments, cells harvested from a 3D substrate can undergo one or more additional cell culture operations (e.g., cell passage) within a solid-state bioreactor. Passaging may include harvesting cells from a culture and transferring the cells, or a subset thereof, under different conditions, such as at a new (e.g., lower or higher) cell concentration or density, or in fresh growth medium, to one or more culture vessels to establish a new culture. Solid-state bioreactor devices and cell handling systems may be configured to harvest cells from a solid-state bioreactor and subsequently re-inoculate new 3D cell cultures within the solid-state bioreactor. In some embodiments, the solid-state bioreactor may be configured with multiple cell handling units, some for the first passage and others for the second or subsequent passages. In some embodiments, the solid-state bioreactor may be configured with cell handling units that can be reused in one or more passages. In some embodiments, the solid-state bioreactor may be configured to re-inoculate passaged cells in a first 3D substrate or in a separate 3D substrate, such as by mixing cells with a fresh liquid formulation of a soluble 3D matrix material and subsequently forming a new (de novo) 3D substrate. In some embodiments, solid-state bioreactors are configured for reseeding or passage programs designed to extend the duration of 3D cell culture, increase the total capacity for cell expansion and proliferation, or tandemly execute multiple cell engineering or other biological processing procedures. In some embodiments, solid-state bioreactors can be configured to perform multiplex automated genome engineering (MAGE) programs, wherein cells are cultured, engineered, harvested, and optionally selected or isolated subsets of cells in two or more passages, such as for the purpose of performing a series of cell engineering procedures or iterative or multiplex genome engineering. For example, in the use of a solid-state bioreactor to passage 3D cell cultures two or more times, cells can be exposed to multiple rounds of genome engineering (such as through the use of CRISPR-Cas9) for the development or manufacture of highly engineered cell products. In one 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 can be used to generate a cell therapy product. The cell therapy product can be generated at a human dose scale.

[0184] Cell processing systems and methods In one aspect, this disclosure provides a system for manufacturing cell products. The system may include one or more compartments configured to hold or retain 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 allow fluid to flow in a first direction, or to allow fluid to flow in a first direction at an angle relative to a second flow direction within the compartment. The angle may be greater than zero degrees. The fluid flow path may be further configured to provide material from the fluid to the compartment, or to provide material from the fluid to the compartment. The material from the fluid may be used to generate the cell product. The system may include an analytical unit coupled to the compartment and the fluid flow path. The analytical unit may be housed within the same housing as the compartment, the fluid flow path, or both the compartment and the fluid flow path. Alternatively, the analytical unit may be housed in a different housing than the compartment, the fluid flow path, or both the compartment and the fluid flow path. The analytical unit may be configured to monitor a plurality of cells or fluid during cell product generation when material is provided to the compartment, or may monitor a plurality of cells or fluid during cell product generation when material is provided to the compartment.

[0185] In another aspect, this disclosure provides a system for cell processing. The system may include a cell processing unit, a preservation unit, an analysis unit, or a combination thereof. The cell processing unit may be configured to preserve and process multiple cells, or to preserve and process multiple 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 the cell product, or to 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 one or more properties of multiple cells in the cell product, or to detect or monitor one or more properties of multiple cells in the cell product.

[0186] In another aspect, this disclosure provides a method for manufacturing a cell product. The method may include supplying a plurality of cells to a system. The system may include one or more compartments holding 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 housed in the same housing as the compartment, the fluid flow path, or both. Alternatively, the analysis unit may be housed in a different housing than the compartment, the fluid flow path, or both. The method may include supplying material from a fluid in the fluid flow path to the compartment to generate the cell product. The fluid flow path may allow the fluid to flow in a direction angled relative to a second flow direction within the compartment. The angle may be greater than zero degrees. The method may include using the analysis unit to monitor the fluid of the plurality of cells during cell product generation as material is supplied to the compartment.

[0187] In another aspect, this disclosure provides methods for cell processing. The methods 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 a combination thereof. The cell processing unit may retain a 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 methods may include processing cells in the cell processing unit to generate a cell product. The methods may include preserving the cell product in the preservation unit. The methods may include detecting or monitoring one or more properties of a plurality of cells in the cell product during cell processing, preservation of the cell product, or both.

[0188] The systems and methods described herein (e.g., integrated cell processing systems) allow the use of 3D matrices and material compositions described elsewhere herein for the purpose of processing cells. Cell processing may include automated cell processing, cell culture, cell manipulation, or any combination thereof for the production of cell products. Cell products may include cell therapies, gene therapies, biologics, biobank cell stocks, or combinations thereof. The systems and methods described herein can be used for manufacturing or research applications (e.g., genetic and environmental manipulation of cells and cell line creation). In some embodiments, the systems and methods may include solid-state bioreactors. Solid-state bioreactors may allow the culture or manipulation of adherent cells, microcarrier cells, or suspension cell cultures. The systems and methods described herein can be used to reproduce cell products with high cell health and viability. Cell products produced using the systems and methods described herein may have viability greater than or equal to about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or higher. In one example, cell products produced using the systems and methods described herein may have cell viability greater than or equal to about 75%. In another example, cell viability may be greater than or equal to about 90%. In another example, cell viability can be greater than or equal to approximately 95%.

[0189] The cell processing system described herein may include one or more receiving units, bioreactors, cell processing units, preservation units, analysis units, control units, thermal control units, fluid units, or any combination thereof. In one example, the cell processing system includes a bioreactor coupled to a receiving unit, a cell processing unit, a preservation unit, or an analysis unit. In another example, the cell processing system includes a bioreactor coupled to a cell processing unit, a preservation unit, and an analysis unit. In one example, the bioreactor is a solid-state bioreactor. The bioreactor may be configured to subject multiple cells to cell culture, membrane transduction, amplification, gene delivery, cell selection, or any combination thereof.

[0190] In one example, the cell processing system includes a receiving unit. The receiving unit may be coupled to a bioreactor, a cell processing unit, a preservation unit, an analysis unit, or any combination thereof. The receiving unit may be configured to receive multiple cells from an environment outside the cell processing system. The receiving unit may be configured to receive multiple cryopreserved (e.g., frozen) cells. The receiving unit may include a thermal unit as described elsewhere herein and may enable controlled thawing of the cryopreserved cells. The receiving unit may be configured to automatically transfer multiple cells to a processing unit or a bioreactor after thawing. Alternatively or supplementarily, the receiving unit may be configured to automatically transfer cells to a thermal unit for thawing or to a cell processing unit upon receipt of multiple cells.

[0191] A cell processing system may include one or more control systems. The control system may include a computer system configured to control or operate the cell processing system. The cell processing system may include 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, an analysis unit, a bioreactor, a thermal control unit, a receiving unit, a storage unit, a fluid flow unit, or any combination thereof. In one example, the one or more computer processors are operatively coupled to a cell processing unit and configured to guide the cell processing unit to automatically process multiple cells within the cell processing unit (e.g., within a 3D matrix), to guide the cell processing unit to separate multiple treated cells from the 3D matrix, and to guide the cell processing unit to collect multiple treated cells. In another example, the one or more computer processors are operatively coupled to a receiving unit and may be programmed to automatically guide the receiving unit to provide multiple cells to the cell processing unit.

[0192] A cell processing system or bioreactor may include one or more cell processing units. In one example, the cell processing system includes one or more bioreactors, which in turn include one or more cell processing units. In another example, the cell processing system includes one or more bioreactors comprising one or more cell processing units, as well as additional cell processing units separate from the bioreactors. In yet another example, the cell processing system may include one or more cell processing units and may not include a bioreactor.

[0193] A cell processing unit may include any component or feature configured or available for processing cells. A cell processing unit may be configured or otherwise used to generate a cell product. A cell processing unit may include compartments configured to hold a plurality of cells (e.g., cell compartments), fluid flow paths configured to supply fluid to the cell compartments, one or more interfaces disposed between the fluid flow path, the cell compartments, or both, or any combination thereof. In one example, the cell processing unit includes at least one cell compartment, at least one fluid flow path, and at least one interface. In another example, the cell processing unit includes at least one cell compartment, at least one fluid flow path, and no interface. In yet another example, the cell processing unit includes at least one compartment, at least one interface, and no fluid flow path. In yet another example, the cell processing unit includes at least one compartment and no fluid flow path or interface.

[0194] A cell processing unit may include at least 1, 2, 3, 4, 5, 6, 8, 10 or more cell compartments. A cell processing unit may include at least 1, 2, 3, 4, 5, 6, 8, 10 or more fluid flow paths. In one example, a fluid flow path is an perfusion compartment. A cell processing unit may include at least 1, 2, 3, 4, 5, 6, 8, 10 or more interfaces. An interface may be located between one cell compartment and another cell compartment or fluid flow path. An interface may be located 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.

[0195] A bioreactor may include one or more cell processing units. A bioreactor may include at least 1, 2, 3, 4, 5, 6, 8, 10 or more cell processing units. A cell processing system may include 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 the bioreactor (e.g., the cell processing system does not include the bioreactor, or the cell processing units are not part of the bioreactor).

[0196] A cell processing unit can be configured to process multiple cells, or can process multiple cells. Cell processing can include, but is not limited to, cell enrichment, cell selection, cell isolation, cell expansion or culture, washing, concentration, buffer exchange, depolymerization, or any combination thereof. A cell processing unit may include compartments (e.g., cell compartments) configured to provide cells with one or more processes. For example, a cell compartment may be configured to subject multiple cells to cell culture, membrane transduction, expansion, gene delivery, cell selection, or any combination thereof.

[0197] Cell preservation The systems and methods described herein can provide integrated cell retention. Cell processing may include supplying culture medium or other liquids to cells, removing culture medium or other liquids from cells, or replacing culture medium or other liquids in the system. Cells may be retained within the system to provide efficient separation of cells from waste culture medium and other fluids, molecules, reagents, and cell byproducts to be removed or harvested from the culture environment. The volume of a cell processing system containing cells may be a subset of the total working volume of the cell processing system. In a cell processing system, at least some other volumes (e.g., not containing cells) may contain other fluid components (such as growth media and buffers) or gases. The former, the cell-containing volume, is referred to herein as a cell compartment. Cell compartments may be located within 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 cells or to process cells.

[0198] Cell compartments can be provided in a single continuous configuration or multiple discrete configurations. Cell compartments provided in discrete configurations can include multiple cell compartments physically separated from each other, for example, by separation via cell-free volumes. Regardless of the configuration, cell compartments can provide essential cell retention by their design or their configuration utilizing a 3D substrate. Cell compartments can be in fluid communication with connected cell-free or substantially cell-free volumes. Cell-free or substantially cell-free volumes can be fluid flow paths (e.g., perfusion compartments), gas exchange compartments, or both. Cell compartments can be containers or other compartments configured to retain a 3D matrix. Alternatively, the 3D matrix can be a cell compartment. Cell compartments can represent a physical 3D volume within a cell processing unit (e.g., a solid-state bioreactor or other cell processing unit) and include a physical form defined by the size and shape of the 3D matrix or the cell occupancy boundary within the cell processing unit.

[0199] In some embodiments, cell compartments include 3D substrates for cell processing (e.g., cell culture, gene delivery, transfection, etc.) as described elsewhere herein. In this configuration, the 3D substrate (such as a 3D matrix) can provide essential cell retention. For example, a 3D substrate can retain cells by means of cell adhesion to the substrate, by encapsulation, by a porosity unsuitable for cell migration, by a flow configuration or hydrodynamics within the 3D substrate during perfusion or fluid flow that prevents cells from substantially migrating from the 3D substrate, or by other physical and biological interactions between the 3D substrate, cells, and fluid, or a combination thereof, as well as the effects of gravity (depending on the configuration). In some embodiments, the interface between cell compartments of a cell processing unit (e.g., a solid-state bioreactor or other cell processing unit) and other volumes (e.g., fluid flow paths) can be defined by the boundaries of the 3D substrate where cells reside. Such interfaces and configurations can be considered phase-separated; for example, a cell compartment is defined as a solid or semi-solid phase containing the 3D substrate, and other compartments are defined as liquid or gas phases containing culture medium, atmosphere, or other aspects 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 due to gravity, attach via cell adhesion, migrate into the 3D substrate, or otherwise be localized to or within the 3D substrate. In some embodiments, cell signaling factors or cell-binding factors are provided to facilitate such cell localization. In some embodiments, a liquid matrix formulation (e.g., a polymer precursor mixture) is loaded into the cell processing unit and allows the formation of the 3D substrate, such as through gel-sol transition, in-situ polymerization, in-situ crosslinking of the matrix, or a combination thereof. In some embodiments, the 3D substrate is configured not to occupy the entire volume of the cell processing unit, for example by loading a suitable volume of liquid matrix such that a “headspace” not occupied by the 3D substrate is formed during matrix formation. The liquid matrix formulation (e.g., a polymer precursor mixture) may contain cells to allow the formation of a cell-loaded 3D substrate during matrix formation. Alternatively, cells may be added to the 3D substrate after matrix formation. In some embodiments, cells can be added prior to matrix formation, and the resulting 3D matrix can subsequently be seeded with additional cells. In some embodiments, the volume and specific spatial configuration of the cell compartments can be further defined by phase separation, such as gases (e.g., “headspaces”) or by using phase-separated liquids. For example, a liquid matrix (e.g., a polymer precursor) can be loaded into the cell processing unit (simultaneously, before, or after) along with a separate immiscible liquid (such as oil), such that during 3D matrix formation, the immiscible phase excludes the matrix from a specific volume of the bioreactor.This phase-separated non-matrix liquid can be removed from the cell processing unit (e.g., washed out), such as to form perfusion or gas exchange volumes within the cell processing unit and interfaces with the 3D substrate. In some embodiments, multiphase liquids are utilized. Multiphase liquids may comprise at least a liquid matrix (e.g., a polymer precursor) and separate immiscible liquids that form two or more discrete phases or volumes separated by one or more interfaces during 3D matrix formation.

[0200] The cell processing unit may include at least one fluid flow path. The fluid flow path may be configured to provide reagents to the 3D matrix and remove waste (e.g., metabolic waste) from the 3D matrix, or may provide reagents to the 3D matrix and remove waste (e.g., metabolic waste) from the 3D matrix, such as... Figure 16A and 16B As shown. In one example, during cell culture, the fluid flow path can deliver reagents to the 3D matrix to provide conditions sufficient for cell culture, such as... Figure 16A As shown. Reagents may include, but are not limited to, glucose, oxygen, buffers, proteins, amino acids, lipids, inorganic salts, trace elements, vitamins, reducing agents, or any combination thereof. The fluidic pathway may be configured to remove waste from or from the 3D matrix to maintain conditions sufficient for cell culture. Removed waste may include, but is not limited to, lactic acid, carbon dioxide, ammonia, other metabolites, or any combination thereof. In another example, during cell activation, the fluidic pathway may be configured to provide growth factors, signaling factors, or both to cells encapsulated within the 3D matrix, or to provide growth factors, signaling factors, or both to cells encapsulated within the 3D matrix. For example, the fluidic pathway 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 fluidic pathway 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 (LNPs), electroporation buffers, adjuvants, or any combination thereof. The fluid flow path can be configured to wash the 3D matrix before, during, or after activation or gene delivery. Washing may include providing the 3D matrix with culture medium or other washing solution to remove growth factors, signaling factors, gene delivery reagents, or any combination thereof. In another example, the cell processing unit may include a fluid flow path configured to deliver liquid nutrients and reagents, and another fluid flow path configured to deliver gaseous nutrients and reagents, such as… Figure 16BAs shown. The 3D matrix can be positioned between a fluid flow path (e.g., a first fluid flow path) configured to provide fluid nutrients (e.g., nutrients disposed in a liquid phase, such as culture medium) to the 3D matrix and another fluid flow path (e.g., a second fluid flow path) configured to provide gaseous nutrients and reagents to the 3D matrix. Either or both of the first or second fluid flow paths can be configured to allow gas exchange liquid (e.g., oxygenated culture medium) to flow to provide gaseous nutrients and reagents to the 3D matrix. In one example, the second fluid flow path can be configured to allow gaseous fluid to flow, such that gas from the gaseous fluid can diffuse through a gas-permeable and liquid-impermeable membrane (e.g., polydimethylsiloxane (PDMS), silicone, etc.) into the 3D matrix or culture medium compartment. In one example, the fluid flow path for delivering liquid and the fluid flow path for delivering gas can be positioned on the same side of the 3D matrix.

[0201] The cell processing unit may include a membrane or interface. The membrane or 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 allow the flow of a fluid (e.g., a liquid or gas). The membrane may be configured to allow material transport across it. For example, the membrane may allow nutrients, reagents, or other components in a liquid or gas phase to be transported across it. In some embodiments, the cell processing unit does not include a membrane or semipermeable membrane between the compartment and the fluid flow path or other volume. In some embodiments, the cell compartment further includes or is coupled to one or more membranes or semipermeable substrates, such as a semipermeable membrane. The membrane may have a thickness that allows fluid or fluid containing nutrients, reagents, or other components to be transported from the fluid flow path to the cell compartment (e.g., a 3D matrix or a 3D matrix within the cell compartment) and vice versa. The membrane can have a thickness 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, 5 mm or more. The membrane can have a thickness of 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. The membrane can have the following thicknesses: approximately 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, and 5 μm to 1 mm.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 to 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 4mm, 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, 600 μm to 2 mm, 600 μm to 3 mm, 600 μm to 4mm, 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 3 mm, 1.5 mm to 2 mm, 1.5 mm to 3 mm, 1.5 mm to 3 mm mm to 4 mm, 1.The thickness can be 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 one example, the semipermeable membrane can have a thickness of about 18 to 25 μm or about 110 to 150 μm.

[0202] Semipermeable membrane materials may include polysulfone (PS / PSF), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), nylon (polyamide), polycarbonate (PC, PCTE), Teflon (PTFE), nitrocellulose, cellulose acetate, mixed cellulose (MCE), glass fiber, polypropylene (PP), polyacrylonitrile (PAN), polyester (PETE), polyethylene (PE), polyetheretherketone (PEEK), thermoplastics, metals (such as silver, aluminum), ceramics, or any combination thereof. The membrane may be surface-treated, such as with a wetting agent. For example, the wetting agent may include a few molecules thick of polyvinylpyrrolidone (PVP) deposited on the membrane surface of a material such as PCTE. As another example, the wetting agent may include the deposition of polymers such as proteins, including but not limited to collagen, albumin, fibronectin, and laminin. For example, collagen treatments (such as adsorption or coating) can increase the contact angle and hydrophilicity of plastic materials, including membranes and other aqueous fluid contact surfaces. Porous or semi-permeable membranes can be fabricated by track etching or as woven meshes, nanofiber meshes, nonwoven matrices, or combinations thereof. Membranes can be selected based on properties including: porosity (the percentage of the total area occupied by pores, e.g., less than about 1% to 16% for track-etched PC and PE, and about 40% to 80% for other materials and fabrication methods), pore size (e.g., pore diameter), hydrophilicity and hydrophobicity, bubble point (the minimum pressure required to push a bubble through the largest pore of the wet membrane), maximum operating temperature, sealing compatibility (e.g., ultrasonic, thermal, radio frequency, insert molding), burst strength, and water flow rate (e.g., mL / min / cm at a given pressure). 2 Air velocity (e.g., L / min / cm at a given pressure) 2 Optical properties (e.g., transparency), solvent resistance, chemical compatibility, peeling or particle shedding, biocompatibility, extractability and adsorption properties, biofouling and protein binding (e.g., some hydrophilic materials such as nylon and nitrocellulose exhibit high protein binding capacity, while regenerated cellulose and cellulose acetate hardly bind protein).

[0203] In one example, the membrane can be disposed between a 3D matrix or cell compartment and a culture medium compartment. The membrane can be a track-etched PC or PETE membrane with a thickness of about 18 to 25 μm. The membrane can have a pore size of about 1 to 12 μm and a porosity of about 1% to 16%. In one 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 yet another example, the membrane has a pore size of less than about 3 μm.

[0204] In another example, the system may include a breathable membrane. The breathable membrane may be dense (e.g., non-porous and without an interconnected network of pores that allows gas molecules to be transported by diffusion) or porous (e.g., by means of hydrophobicity, a porous membrane may prevent liquid from permeating into the gaseous pores of the membrane under pressure until a certain critical pressure is reached). The breathable membrane may be made of 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 intrinsically microporous polymers (PIMs), thermally rearranged (TR) polymers, and heterogeneous membranes, including thin-film composite (TFC) membranes (e.g., ultrathin dense polymer layers formed or deposited on a rigid membrane support, allowing for the simultaneous integration of the selectivity / capacitance of the dense membrane and the mechanical strength of the support layer) and hybrid matrix membranes (MMMs, composed of organic polymers blended with inorganic additives such as zeolites, metal-organic frameworks (MOFs), carbon nanoparticles, and other nanoparticles, wherein the inorganic materials enhance gas selectivity while the polymers provide mechanical stability). The membrane or membrane material may be treated (e.g., by ionization) to further enhance membrane properties. In one example, a gas-permeable, liquid-impermeable membrane may contain PDMS and may have a thickness of at least about 20 μm, 100 μm, 0.25 mm, 0.5 mm, or greater.

[0205] The interface or membrane can be configured to allow reagents of selected sizes to be delivered to or removed from the 3D matrix. Example reagent sizes are shown below. Figure 17As shown. The properties of the interface or membrane can be modified for size selection. A semipermeable membrane can provide cell retention or 3D substrate retention, or enhance cell retention (e.g., as described elsewhere herein, it contributes to cell retention when used in conjunction with a 3D substrate that includes cell retention properties). In some embodiments, the semipermeable membrane provides an interface between a cell compartment and one or more other compartments or volumes within a cell processing unit. The semipermeable membrane can be configured to allow the transfer of substances between the cell compartment and another compartment (e.g., a fluid flow pa...

Claims

1. A system for manufacturing cell products, comprising: The compartments are configured to hold multiple cells; A fluid flow path in fluid communication with the compartment, wherein the fluid flow path is configured to (i) allow fluid to flow in a first direction, the first direction being at an angle relative to a second flow direction within the compartment, wherein the angle is greater than zero degrees, and (ii) supply material from the fluid to the compartment to generate the cell product; and An analysis unit, coupled to and located within the same housing as the compartment and the fluid flow path, wherein the analysis unit is configured to monitor the plurality of cells or the fluid during the generation of the cell product, as the material is supplied to the compartment.

2. The system according to claim 1, wherein the angle is greater than 45 degrees.

3. The system according to any one of claims 1 or 2, wherein the cell product is a cell therapy product.

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

5. The system according to any one of claims 1-4, wherein the analysis unit is configured to monitor the plurality of cells in the compartment.

6. The system according to any one of claims 1-5, the system further comprising a cell processing unit in fluid communication with the compartment.

7. The system according to any one of claims 1-6, wherein the compartment is configured to provide the plurality of cells with one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection.

8. The system according to any one of claims 1-7, wherein the housing has a volume of about 0.01 cubic meters (m³). 3 ) to about 2m 3 The volume.

9. The system according to any one of claims 1-8, wherein the analysis unit comprises one or more sensors configured to monitor an environment containing the plurality of cells.

10. The system according to any one of claims 1-9, wherein the analysis unit includes one or more sensors configured to analyze the cell product.

11. A system for cell treatment, comprising: A cell processing unit configured to retain and process multiple cells to generate cell products; A storage unit coupled to the cell processing unit, wherein the storage unit is configured to store 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.

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

13. The system of claim 11 or claim 12, further comprising a receiving unit configured to receive the plurality of cells.

14. The system of claim 13, wherein the plurality of cells are a plurality of cryopreserved cells.

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

16. The system of claim 15, wherein the receiving unit is configured to automatically transfer the plurality of thawed cells to the cell processing unit.

17. The system according to any one of claims 11-16, wherein the preservation unit is configured to generate cryopreserved cell products.

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

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

20. The system of claim 18, wherein the bioreactor is configured to provide the plurality of cells with one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection.

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

22. The system according to any one of claims 11-21, wherein the analysis unit is configured to provide release testing of the cell product.

23. The system according to any one of claims 11-22, wherein the cell processing unit, the analysis unit, and the preservation unit are integrated within a housing.

24. The system of claim 23, wherein the housing has a volume of approximately 0.01 cubic meters (m³). 3 (approximately 2 m) 3 The volume.

25. The system according to any one of claims 11-24, wherein the analysis unit comprises one or more sensors configured to monitor an environment containing the plurality of cells.

26. The system according to any one of claims 11-25, wherein the analysis unit includes one or more sensors configured to analyze the cell product.

27. A method for manufacturing cell products, comprising: (a) Providing a plurality of cells to a system, the system comprising (i) a compartment for retaining the plurality of cells, (ii) a fluid flow path in fluid communication with the compartment, and (iii) an analysis unit coupled to the compartment and the fluid flow path and located within the same housing as the compartment and the fluid flow path; (b) Providing material from the fluid in the fluid flow path to the compartment to generate the cell product, wherein the fluid flow path causes the fluid to flow in a first direction, the first direction being at an angle relative to a second flow direction within the compartment, wherein the angle is greater than zero degrees; as well as (c) Using the analytical unit to monitor the plurality of cells or the fluid during the generation of the cell product, when the material is provided to the compartment.

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

29. The method according to any one of claims 27 or 28, wherein the cell product is a cell therapy product.

30. The method of claim 29, wherein the cell therapy product is produced at a scale equivalent to at least one human dose.

31. The method according to any one of claims 27-30, wherein the analysis unit monitors the plurality of cells in the compartment.

32. The method according to any one of claims 27-31, wherein the analysis unit monitors the plurality of cells in real time.

33. The method according to any one of claims 27-32, further comprising using a cell processing unit to generate the cell product.

34. The method according to any one of claims 27-33, further comprising subjecting the plurality of cells to one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection.

35. The method according to any one of claims 27-34, wherein the housing has a volume of about 0.01 cubic meters (m³). 3 (approximately 2 m) 3 The volume.

36. A method for cell treatment, comprising: (a) Providing a plurality of cells to a system, the system comprising (i) a cell processing unit holding the plurality of cells, (ii) a storage unit coupled to the cell processing unit, and (iii) an analysis unit coupled to the cell processing unit or the storage unit; (b) The plurality of cells are processed in the cell processing unit to generate a cell product; (c) The cell product is stored in the storage unit; as well as (d) During the processing or preservation, detect or monitor one or more properties of the plurality of cells or the cell product.

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

38. The method of claim 36 or 37, further comprising providing the plurality of cells to a receiving unit coupled to the cell processing unit, the storage unit, or the storage unit.

39. The method of claim 38, wherein the plurality of cells provided to the receiving unit are a plurality of cryopreserved cells.

40. The method of claim 39, wherein the receiving unit causes the plurality of cryopreserved cells to undergo controlled thawing to generate a plurality of thawed cells.

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

42. The method according to any one of claims 36-41, wherein the preservation unit generates cryopreserved cell products.

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

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

45. The method of claim 43, further comprising using the bioreactor to subject the plurality of cells to one or more processes selected from: cell culture, membrane transduction, amplification, gene delivery, and selection.

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

47. The method according to any one of claims 36-46, further comprising using the analytical unit to perform release testing of the cell product.

48. A cell treatment system comprising: Multiple three-dimensional (3D) matrices, wherein the 3D matrices in the multiple 3D matrices are configured to contain or encapsulate cell populations; and Multiple fluid flow paths, wherein one of the multiple fluid flow paths is independent of and adjacent to the 3D matrix, and wherein the fluid flow path is configured to deliver fluid material to the 3D matrix to process the cell population into a cell product.

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

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

51. The system according to any one of claims 48-50, wherein the 3D matrix is ​​configured as a flat plate, the flat plate comprising a first dimension and a second dimension greater than a third dimension.

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

53. The system according to any one of claims 48-52, wherein the 3D matrix is ​​configured as a fiber, the fiber comprising a diameter smaller than the length dimension.

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

55. The system according to any one of claims 48-54, wherein the 3D matrix is ​​configured as a sphere, and wherein the tangential flow of the fluid flows along the outer circumference of the sphere.

56. The system according to any one of claims 48-55, wherein the plurality of 3D matrices are fluidly coupled to each other.

57. The system according to any one of claims 48-56, wherein the fluid flow path is disposed between the first 3D matrix and the second 3D matrix.

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

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

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

61. The system according to any one of claims 48-60, wherein the interface area between the fluid flow path and the 3D matrix is ​​greater than or equal to about 5 square centimeters (cm²). 2 ).

62. The system according to any one of claims 48-61, wherein the 3D matrix comprises a porous solid matrix having interconnected pores with an average pore size sufficient to allow lymphocytes to grow within the 3D matrix.

63. The system according to any one of claims 48-62, wherein the 3D matrix is ​​a programmable 3D matrix.

64. The system according to any one of claims 48-63, wherein the 3D matrix has a surface area to volume ratio greater than or equal to about 0.

1.

65. The system according to any one of claims 48-64, wherein the 3D matrix has about 10 -5 Siemens / m (S / m) to approximately 10 -1 Conductivity in S / m.

66. The system according to any one of claims 48-65, wherein the 3D matrix is ​​configured to provide activation of the plurality of cells via membrane signal transduction in the 3D matrix.

67. A method for cell treatment, comprising: (a) Providing (i) a cell population, (ii) a plurality of three-dimensional (3D) matrices, and (iii) a plurality of fluid flow paths, wherein the cell population is contained or encapsulated within the 3D matrices, and wherein the fluid flow paths are independent of and adjacent to the 3D matrices; (b) Using the fluid flow path to deliver fluid material to the 3D matrix; as well as (c) In the presence of the fluid material delivered to the 3D matrix, the cell population is processed into a cell product.

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

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

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

71. The method of claim 70, wherein the functionalization comprises coupling one or more biomolecules to the 3D matrix.

72. The method of claim 71, wherein the one or more biomolecules are used for membrane signal transduction.

73. The method of claim 70, further comprising polymerizing a prefunctionalized polymer precursor to generate the 3D matrix.

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

75. The method according to any one of claims 67-74, wherein the 3D matrix and the fluid flow path are separated by a membrane, and wherein the fluid material is transported through the membrane.

76. The method according to any one of claims 67-75, wherein the 3D matrix has a size of less than or equal to about 10 millimeters (mm).

77. The method according to any one of claims 67-76, wherein the plurality of 3D matrices are fluidly coupled to each other.

78. The method according to any one of claims 67-77, wherein the fluid flow path is disposed between the first 3D matrix and the second 3D matrix among the plurality of 3D matrices.

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

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

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

82. The method according to any one of claims 67-81, wherein the interface area between the fluid flow path and the 3D matrix is ​​greater than or equal to about 5 square centimeters (cm²). 2 ).

83. The method according to any one of claims 67-82, wherein the 3D matrix comprises a porous solid matrix having interconnected pores with an average pore size sufficient to allow lymphocytes to grow within the 3D matrix.

84. The method according to any one of claims 67-83, wherein the 3D matrix has a surface area to volume ratio greater than or equal to about 0.

1.

85. The method according to any one of claims 67-84, wherein the 3D matrix is ​​a programmable 3D matrix.

86. A method for cell treatment, comprising: (a) Providing a three-dimensional (3D) matrix in the cell processing unit to accommodate multiple cells; (b) In the cell processing unit, automatically: (i) Treating the plurality of cells within the 3D matrix to produce a plurality of treated cells; and (ii) Isolate the plurality of treated cells from the 3D matrix; as well as (c) Collect the plurality of treated cells.

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

88. The method of claim 86 or 87, wherein the plurality of treated cells are cell therapy products.

89. The method of claim 88, wherein the cell therapy product is produced at a scale equivalent to at least one human dose.

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

91. The method of claim 90, further comprising, 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.

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

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

94. The method of claim 91, wherein the first stimulus is an enzyme stimulus.

95. The method of claim 91, further comprising functionalizing the 3D matrix with one or more biomolecules while applying the first stimulus to generate the 3D matrix.

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

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

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

99. The method of claim 96, wherein the second stimulus is an enzyme stimulus.

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

101. The method of claim 100, wherein (a) comprises loading the 3D matrix encapsulating the plurality of cells into a compartment in the cell processing unit.

102. The method according to any one of claims 86-101, wherein (a) comprises providing the plurality of cells and the 3D matrix to the cell processing unit respectively.

103. The method of claim 102, wherein the 3D matrix is ​​generated in a compartment in the cell processing unit.

104. The method of claim 102, wherein the 3D matrix is ​​generated outside the cell processing system and subsequently provided to the cell processing unit.

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

106. The method of claim 105, wherein the functionalization comprises coupling one or more biomolecules to the 3D matrix.

107. The method of claim 106, wherein the one or more biomolecules are used for membrane signal transduction.

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

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

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

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

112. The method according to any one of claims 86-111, wherein the 3D matrix has about 10 -5 Siemens / m (S / m) to approximately 10 -1 Conductivity in S / m.

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

114. A cell processing system comprising: A cell processing unit configured to retain a three-dimensional (3D) matrix, wherein the 3D matrix is ​​configured to contain or encapsulate multiple 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 treated cells, (ii) direct the cell processing unit to separate the plurality of treated cells from the 3D matrix, and (iii) direct the cell processing unit to collect the plurality of treated cells.

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

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

117. The system of claim 115, wherein 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.

118. The system according to any one of claims 114-117, further comprising an analysis unit configured to monitor the plurality of cells during processing of the plurality of cells.

119. The system of claim 118, wherein the analysis unit includes one or more sensors configured to monitor an environment containing the plurality of cells.

120. The system of claim 118, wherein the analysis unit includes one or more sensors configured to analyze the cell product.

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

122. The system of claim 121, wherein the housing has a volume of approximately 0.01 cubic meters (m³). 3 (approximately 2 m) 3 The space it occupies.

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

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

125. The system of claim 124, wherein the cell processing unit is configured to receive the preformed 3D matrix comprising the plurality of cells.

126. The system of claim 125, wherein the cell processing unit is configured to encapsulate the plurality of cells in the preformed 3D matrix.

127. The system according to any one of claims 114-126, wherein the 3D matrix has about 10 -5 Siemens / m (S / m) to approximately 10 -1 Conductivity in S / m.

128. The system according to any one of claims 114-127, wherein the 3D matrix is ​​configured to provide activation of the plurality of cells via membrane signal transduction in the 3D matrix.

129. A method for cell treatment, comprising: (a) Providing multiple cells in vitro to a cell processing unit; (b) In the cell processing unit, the plurality of cells are contacted with a selective reagent, wherein upon contact, the selective reagent (i) is cell-coupled to a subpopulation of the plurality of cells, and (ii) the state of one or more cells in the subpopulation is perturbed to produce (A) a perturbed cell population and (B) an unperturbed cell population; and (c) Process the disturbed cell population or the undisturbed cell population into a cell therapy product.

130. The method of claim 129, wherein the cell therapy product is produced at a scale equivalent to at least one human dose.

131. The method of claim 129 or claim 130, wherein the disturbed cell population has an altered growth rate or vigor compared to the undisturbed cell population.

132. The method according to any one of claims 129-131, wherein the cell processing unit comprises a three-dimensional (3D) matrix encapsulating the plurality of cells.

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

134. The method according to any one of claims 129-133, wherein the cell processing unit comprises a container containing the plurality of cells.

135. The method of claim 134, further comprising contacting the plurality of cells with the selection reagent within the container.

136. The method according to any one of claims 129-135, wherein the selection agent comprises a binder and a selector.

137. The method of claim 136, wherein the selector is a cytotoxic agent.

138. The method of claim 136, wherein the binder is coupled to the selector via a connector.

139. The method of claim 138, wherein the connector is a cuttable connector.

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

141. The method of claim 136, wherein the binder is an aptamer.

142. The method according to any one of claims 129-141, wherein the disturbed cell population comprises dead cells or cell degradation products.

143. The method according to any one of claims 129-142, wherein the disturbed cell population has altered cell morphology compared to the undisturbed cell population.

144. A system for manufacturing cell therapy products, comprising: A three-dimensional (3D) matrix configured with more than approximately 80 million cells per cubic centimeter (cells / cm³). 3 The system can contain or encapsulate cell populations at a density of 3D matrix, wherein the system is capable of processing the cells of the cell population into the cell therapy product by means of the 3D matrix.

145. The system of claim 144, wherein the density is at least 100 million cells / cm³. 3 .

146. The system of claim 144 or claim 145, wherein the 3D matrix has a surface area to volume ratio greater than or equal to 0.

1.

147. The system according to any one of claims 144-146, wherein the 3D matrix has a porosity of at least about 80%.

148. The system according to any one of claims 144-147, further comprising a plurality of 3D matrices including the 3D matrix.

149. The system according to any one of claims 144-148, further comprising a fluid flow path in fluid communication with the 3D matrix, wherein the fluid flow path is adjacent to and independent of the 3D matrix.

150. The system of claim 149, further comprising a membrane disposed between the fluid flow path and the 3D matrix.

151. The system according to any one of claims 144-150, wherein the 3D matrix is ​​programmable.

152. A method for manufacturing a cell therapy product, comprising (i) providing a concentration of more than about 80 million cells / cm³ (cells / cm²). 3 (ii) The density of the cell population is preserved within a three-dimensional (3D) matrix, and the cells of the cell population are processed into the cell therapy product by means of the 3D matrix.

153. The method of claim 152, wherein the treatment of the cells comprises culturing the cell population.

154. The method of claim 152 or 153, wherein the treatment of the cell comprises in situ electroporation of the cell.

155. The method of claim 154, wherein the 3D matrix has about 10 -5 Siemens / m (S / m) to approximately 10 -1 Conductivity in S / m.

156. The method according to any one of claims 152-155, wherein the treatment of the cells comprises activating the cells in the 3D matrix.

157. A bioreactor for processing a cell population, comprising a three-dimensional (3D) matrix configured to retain the cell population, wherein the bioreactor has a maximum of about 2 cubic meters (m³). 3 The volume of the cell population is configured to process the cell population into a cell product with high reproducibility using the 3D matrix.

158. The bioreactor of claim 157, wherein the cell product has a viability of more than or equal to about 75%.

159. The bioreactor according to claim 157 or 158, wherein the volume is at most 1 m³. 3 .

160. A method for processing a cell population, comprising (i) providing a cell population retained within a three-dimensional (3D) matrix of a bioreactor, wherein the bioreactor has a mass of up to 2 cubic meters (m³). 3 (ii) the volume of the cell population and (iii) the cell population is processed into cell products with high reproducibility using the 3D matrix.

161. The method of claim 160, wherein the cell product has a viability of more than or equal to about 75%.

162. The method according to claim 160 or 161, further comprising generating the 3D matrix in the presence of the cell population prior to (i).

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

164. A system for manufacturing cell therapy, comprising: A three-dimensional (3D) matrix configured with at least approximately 5 million cells per cubic centimeter (cells / cm³). 3 The system preserves a density of cell populations, wherein the system is capable of treating the population of cells within the 3D matrix for a period of up to approximately 48 hours to generate a cell product, and wherein the cell product can be used to generate cell therapies.

165. The system of claim 164, wherein the density is at least 10 million cells / cm³. 3 .

166. The system according to any one of claims 164 or 165, wherein the 3D matrix has a surface area to volume ratio greater than or equal to 0.

1.

167. The system according to any one of claims 164-166, wherein the 3D matrix has a porosity of at least about 80%.

168. The system according to any one of claims 164-167, wherein the 3D matrix has a size of less than about 200 micrometers (μm).

169. The system according to any one of claims 164-168, wherein the 3D matrix comprises a porous solid matrix having interconnected pores with an average pore size sufficient to allow lymphocytes to grow within the 3D matrix.

170. The system according to any one of claims 164-169, further comprising a plurality of 3D matrices including the 3D matrix.

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

172. The system of claim 171, further comprising a membrane disposed between the fluid flow path and the 3D matrix.

173. A method for manufacturing a cell therapy, comprising (i) providing at least about 5 million cells / cm³ (cells / cm²) 3 (ii) Preserving the density of the cell population within a three-dimensional (3D) matrix; (iii) treating the cells of the cell population within the 3D matrix for a period of up to approximately 48 hours to generate a cell product; and (iv) using the cell product to generate the cell therapy.

174. The method of claim 173, wherein the treatment of the cells comprises culturing the cell population.

175. The method of claim 173 or 174, wherein the treatment of the cells comprises in situ electroporation of the cell population.

176. The method of claim 174, wherein the 3D matrix has about 10 -5 Siemens / m (S / m) to approximately 10 -1 Conductivity in S / m.

177. The method according to any one of claims 173-176, wherein the treatment of the cell comprises activation via membrane signal transduction in the 3D matrix.

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