Continuous flow micro-bioreactor

Abstract

The present disclosure contemplates components, systems, and methods for bioreactors that can be used to produce and maintain cells, optimize cell growth, and optimize the production of products from these cells, for the production and separation of cells and the products produced by these cells. The systems, components, and methods herein address scale, cost, efficiency, and consistency and are suitable for customized cell and bioproduct production.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 743,974, filed Oct. 10, 2018, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] The production of biological products, including cells, proteins, and small and large chemical molecules, has increasingly become a focus in providing medical, food, industrial, and other types of products. Product consistency, the ability to scale production, and the flexibility to adjust production for different sites and environmental conditions are important factors in production.

[0004] Bioreactors provide an environment for large-scale cell production and the production of proteins and other molecules from these cells. Many bioreactors require a large capital investment and also require a large physical space. In addition, the environment of large bioreactors may be environmentally different from smaller-scale growth chambers and may thus result in suboptimal growth and production conditions. Larger-scale bioreactors may also make it difficult to study growth conditions at the single-cell level. This can lead to cell population heterogeneity and can also affect the quality, purity, and yield of biological products produced by the cells. SUMMARY OF THE INVENTION

[0005] Systems, components, and methods are provided herein for producing and maintaining cells, for producing and separating cells, and for producing products from these cells. The systems, components, and methods herein address scale, cost, efficiency, and consistency.

[0006] In one aspect, the present disclosure provides a bioreactor comprising: an inlet configured to receive a plurality of cells; a plurality of minimodules in fluid communication with the inlet, wherein a minimodule of the plurality of minimodules comprises a double gyroid structure or a modified double gyroid structure, wherein the plurality of minimodules are fluidly interconnected to provide at least one microchannel configured to flow the plurality of cells; and an outlet in fluid communication with the plurality of minimodules, the outlet being configured to direct the plurality of cells or derivatives thereof out of the at least one microchannel.

[0007] In some embodiments, the micro modules are interconnected such that at least two non-overlapping microchannels each having a constant mean curvature are provided. In some embodiments, a first microchannel of the at least two non-overlapping microchannels is configured to allow a liquid culture medium to flow, and wherein a second microchannel of the at least two non-overlapping microchannels is configured to allow a gas component to flow. In some embodiments, the at least two non-overlapping microchannels provide a liquid. In some embodiments, the at least two non-overlapping microchannels are separated by a porous membrane. In some embodiments, the area of the first microchannel is equal to the area of the second microchannel, and wherein the area of the porous membrane is the sum of the areas of the first microchannel and the second microchannel. In some embodiments, the plurality of micro modules are assembled into a macrostructure. In some embodiments, the macrostructure is selected from the group consisting of a pyramid, a hollow pyramid, a layered pyramid, a layered structure, a checkerboard arrangement, and a log. In some embodiments, the plurality of micro modules are arranged in layers within the macrostructure, and wherein the layers are configured such that the velocity of the liquid culture medium is substantially the same in each layer. In some embodiments, the plurality of micro modules are arranged in layers within the macrostructure, and wherein the layers are configured such that the velocity of the liquid culture medium varies throughout the layer. In some embodiments, the velocity of the liquid culture medium flowing through the at least one microchannel is greater than the free sedimentation velocity of the cells flowing through the at least one microchannel. In some embodiments, the bioreactor further includes a gas input at the base of the macrostructure and a gas output at the top of the macrostructure. In some embodiments, the bioreactor further includes a cell input at the top of the macrostructure and a cell collection device at the base of the macrostructure, the cell input being configured to provide a plurality of cells and the cell collection device being configured to harvest the plurality of cells. In some embodiments, the bioreactor further includes a liquid culture medium input device configured to allow the liquid culture medium to flow into each layer of the plurality of micro modules. In some embodiments, the volume of the liquid culture medium provided to each layer by the liquid culture medium device maintains a substantially constant cell density in each layer. In some embodiments, the velocity of the liquid culture medium through each micro module depends on the cell division rate such that the time required for the cells to traverse a single micro module or a layer of micro modules is substantially the same as or proportional to the cell division rate. In some embodiments, the bioreactor is interconnected with a sandbox module. In some embodiments, the bioreactor is interconnected with a cell chip module.

[0008] In another aspect, the present disclosure provides a system for cell production, the system comprising: a first module including a cell chip configured to accommodate a plurality of cells; a second module in fluid communication with the first module, wherein the second module includes a sandbox bioreactor configured to: (i) interface with the cell chip, (ii) direct a cell subset from the plurality of cells to different sections, wherein cell growth conditions are independently configurable in the different sections, and (iii) iteratively generate a set of growth conditions for the plurality of cells; and a third module in fluid communication with the first module and the second module, wherein the third module includes a bioreactor configured to: (i) interface with the second module, (ii) receive the cell subset, and (iii) produce copies of the cell subset under the set of growth conditions.

[0009] In some embodiments, the first module, the second module, and the third module are fluidly interconnected. In some embodiments, the system further includes pumps corresponding to each module, wherein the pumps are configured to supply a liquid culture medium to the corresponding module at a certain flow rate or pressure. In some embodiments, the pumps are syringe pumps, peristaltic pumps, or pressure pumps. In some embodiments, the system further includes components selected from the group consisting of: a culture medium preparer, an electroporator, a reservoir, a pump, a bubble sensor, a bubble trap, and combinations thereof. In some embodiments, the system further includes at least one sensor. In some embodiments, the at least one sensor is an on-line sensor. In some embodiments, the at least one sensor measures biological parameters, physical parameters, or chemical parameters. In some embodiments, the biological parameters are selected from the group consisting of: cell division rate, cell growth rate, cell stress response, cell protein content, cell carbohydrate content, cell lipid content, and cell nucleic acid content. In some embodiments, the physical parameters are selected from the group consisting of: cell size, cell density, cell flow rate, liquid culture medium flow rate, mixing rate, turbidity, temperature, and pressure. In some embodiments, the chemical parameters are selected from the group consisting of: pH, liquid culture medium composition, concentration of each liquid culture medium component, gas composition and gas concentration, and dissolved gas concentration. In some embodiments, the system further includes a camera device. In some embodiments, the camera device is configured to count cells from an output of the sandbox bioreactor or the bioreactor. In some embodiments, the camera device is configured to capture at least one additional parameter associated with each cell from an output of at least one bioreactor module, and wherein the additional parameter is a biological, chemical, or physical characteristic of the cell.

[0010] In another aspect, the present disclosure provides a cell chip module, the cell chip module comprising: a layered structure including at least one fluid circuit; a cell accommodation region in fluid communication with the at least one fluid circuit, wherein the cell accommodation region includes at least one first trap configured to accommodate a plurality of cells; an inlet port in fluid communication with the layered structure and configured to input a liquid culture medium into the cell accommodation region; and an outlet port in fluid communication with the layered structure and configured to collect used or excess culture medium and cells.

[0011] In some embodiments, the at least one fluid circuit is configured to direct gas flow to the cell accommodation region. In some embodiments, the at least one fluid circuit is configured to direct liquid culture medium flow to the cell accommodation region. In some embodiments, the at least one trap includes a suction trap. In some embodiments, the at least one trap includes a gate trap. In some embodiments, the cell accommodation region further includes a second trap. In some embodiments, the second trap is an overflow trap. In some embodiments, the at least one trap and the second trap are connected in series with each other. In some embodiments, the cell accommodation region includes at least one gate trap and two or more overflow traps. In some embodiments, the cell chip module further includes one or more cells in a storage mode. In some embodiments, the storage mode is selected from cells that are dry, lyophilized, frozen, or suspended in a liquid. In some embodiments, the cell chip module further includes one or more physical barriers to distribute cells flowing through the cell chip module.

[0012] In another aspect, the present disclosure provides a sandbox bioreactor module, which includes a series of segments, wherein the segments in the series of segments include at least two microchannels configured to transport at least one cell from one end of the microchannel in the at least two microchannels to the other end of the microchannel in the at least two microchannels, wherein the one end of the microchannel is configured to input a liquid culture medium and the at least one cell, wherein the other end of the microchannel is configured to output the liquid culture medium and the at least one cell, and wherein growth conditions are independently configurable in the series of segments.

[0013] In some embodiments, the first and second segments of the series of segments are arranged in series such that cells are transported from the microchannels of the first segment to the microchannels of the second segment. In some embodiments, the microchannels of the first segment bifurcate at an output end into at least two microchannels of the second segment, wherein the at least two microchannels are arranged in a parallel configuration such that a cell output from the first segment is input into one of the at least two microchannels and another cell output from the first segment is input into another one of the at least two microchannels. In some embodiments, the sandbox bioreactor further includes a first inlet configured to provide a liquid culture medium to the series of segments. In some embodiments, the length of the microchannels depends on the cell division rate such that the cells divide zero, one, two, three, four, five, or more than five times during transport from one end of the microchannel to the other end. In some embodiments, the diameter of the microchannels depends on the cell size, the mixing rate of the transported liquid, or a combination thereof. In some embodiments, the sandbox bioreactor further includes at least one sensor for measuring parameters of the cell environment of the sandbox bioreactor. In some embodiments, the sandbox bioreactor further includes a controller configured to change an input to the sandbox bioreactor in response to measurements from the at least one sensor. In some embodiments, the parameters are selected from the group consisting of: biological parameters, physical parameters, and chemical parameters. In some embodiments, the parameters are selected from the group consisting of: gas content, gas concentration, pH, optical density, and temperature. In some embodiments, the parameters are selected from the group consisting of: cell division rate, cell density, cell stress response, or cell metabolites. In some embodiments, the sandbox bioreactor further includes a sample collection chamber located at an outlet of the microchannels of the last segment in the series of segments. In some embodiments, the sandbox bioreactor is interconnected with a cell chip module.

[0014] In another aspect, the present disclosure provides a method of culturing and storing cells, comprising: inoculating at least one cell into a cell chip module, wherein the cell chip module includes: a layered structure having at least one fluid circuit, a cell accommodation region in fluid communication with the at least one fluid circuit, an inlet port in fluid communication with the layered structure, and an outlet port in fluid communication with the layered structure; providing a liquid culture medium to the inlet such that the at least one cell is retained in a first trap of the cell accommodation region; incubating the cell chip for a period of time under conditions sufficient to allow cell division such that the dividing cells are retained in the first trap; and placing the cells in a storage mode after the period of cell division.

[0015] In some embodiments, the storage mode is selected from the group consisting of: drying, lyophilization, freezing, or suspension in a liquid. In some embodiments, the method further comprises providing fresh liquid medium to the inlet and incubating the cell chip for a period of time under conditions that permit cell division to reactivate the cell division. In some embodiments, the period of cell division produces a sufficient number of cells such that a large number of cells leave the first trap and enter a second trap in the cell chip module. In some embodiments, the cells are further incubated for a second period of time for cell division, and wherein the second period of time produces a sufficient amount of cells such that a large number of cells leave the second trap and flow from the cell chip module to the outlet for collection. In some embodiments, the cells from the outlet of the cell chip module are provided to an interconnected sandbox or bioreactor module. In some embodiments, the second trap is a suction trap or an overflow trap. In some embodiments, the first trap is a gate trap or a suction trap.

[0016] In another aspect, the present disclosure provides a method of selecting cell growth conditions, the method comprising: introducing a first group of cells into a sandbox bioreactor, the sandbox bioreactor comprising a series of sections, wherein the sections in the series of sections are independently configurable; incubating the first group of cells in a first section of the series of sections under a first set of growth conditions; monitoring a first parameter of the first group of cells in the first section; and changing the set of growth conditions in response to monitoring the first parameter in the first section to create a second set of growth conditions in a second section of the series of sections.

[0017] In some embodiments, the first group of cells moves to the second section, and wherein a second group of cells is introduced into the first section. In some embodiments, the first group of cells is introduced into the sandbox bioreactor from a cell chip module. In some embodiments, the sandbox reactor is interconnected with a bioreactor module. In some embodiments, the second set of growth conditions is applied to the bioreactor. In some embodiments, the flow rate of cells from one end to the other end of each section depends on the cell division rate. In some embodiments, the cells divide once during the period of time in which the cells are transported from one end of the section to the other end. In some embodiments, the liquid medium flow through each section of the series of sections is laminar flow.

[0018] In another aspect, the present disclosure provides a method for large-scale cell production, the method comprising: introducing a plurality of cells into an inlet of a bioreactor, wherein the bioreactor comprises a collection of micro-modules having a double-helical icosahedral structure or a modified double-helical icosahedral structure, wherein the micro-modules are arranged in layers within a macroscopic structure, the macroscopic structure comprising an inlet and an outlet; flowing a liquid culture medium into the bioreactor; supplying a gas component into the bioreactor; and collecting the plurality of cells from the outlet; wherein the plurality of cells are transported between the micro-modules, and wherein the plurality of cells are transported from the inlet end of the macroscopic structure to the outlet end of the macroscopic structure.

[0019] In some embodiments, the plurality of cells divide on average once during transport from one layer of micro-modules to the next layer of micro-modules. In some embodiments, the amount of liquid culture medium flowing to each layer of micro-modules is such that a substantially constant cell density is maintained in each layer. In some embodiments, the velocity of the liquid culture medium flowing through the bioreactor exceeds the free sedimentation velocity of the cells. In some embodiments, the plurality of cells are introduced into the bioreactor from a cell chip or a sandbox module. In some embodiments, a portion of the plurality of cells is collected from the outlet end of the macroscopic structure. In some embodiments, the plurality of cells produce at least one bioproduct, and wherein the bioproduct is collected from the outlet end of the macroscopic structure. In some embodiments, the bioproduct is selected from the group consisting of: small molecules, proteins, antibodies, macromolecules, and metabolites.

[0020] In another aspect, the present disclosure provides a method for customized cell production, comprising: introducing a selected type of cells into a cell chip module; culturing the cells in the cell chip module; transporting the cells from the cell chip module to a sandbox bioreactor; and selecting at least one growth condition from a first set of growth conditions in the sandbox bioreactor to generate a second set of growth conditions.

[0021] In another aspect, the present disclosure provides a method for culturing cells, comprising: providing a plurality of cells to an adherent bioreactor, the adherent bioreactor comprising at least one channel and a microporous membrane; allowing at least a portion of the plurality of cells to adhere to the surface of the at least one channel, such that at least a portion of the plurality of cells replicate on the surface of the at least one channel to generate adherent cells; flowing a liquid culture medium from the at least one channel through the microporous membrane to (i) wash the adherent cells, (ii) detach the adherent cells to produce suspended cells, and (iii) wash the suspended cells; and optionally, collecting the suspended cells.

[0022] In some embodiments, the at least one channel comprises a material suitable for attachment of at least a portion of the plurality of cells. In some embodiments, the method further comprises flowing additional liquid medium through the at least one channel to (i) provide medium to allow growth and / or replication of at least a portion of the plurality of cells, (ii) detach the adherent cells from the at least one channel, or (iii) flow the suspension cells from the at least one channel to a collection area. In some embodiments, the adherent bioreactor is in fluid communication with a cell chip module, and wherein the cell chip module provides the plurality of cells to the adherent bioreactor. In some embodiments, the adherent bioreactor is in fluid communication with a bioreactor, and wherein the adherent bioreactor provides the suspension cells to the bioreactor. In some embodiments, the plurality of cells are selected from the group consisting of: bacterial cells, fungal cells, yeast cells, eukaryotic cells, plant cells, and algal cells. In some embodiments, the plurality of cells are recombinant cells.

[0023] In some embodiments, the method further comprises culturing a sample of the selected type of cells in the bioreactor using the second set of growth conditions. In some embodiments, the selected type of cells or a portion thereof is collected from the bioreactor. In some embodiments, the selected type of cells are chimeric antigen receptor T (CAR-T) cells, stem cells, or differentiated cells. In some embodiments, the selected type of cells produce at least one biological product, and wherein the biological product is collected from the bioreactor.

[0024] In another aspect, the present disclosure provides a system comprising a plurality of fluid flow paths having a substantially constant cross-section, wherein a first fluid flow path of the plurality of fluid flow paths is in fluid communication with a second fluid flow path of the plurality of fluid flow paths to allow gas to flow from the first fluid flow path to the second fluid flow path at a substantially constant rate along the first fluid flow path, and wherein the first fluid flow path is configured to allow cell culture.

[0025] In some embodiments, the plurality of fluid flow paths include: a spiral icosahedron structure, a double spiral icosahedron structure, a modified double spiral icosahedron structure, a triply periodic minimal surface, or a combination thereof.

[0026] In another aspect, the present disclosure provides a method for processing multiple cells, comprising: (a) providing a bioreactor, the bioreactor comprising: (i) an inlet, (ii) a plurality of micro modules fluidly connected to the inlet, wherein the micro modules in the plurality of micro modules comprise a double helix icosahedron structure or a modified double helix icosahedron structure, wherein the plurality of micro modules are fluidly interconnected to provide at least one microchannel; and (iii) an outlet fluidly connected to the plurality of micro modules; and (b) guiding the multiple cells to the inlet and guiding the multiple cells or their derivatives from the inlet through the at least one microchannel to the outlet.

[0027] In some embodiments, the bioreactor comprises at least two microchannels. In some embodiments, a first microchannel of the at least two microchannels allows a liquid culture medium to flow. In some embodiments, a second microchannel of the at least two microchannels allows a gas component to flow. In some embodiments, the at least two microchannels are separated by a porous membrane. In some embodiments, the plurality of micro modules are assembled into a macroscopic structure.

[0028] In another aspect, the present disclosure provides a method for preparing a bioreactor, the bioreactor comprising: an inlet configured to receive multiple cells; a plurality of micro modules fluidly connected to the inlet, wherein the micro modules in the plurality of micro modules comprise a double helix icosahedron structure or a modified double helix icosahedron structure, wherein the plurality of micro modules are fluidly interconnected to provide at least one microchannel configured to allow the multiple cells to flow; and an outlet fluidly connected to the plurality of micro modules, the outlet being configured to guide the multiple cells or their derivatives out of the at least one microchannel.

[0029] In some embodiments, the bioreactor is prepared by three-dimensional (3-D) printing the plurality of micro modules.

[0030] In some embodiments, a system for cell production is provided, the system comprising: a first module including a cell chip configured to accommodate multiple cells; a second module including a sandbox bioreactor configured to: (i) interface with the cell chip, (ii) guide cells from the multiple cells to different sections, wherein cell growth conditions are independently configurable in the different sections, and (iii) iteratively generate a set of growth conditions for the multiple cells; and a third module including a production bioreactor configured to: (i) interface with the second module, (ii) receive the cells, and (iii) produce copies of the cells under the set of growth conditions.

[0031] In some embodiments, the first module, the second module, and the third module of the system are functionally interconnected. In some embodiments, the system further includes pumps corresponding to each module, wherein the pumps supply a liquid culture medium to the corresponding module at a certain flow rate or pressure. In some embodiments, the pumps are syringe pumps, peristaltic pumps, or pressure pumps.

[0032] Also provided herein is such a system, which further includes components selected from the group consisting of: a culture medium preparer, an electroporator, a reservoir, a pump, a bubble sensor, a bubble trap, and combinations thereof. In some embodiments, the system further includes at least one sensor, such as an on-line sensor. In some embodiments, the at least one sensor measures biological parameters, physical parameters, or chemical parameters, which may include, for example, biological parameters selected from the group consisting of: cell division rate, cell growth rate, cell stress response, cell protein content, cell carbohydrate content, cell lipid content, and cell nucleic acid content; physical parameters selected from the group consisting of: cell size, cell density, cell flow rate, liquid culture medium flow rate, mixing rate, turbidity, temperature, and pressure; chemical parameters selected from the group consisting of: pH, liquid culture medium composition, concentration of each liquid culture medium component, gas composition and gas concentration, and dissolved gas concentration; and combinations thereof.

[0033] Also provided herein is such a system, which further includes a camera device. In some embodiments, the camera device counts the cells at the output of the sandbox bioreactor or the production bioreactor. In some embodiments, the camera device captures at least one additional parameter associated with each cell at the output of at least one bioreactor module, and wherein the additional parameter is a biological, chemical, or physical characteristic of the cell.

[0034] In some embodiments, a cell chip module is also provided, which includes: a cell accommodation area, the cell accommodation area including at least one first trap for accommodating cells, and optionally at least one second trap for accommodating cells; an inlet port configured to input a liquid culture medium into the cell accommodation area; and an outlet port configured to collect the used or excess culture medium and cells. In some embodiments, the first trap may be a suction trap, a gate trap, an overflow trap, or a combination thereof. In some embodiments, the first trap and the second trap are connected in series with each other. In some embodiments, the cell chip module includes at least one gate trap and two or more overflow traps. In some embodiments, the cell chip module may include one or more physical barriers for distributing the cells flowing through the module.

[0035] In some embodiments, the cell chip module further includes one or more cells in a storage mode, which can be, for example, dried, lyophilized, frozen, or cells suspended in a liquid.

[0036] In some embodiments herein, a sandbox bioreactor module is provided, which includes a series of sections, wherein the sections in the series of sections include at least one microchannel configured to transport cells from one end of the microchannel to the other end of the microchannel, wherein one end of the microchannel is configured to input a liquid culture medium and at least one cell, and wherein the other end of the microchannel is configured to output the liquid culture medium and the at least one cell.

[0037] In some embodiments of the sandbox bioreactor, a first section and a second section are arranged in series such that cells are transported from one of the microchannels of the first section to one of the microchannels of the second section. In some embodiments, the microchannel of the first section branches at an output end into at least two microchannels of the second section, wherein the at least two microchannels are arranged in a parallel configuration such that a cell output from the first section is input into one of the at least two microchannels, and another cell output from the first section is input into another one of the at least two microchannels.

[0038] In some embodiments, the sandbox bioreactor further includes a first inlet suitable for providing a liquid culture medium to the section. In some embodiments, the length of each microchannel of the sandbox bioreactor depends on the cell division rate such that the cells divide zero, one, two, three, four, five, or more than five times during the transport from one end of the microchannel to the other end of the microchannel. In some embodiments, the diameter of each microchannel depends on the cell size, the mixing rate of the transported liquid, or a combination thereof.

[0039] In some embodiments, the sandbox bioreactor includes at least one sensor for measuring parameters of the cell environment of the bioreactor, and optionally further includes a controller for changing the input to the bioreactor in response to measurements from the at least one sensor. In some embodiments, the sensor measures parameters such as biological parameters, physical parameters, and / or chemical parameters, such as gas content, gas concentration, pH, optical density, temperature, cell division rate, cell density, cell stress response, cell metabolites, or a combination thereof.

[0040] In some embodiments, the sandbox bioreactor includes a sample collection chamber located at the outlet of the microchannel of the last section in the series of sections. In some embodiments, the sandbox bioreactor is interconnected with a cell chip module (such as any of the cell chip modules described herein).

[0041] In some embodiments herein, a production bioreactor is provided that includes a plurality of micro modules, wherein each of the plurality of micro modules includes a double helix icosahedral shape or structure, and wherein the plurality of micro modules are interconnected to provide microchannels. In some embodiments, the interconnection of the micro modules results in two non-overlapping channels each having a constant mean curvature. In some embodiments, one microchannel of the production bioreactor provides a liquid medium and a second microchannel provides a gas component. In some embodiments, both microchannels provide liquid. In some embodiments, the microchannels are separated by a porous membrane. In some embodiments, the area of the first microchannel is equal to the area of the second microchannel, and the area of the membrane is the sum of the areas of the first and second microchannels.

[0042] In some embodiments, the production bioreactor may include micro modules assembled into a macroscopic structure, which may be, for example, a pyramid, a hollow pyramid, a layered pyramid, a layer, a checkerboard arrangement, or a log-like shape. In some embodiments, the micro modules are arranged in a hierarchy within the macroscopic structure, and wherein the velocity of the liquid medium is substantially the same in each hierarchy. In some embodiments, the velocity of the liquid medium varies throughout the hierarchy. In some embodiments, the velocity of the liquid medium flowing through the microchannel is greater than the free settling velocity of the cells flowing through the microchannel.

[0043] In some embodiments, the production bioreactor includes a gas input at the base of the macroscopic structure and a gas output at the top of the macroscopic structure. In some embodiments, a cell input is provided at the top of the macroscopic structure and a cell collection device is provided at the base of the macroscopic structure.

[0044] In some embodiments, the production bioreactor includes a liquid medium input device, wherein the liquid medium flows into each hierarchy of the micro modules. In some embodiments, the volume of the liquid medium provided to each hierarchy by the liquid medium device maintains a substantially constant cell density in each hierarchy. In some embodiments, the velocity of the liquid medium flowing through each micro module depends on the cell division rate such that the time required for the cells to cross a single micro module or a hierarchy of micro modules is substantially the same as or proportional to the cell division rate.

[0045] In some embodiments, the production bioreactor is interconnected with a sandbox module and a cell chip module, or is interconnected with a system that includes both a cell chip module and a sandbox module. In this document, in some embodiments, a system is provided that includes a plurality of fluid flow paths having a substantially constant cross-section, wherein a first fluid flow path among the plurality of fluid flow paths is in fluid communication with a second fluid flow path among the plurality of fluid flow paths to allow gas to flow from the first fluid flow path to the second fluid flow path at a substantially constant rate along the first fluid flow path. In some embodiments, the plurality of fluid flow paths include: a spiral icosahedron shape or structure, a modified spiral icosahedron shape or structure, a triply periodic minimal surface, or a combination thereof.

[0046] Also provided herein are methods for culturing and storing cells, including: inoculating at least one cell into a cell chip module; providing a liquid culture medium to a liquid inlet of the cell chip such that the at least one cell remains in a first trap; incubating the cell chip for a period of time under conditions that permit cell division such that the dividing cells remain in the first trap; and, after the period of cell division, placing the cells in a storage mode. In some embodiments, the storage mode is selected from the group consisting of: drying, lyophilization, freezing, or suspension in a liquid. In some embodiments, the method further includes providing a fresh liquid culture medium to the liquid inlet of the cell chip and incubating the cell chip for a period of time under conditions that permit cell division to reactivate cell division. In some embodiments, during the period of cell division, sufficient cells are produced such that a large number of cells leave the first trap and enter a second trap in the cell chip module. In some embodiments, the cells are further incubated for a second period of cell division, and wherein the second period of cell division produces a sufficient amount of cells such that a large number of cells leave the second trap and flow from the cell chip to an outlet for collection. In some embodiments, the cells from the outlet of the cell chip are provided to an interconnected sandbox or production bioreactor module. In some embodiments of the method, the second trap is a suction trap or an overflow trap. In some embodiments, the first trap is a gated trap or a suction trap.

[0047] Methods for optimizing the cellular environment are provided herein, the method comprising: introducing a first set of cells into a sandbox bioreactor, the sandbox bioreactor comprising a series of sections; incubating the first set of cells in a first section under a first environment; monitoring a first parameter of the cells in the first section; and changing the first environment in response to the monitoring of the first parameter to create a second environment. In some embodiments of the method, the first set of cells is moved to a second section, and a second set of cells is introduced into the first section. In some embodiments, the cells are introduced into the sandbox bioreactor from a cell chip module. In some embodiments, the sandbox reactor is interconnected with a production bioreactor module. In some embodiments of the method, the second environment is applied to the production bioreactor.

[0048] In some embodiments of the method, the flow rate of the cells from one end to the other end of each section depends on the cell division rate. In some embodiments, the cells divide once during the period in which the cells are transported from one end of the section to the other end of the section. In some embodiments, the liquid culture medium flow through each section is laminar flow.

[0049] Methods for large-scale production of cells are also provided herein, the method comprising: introducing cells into a production bioreactor, wherein the production bioreactor comprises a collection of micro-modules having a double-helical icosahedral shape or structure, wherein the micro-modules are arranged in a hierarchy within a macroscopic structure; flowing a liquid culture medium into the production bioreactor; and supplying a gas component into the production bioreactor; wherein the cells are transported between the micro-modules, and wherein the cells are transported from an inlet end of the macroscopic structure to an outlet end of the macroscopic structure. In some embodiments of the method, the cells divide on average once during the transfer from one hierarchical micro-module to the next hierarchical micro-module. In some embodiments, the amount of the liquid culture medium flowing to each hierarchical micro-module is such that a substantially same cell density is maintained in each hierarchy. In some embodiments, the velocity of the liquid culture medium flowing through the production reactor exceeds the free sedimentation velocity of the cells.

[0050] In some embodiments of the methods herein, the cells are introduced into the bioreactor from a cell chip or sandbox module. In some embodiments, a portion of the cells is collected from the outlet end of the macroscopic structure. In some embodiments, the cells produce at least one bioproduct, and wherein the bioproduct is collected from the outlet end of the macroscopic structure, for example, small molecules, proteins, antibodies, macromolecules, metabolites, or combinations thereof produced by the cells.

[0051] Methods for customizing cell production are provided herein, including: introducing a selected type of cell into a cell chip; culturing the cell in the cell chip; transporting the cell from the cell chip module to a sandbox bioreactor; and optimizing at least one parameter of a first cell environment in the sandbox bioreactor to establish a second cell environment. In some embodiments, the method further includes culturing a sample of the selected type of cell using the second cell environment in a production bioreactor. In some embodiments, the cell or a portion thereof is harvested from the production bioreactor. In some embodiments, the cells used in the method are CAR-T cells, stem cells, differentiated cells, or combinations thereof. In some embodiments, the cell produces at least one bioproduct, and the bioproduct is collected from the production bioreactor.

[0052] Methods for culturing cells are provided herein, including culturing cells in the cell chip module described herein. In some embodiments, the method further includes placing the cells in a storage mode. Methods for optimizing cell environmental conditions are provided herein, including: culturing cells in the sandbox bioreactor described herein, and optimizing at least one parameter of the cell environment. Methods for culturing cells are also provided herein, including culturing cells in the production bioreactor described herein. In some embodiments, the method is a method for large-scale cell production. In some embodiments of the methods for culturing and producing cells, the cells are selected from the group consisting of: bacterial cells, fungal cells, plant cells, animal cells, avian cells, mammalian cells, human cells, and transgenic cells. In some embodiments, the systems, devices, and methods described herein can be used under zero gravity or microgravity conditions to culture cells under zero gravity or microgravity conditions.

[0053] Based on the following detailed description, additional aspects and advantages of the present disclosure will become apparent to those skilled in the art, where only illustrative exemplary embodiments of the present disclosure are shown and described. As will be recognized, the present disclosure is capable of having other and different embodiments, and several details thereof can be modified in various obvious aspects, and all without departing from the content of the present disclosure. Accordingly, the accompanying drawings and the specification should be regarded as illustrative in nature and not restrictive.

[0054] Incorporation by reference

[0055] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the incorporated publications and patents or patent applications conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth exemplary embodiments, and the accompanying drawings (also referred to herein as "drawings" and "figures"), in which the principles of the invention are employed:

[0057] Figure 1 Showing an exemplary embodiment of a system having three modules;

[0058] Figure 2A Showing an exemplary embodiment of the cell chip module 201; Figure 2B Showing an exemplary embodiment of the cell environment 205, the cell chip culture medium circuit 230, and the cell chip gas circuit 250 as respective "layers" of the cell chip; Figure 2C Showing a side profile of the layers of the cell chip;

[0059] Figure 3A-3F Providing an exemplary embodiment of a cell trap for use with the cell chip module; Figure 3A-3B Showing an exemplary embodiment of a gate trap; Figure 3C-3D Showing an exemplary embodiment of an overflow trap; and Figure 3E-3F Showing an exemplary embodiment of a suction trap;

[0060] Figure 4A-4E Providing an exemplary embodiment of a sandbox bioreactor;

[0061] Figure 5 Providing an exemplary embodiment of a production bioreactor;

[0062] Figure 6A-6C Providing an exemplary embodiment of the macroscopic structure of a production bioreactor;

[0063] Figure 7A Showing an exemplary embodiment of a modified double-helix icosahedron shape or structure, and assembled into stacked channels such as for use in the production bioreactor described herein; Figure 7B Providing an example of a modified double-helix icosahedron shape or structure assembled into two overlapping channel networks; Figure 7CExamples of curvature resulting from channels formed by modified double helix icosahedrons are provided;

[0064] Figure 8 Schematic diagrams of methods such as cell culture, storage, environmental optimization, and large-scale production are shown;

[0065] Figure 9A-9F Exemplary schematic diagrams showing the assembly of micro-modules into a macroscopic structure are shown; Figure 9A Examples of micro-modules are shown; Figure 9B Examples of the assembly of micro-modules into an exemplary three-dimensional matrix are shown; Figure 9C An exemplary three-dimensional matrix is shown; Figure 9D Exemplary layers of the three-dimensional matrix are shown; Figure 9E-9F Exemplary assemblies are shown, which include multiple layers of three-dimensional layers;

[0066] Figure 10A-10F Examples of layer assemblies of various shapes such as square and square-like assembly shapes are shown;

[0067] Figure 11A-11F Examples of module layers connected to an exemplary feed circuit are shown;

[0068] Figure 12 An exemplary layer for a hollow pyramid shape is shown;

[0069] Figure 13A and 13B Examples of growth for a hollow pyramid shape are provided;

[0070] Figure 14 Examples of an external feed circuit for a hollow pyramid shape are shown;

[0071] Figure 15 Examples of layered macroscopic structures are shown;

[0072] Figure 16 Examples of layered macroscopic structures with a feed circuit are shown;

[0073] Figure 17 An exemplary macroscopic structure is shown;

[0074] Figure 18 Exemplary feed and collection arrangements are shown;

[0075] Figure 19A-19E An exemplary connection system is shown; Figure 19A A general diagram of an exemplary connection system is shown, which includes a connector between a cell chip module and a fluid source; Figure 19B An exemplary connection system with input and output needles is shown; Figure 19C Exemplary connections achieved by an exemplary connector system are shown;Figure 19D An exemplary embodiment of a connection system is shown, where a needle pierces a chamber in an exemplary cell chip module; Figure 19E An exemplary connection system is shown, where a needle pierces a second chamber;

[0076] Figure 20 An exemplary multi-layer module for adherent cells is shown;

[0077] Figure 21A An embodiment of a layer module for adherent cells is shown; Figure 21B An example of cells attached to a layered structure is shown; Figure 21C An example of trypsin washing for cell detachment is shown; Figure 21D An example of cell detachment is shown; Figure 21E An example of a detached cell stream is shown;

[0078] Figure 22A A cross-sectional view of an exemplary micro-module is shown; Figure 22B A cross-sectional view of an exemplary micro-module with enhanced mixing is shown;

[0079] Figure 23A An exemplary assembly of ten micro-modules is shown; Figure 23B An exemplary assembly of ten micro-modules with enhanced mixing is shown;

[0080] Figure 24 An exemplary bioreactor design with a macroscopic structure is shown;

[0081] Figure 25A An isometric view of an exemplary macroscopic structure is shown; Figure 25B A printed exemplary macroscopic structure is shown; Figure 25C A printed exemplary macroscopic structure made of a commercial resin is shown;

[0082] Figure 26A An exemplary bioreactor loop saturated with a dye is shown; Figure 26B An exemplary double-helix icosahedron structure saturated with a dye is shown;

[0083] Figure 27 An exemplary design of an exemplary cell line on a chip is shown;

[0084] Figure 28 An example of particle flow in a chamber is shown;

[0085] Figure 29 An example of particle flow over time is shown;

[0086] Figure 30A An exemplary sandbox unit with a mixing module is shown; Figure 30B-30DShows an exemplary sandbox unit formed of polydimethylsiloxane;

[0087] Figure 31 Shows the gas circuit and culture circuit of an exemplary sandbox bioreactor;

[0088] Figure 32 Shows an exemplary printed culture layer of a sandbox;

[0089] Figure 33 Shows the assembly layer of an exemplary sandbox; and

[0090] Figure 34 Shows a computer system that is programmed or otherwise configured to implement the methods provided herein. Detailed Description

[0091] Although various embodiments of the present invention have been shown and described herein, these embodiments are provided by way of example only to those skilled in the art. Many variations, changes, and alternatives can be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives of the embodiments of the present invention described herein can be employed.

[0092] Systems, components, and methods for producing and maintaining cells and for producing and separating cells and products made from cells are provided herein. The systems, components, and methods herein provide flexibility to tailor production for different types of cells, types of cell environments, and types of molecules produced. The systems, components, and methods also provide flexibility in scale. For example, the systems, components, and methods described herein can provide scale-up production without changing or significantly changing the growth conditions at the laboratory scale.

[0093] One or more bioreactors for culturing cells are included in the systems and components. The bioreactors are at the micro - bioreactor scale, such that the systems can be constructed as bench - top bioreactors, having the ability to culture and produce cells and / or cell products in both small and large quantities. The advantages of this system and method of use are their scalability, flexibility, and resource conservation.

[0094] The term "cell chip" or "cell chip module" as used herein generally refers to a device suitable for growing, culturing, and / or storing cells, which may include one or more channels or other openings for introducing cells, for providing liquid culture medium and other cell environmental factors, and optionally also includes one or more structures for trapping, containing, or guiding a cell stream within the growth / culture environment of the cell chip or its compartments.

[0095] As used herein, the term "sandbox bioreactor" generally refers to a device for growing cells, and the device allows for testing the effects of one or more cell environmental conditions on one or more types of cells in an iterative manner. The sandbox reactor can include a design to correlate the cell environment therein and its effect on cells with the cell environment and effects for scaling up and producing cells and biological products. The sandbox reactor can include a single test environment or can include multiple sections within which one or more environmental conditions can be tested.

[0096] As used herein, the term "production bioreactor" or "bioreactor" generally refers to a bioreactor device suitable for scaling up the production of cells and / or products produced by cells. The production bioreactor can include: one or more channels or other openings for inputting cells to provide liquid medium, gas components, and other cell environmental factors; and one or more channels for harvesting cells and / or products produced by cells.

[0097] As used herein, the term "media formulator" generally refers to a component or device for mixing the components of a medium used for culturing cells.

[0098] As used herein, the term "trapper" generally refers to a structure for trapping, containing, and / or guiding a cell stream within a physical area. Trappers can include, but are not limited to: gated trappers, overflow trappers, suction trappers, and porous membrane trappers, such as, but not limited to, those described herein. In some embodiments, the trapper size depends on the cell size, volume, and / or diameter such that the trapper slows, impedes, or prevents cells from moving from one physical space to another physical space. In some embodiments, when the cell number or density increases or when the velocity of the cell or liquid medium stream increases, the ability of the trapper to slow, impede, or prevent cell movement can be reduced or overcome.

[0099] As used herein, the term "micro module" generally refers to a section of a production bioreactor that can be interconnected and assembled into a larger structure (e.g., a macroscopic structure or macroscopic shape) to form at least a part or the whole of the production bioreactor.

[0100] The term "gyroid" as used herein generally refers to a connected periodic minimal surface that does not contain straight lines. Such a surface can have a mathematically infinite number of connections. In some examples, the gyroid is the only non-trivial embedded member of an associated family of Schwarz P and D surfaces with an angle of association of approximately 38.01°. The gyroid can be configured as a single gyroid or a double gyroid. The double gyroid can be oriented and configured for specific applications in microfluidic devices. The double gyroid can be configured by balancing geometric aspects related to hydrodynamic performance observed in microscale modules and macroscale structures (e.g., macroscale shapes) such as double gyroid crystallographic structures and space groups. The gyroid or double gyroid can be implemented in various crystallographic structures.

[0101] Whenever the term "at least", "greater than", or "greater than or equal to" precedes the first value in a series of two or more numerical values, the term "at least", "greater than", or "greater than or equal to" applies to each 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.

[0102] Whenever the term "not greater than", "less than", or "less than or equal to" precedes the first value in a series of two or more numerical values, the term "not greater than", "less than", or "less than or equal to" applies 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.

[0103] Bioreactor module

[0104] In one aspect, the present disclosure provides systems and methods for producing modular and interconnected bioreactor components for cells. The cells can be bacterial cells, fungal cells, yeast cells, eukaryotic cells, plant cells, or algal cells. The cells can be recombinant cells. The modular system can include a first module, a second module, and a third module. The first module can be a cell chip configured to accommodate a plurality of cells. The second module can be in fluid communication with the first module and can be a sandbox reactor. The sandbox reactor can be configured to: (i) interface with the cell chip, (ii) direct a cell subset from the plurality of cells to different sections of the sandbox, and (iii) iteratively generate a set of growth conditions for the plurality of cells. The different sections can be independently configurable. The third module can be in fluid communication with the first module and the second module. The third module can include a bioreactor configured to: (i) interface with the second module, (ii) receive the cell subset, and (iii) produce copies of the cell subset under the set of growth conditions. In some embodiments, the third module can include a bioreactor and can be in fluid communication with the cell chip module.

[0105] The systems, components, and methods herein are modular and interconnected. In some embodiments, the system includes one or more modules, each module including at least one bioreactor. In some embodiments, the system includes at least one, two, three, or more modules. In some embodiments, the system includes more than three modules. Each module is configured for laminar flow of liquids (including culture media and / or solvents), and additionally, in some embodiments, for unidirectional flow of cells. In some embodiments, one or more modules are configured for transitional or turbulent flow of liquids (e.g., culture media and / or solvents).

[0106] In some embodiments, the system includes at least 1, 2, 3, or more modules that are interconnected. In some embodiments, the modules include one or more of a cell chip module, a sandbox bioreactor module, and a production bioreactor module. In some embodiments, the cell chip module is interconnected (e.g., fluidly connected) with the sandbox bioreactor module such that the cell chip supplies cells to initially inoculate the sandbox reactor. In some embodiments, the cell chip module is interconnected (e.g., fluidly connected) with the production bioreactor such that the cell chip supplies cells to initially inoculate the production bioreactor. In some embodiments, the sandbox bioreactor is interconnected (e.g., fluidly connected) with the production bioreactor, and cells flow from the sandbox bioreactor into the production bioreactor to scale up. In some embodiments, the system connects the cell chip to the sandbox bioreactor and to the production bioreactor in a series configuration. Alternatively, or additionally, the system connects the cell chip to the sandbox bioreactor and the production bioreactor in a parallel configuration. Cells can be initially cultured in the cell chip, tested for optimized environmental conditions in the sandbox bioreactor, and produced at scale in the production bioreactor.

[0107] In some embodiments, the system includes a module that is a cell housing and storage chip (also referred to herein as a cell chip) in which one or more cells are introduced and proliferated by cell growth and division. The cell chip can be a consumable (e.g., discarded after being used once or several times). The cell chip can be provided with one or more cell lines. The cell chip can include an input channel for a liquid medium to flow in and an output channel for the used medium and optionally cells to flow out of the cell chip. The cell chip can have at least 1, 2, 3, 4, 5, 6, 8, 10, or more input channels. The cell chip can have at least about 1, 2, 3, 4, 5, 6, 8, 10, or more output channels. The cell chip can have an equal number of input channels and output channels or can have a different number of input channels and output channels. Each input channel can flow a single component (e.g., a single liquid medium), or each input channel can flow different components (e.g., different types of liquid media). The output channel can flow both the used or excess medium and cells. Alternatively, or additionally, the cells can be separated from the medium flow and output from the cell chip through one or more output channels. The cell chip can include at least one mechanism to trap cells within the module.

[0108] In some embodiments, the mechanism for trapping cells is a gated trap that collects cells within the trap as the cells flow into the trap along with the laminar flow of the liquid culture medium within the cell chip. The gated trap can prevent the cells from freely flowing within the chip, confine them to a protected environment, relieve them from the inertial forces of the flow, while enabling proper access to nutrients and gases. The gated trap can have an inoculation port to enable inoculation of cells within the internal space of the gated trap. The gated trap can have one or more flow rate protectors shaped to relieve the internal cells from the inertial forces of the flow. Exemplary shapes or structures that can serve as one or more flow rate protectors include triangles, squares, pentagons, hexagons, circles, and combinations thereof. The flow rate protectors can be interspersed with openings to allow gases and nutrients to enter and to enable the cells to escape from the gated trap in a controlled manner. In some embodiments, the opening size is larger than the diameter of the cells within the flow rate protector. In some cases, the opening size is twice the diameter of the cells within the flow rate protector. In some cases, the opening can be at least 2, 3, 4, 5, 7, or 10 times the diameter of the cells within the flow rate protector. In some embodiments, the openings are uniformly distributed throughout the shape or structure of the flow rate protector. In some embodiments, the openings are arranged according to the desired flow direction and velocity. In some embodiments, the openings can be more concentrated on the second half of the flow rate protector shape that is against (counting) the overall direction of the flow. In some embodiments, the diameter of the gated trap is from about 20 microns to about 1000 microns. In some embodiments, the diameter of the gated trap is about greater than or equal to 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, or greater. In some embodiments, the diameter of the gated trap is about 100 microns, 200 microns, 300 microns, 400 microns, 500 microns, 600 microns, 700 microns, 800 microns, 900 microns, 1000 microns, or greater. In some embodiments, the diameter of the gated trap is less than or equal to about 1000 microns, 900 microns, 800 microns, 700 microns, 600 microns, 500 microns, 400 microns, 300 microns, 200 microns, 100 microns, 90 microns, 80 microns, 70 microns, 60 microns, 50 microns, 40 microns, 30 microns, or less.

[0109] In some embodiments, the mechanism for trapping cells is an overflow trap. The overflow trap can prevent cells from flowing freely within the chip, confining them to a protected environment by physically impeding their escape and using inertial flow forces to enhance their confinement while enabling access to nutrients and gases. In some embodiments, the overflow trap flow rate protector includes one or more canalized wall arrangements to physically confine the cells within the trap. In some embodiments, the canalized walls have openings therebetween. In some embodiments, the openings are smaller than the diameter of the internal cells. In some embodiments, the canalized walls have no openings therebetween. In some embodiments, an opening is provided between the top of the chip and the height of the canalized walls. In some embodiments, the opening between the height of the canalized walls and the top is smaller than the diameter of the cells within the trap. When the volume of the cells exceeds the volume capacity of the gated trap, the cells flow out of the trap. In some embodiments, the diameter of the overflow trap is from about 50 microns to about 2000 microns. In some embodiments, the diameter of the overflow trap is greater than or equal to about 180 microns or 210 microns. In some embodiments, the diameter of the overflow trap is less than or equal to about 210 microns or 180 microns. In some embodiments, the diameter of the overflow trap is greater than or equal to about 50 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, 600 microns, 800 microns, 1000 microns, 1200 microns, 1500 microns, 2000 microns or greater. In some embodiments, the diameter of the overflow trap is less than or equal to about 2000 microns, 1500 microns, 1200 microns, 1000 microns, 800 microns, 600 microns, 500 microns, 450 microns, 400 microns, 350 microns, 300 microns, 250 microns, 200 microns, 150 microns, 100 microns, 50 microns or smaller.

[0110] In some embodiments, the cell trap includes a suction trap. The suction trap can be designed by vertically separating the liquid culture medium region from the cell growth region with a porous membrane. The suction trap can be designed to have one or more arms, into which cells are introduced and in which cells grow and divide. The liquid culture medium travels through the chip and can pass through the porous membrane to reach the lower region, while the cells are retained in the upper region due to the pore size being smaller than the cell size. The movement of the liquid culture medium from top to bottom creates suction, thereby retaining the cells within one or more arms. When the cells divide to a certain number or density, the suction no longer retains all the cells within the arms, and the cells can flow out of the arms at the upper level and flow towards the output port of the cell chip. The suction trap can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more arms. In some embodiments, the porous membrane used can be varied according to the cell type. In some embodiments, the porous membrane can have a pore size smaller than the cell diameter. In some embodiments, the pore size of the porous membrane can be greater than or equal to 0.22 microns, 1 micron, 3 microns, 5 microns, 7 microns or larger. In some embodiments, the pore size is less than or equal to about 7 microns, 5 microns, 3 microns, 1 micron, 0.22 microns or smaller. In some embodiments, the porous membrane used has pores of a discrete selected specific pore size; this can be achieved by implementing track etching technology. In some embodiments, the pores of the porous membrane are randomly distributed. Alternatively or additionally, the pores can be arranged or patterned. In some embodiments, the material used for the porous membrane is a high-quality polycarbonate membrane. In some embodiments, the material used for the porous membrane is polysulfone, polyethylene, polytetrafluoroethylene, polypropylene, nitrocellulose, nanocellulose, nylon, ceramic foam or carbon nanotubes.

[0111] In some embodiments, the mechanism for trapping cells in a cell chip includes a combination of trap types, including gated traps, suction traps, and overflow traps. In some embodiments, the trap types are in series such that when cells are released from one type of trap, they flow into and are trapped in another type of trap. Alternatively or additionally, the traps can be arranged in a parallel configuration. In some embodiments, the traps are arranged in series and parallel configurations. The number and arrangement of traps can be adjusted for the cell type, as well as the size and volume of the cell chip and the liquid flow rate in the cell chip. In some embodiments, the cell chip includes greater than or equal to 1, 2, 3, 4, 5, 10, 20, 50, 100, or more gated traps, either alone or in combination with one or more overflow traps, or in combination with one or more suction traps. In some embodiments, the cell chip includes greater than or equal to about 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 300, 400, 500, 1000, or more overflow traps, either alone or in combination with one or more gated traps or one or more suction traps.

[0112] In some embodiments, the mechanism for trapping cells is a porous membrane trap. This type of trap allows a liquid culture medium to flow over the upper side of the porous membrane, and cells are introduced and grow in a chamber below the porous membrane. As the culture medium is infused into the cell chamber and as the cell density increases and the cells grow closer to the membrane, the cells are drawn through the pores of the membrane into the upper culture medium lane, where the cells can then move with the liquid culture medium flow and thus leave the trap. The pore size of the membrane in the trap can be adjusted according to the type and size of the cells. In some embodiments, the pore size is from about 0.22 microns to about 5 microns. In some embodiments, the pore size is greater than or equal to about 0.22 microns, 1 micron, 3 microns, 5 microns, 7 microns, or greater. In some embodiments, the pore size is less than or equal to about 7 microns, 5 microns, 3 microns, 1 micron, 0.22 microns, or less. The size and volume of the chamber below the membrane can be adjusted according to the type and size of the cells. The number of cell chambers can be adjusted for any throughput. In some embodiments, the number of cell chambers is greater than or equal to 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000, or more.

[0113] In some embodiments, after the cells have grown in the cell chip, the cell chip stores the cells trapped therein for subsequent use. The cells can be stored by various methods, including, for example, freezing, drying, lyophilization, or storing in a liquid culture medium, or modifying the liquid culture medium such that the cells can be stored at room temperature or other temperatures.

[0114] In some embodiments, the system includes a sandbox bioreactor. The sandbox reactor can be a consumable (e.g., discarded after being used once or a few times). Alternatively or additionally, the sandbox reactor can not be a consumable. The sandbox bioreactor module is composed of a series of sections, where each section has at least one microchannel suitable for transporting cells from one end of the microchannel to the other end of the microchannel. In some embodiments, the section has two or more microchannels functioning in parallel with each other, so that cells in one microchannel in the same section may not be transported to another microchannel. In some embodiments, the section has greater than or equal to 2, 4, 8, 16, 32, 64, 128 or 2 n parallel microchannels. The length and diameter of the microchannels can be adjusted according to cell type, cell size, and the transport speed of cells and liquid medium in the microchannels. In some embodiments, the channels can have a diameter greater than or equal to about 20 microns, 50 microns, 70 microns, 120 microns, 200 microns, 600 microns, 1500 microns, 2000 microns, and have a length of about 1 mm, 2 mm, 4 mm, 10 mm, 22 mm, 40 mm, 120 mm, 200 mm or greater. In some embodiments, the length of the microchannel is adjusted according to the cell division rate of the cells transported through the microchannel such that the cells divide once during the transit time from entry to exit of the microchannel. The diameter of each microchannel can be adjusted according to cell size, mixing rate of the transported liquid, gas exchange rate, or a combination thereof.

[0115] In some embodiments, a section of the sandbox bioreactor includes two or more compartments. One compartment may include a mixing module while another compartment may include a growth chamber. The mixing module and the growth chamber may be in a series or parallel configuration. In one embodiment, the mixing module and the growth chamber are in a series configuration. The mixing module may be fluidly connected to greater than or equal to 1, 2, 4, 6, 8, 10 or more input channels. The input channels may include channels for flowing a culture medium, gas, and / or cells. In one embodiment, the mixing module is fluidly connected to two input channels such that one channel provides the culture medium while the other channel provides cells to the mixing module. The culture medium and the cells may be mixed in the mixing module. The mixing module may include straight channels or may include channels having a serpentine or tortuous geometry. The diameter of the channels of the mixing module may be greater than or equal to about 20, 50, 70, 120, 200, 600, 1500 or 2000 microns and the length is or about 1, 2, 4, 10, 22, 40, 120, 200 millimeters. The mixing module may provide the mixed cells and culture medium to the growth chamber. The growth chamber may include an expansion band that increases the diameter of the section. The growth chamber may have a diameter greater than or equal to 20, 50, 70, 120, 200, 600, 1500 or 2000 microns and a length that is or about 1, 2, 4, 10, 22, 40, 120 or 200 millimeters.

[0116] In some embodiments, the sandbox bioreactor has two or more sections that are in series with each other such that cells in the microchannels from one section flow into the microchannels of the next section. In some embodiments, the microchannels from a first section are interconnected with two microchannels within a second section, the second section being in series with the first section. The length of the microchannels of the first section is adjusted such that the cells divide at most once during the transit time from entry to exit of the first microchannel and the two cells resulting from that division are separated and flow separately into the two microchannels of the second section. The two microchannels of the second section may be arranged in a parallel configuration. In some embodiments, the second section microchannels are interconnected in series with the microchannels of the next section in a similar manner. In some embodiments, the cells do not divide during their transit through each microchannel. In other embodiments, the cells may divide greater than or equal to 1, 2, 3, 4, 5 or more times during the time they pass through the length of the microchannel. The sections may be in series of greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sections, each section including one or more microchannels. Wherein the microchannels are interconnected from one section to the next and separate dividing cells when the cells transit from the microchannels of one section to the next, the number of microchannels may increase in multiples of 2 n where n = 1, 2, 3, 4, 5, 6 or greater than 6.

[0117] A sandbox bioreactor can allow testing and measuring the effects of different environmental conditions on cells. Different environmental conditions can include differences in culture medium components, pH, temperature, gas composition, gas exchange, cell density, cell flow rate, and / or liquid culture medium flow rate. In some embodiments, the sandbox bioreactor has an inlet leading to a first section through which a liquid culture medium is provided and flows through the first section into downstream sections connected in series and / or in parallel. The composition and / or flow rate of the culture medium can be changed over time or can be kept constant.

[0118] A sandbox bioreactor can include one or more sensors and / or one or more sample collection devices. The sensors and / or sample collection devices can be used to monitor the response of cells to environmental changes. In some embodiments, one or more of the sensors are in-line sensors. In some embodiments, the sensors are off-line and receive samples of cells, culture medium, or a combination thereof. The sensors can measure biological parameters, physical parameters, and / or chemical parameters. Exemplary parameters include: pH, cell division rate, cell growth rate, cell density, temperature, optical density, gas composition, and / or gas exchange rate. Exemplary parameters also include: fingerprint analysis, single-species discrimination, and quantification of one or more of cell metabolites, proteins, nucleic acids, lipids, small molecules, or biomolecules, or a combination thereof. In some embodiments, the sensors measure cell stress responses and cell metabolites as a response to environmental changes.

[0119] The sandbox reactor can further include one or more controllers. The controllers can control the cell environment in one or more sections of the sandbox bioreactor. In some embodiments, the controllers communicate with and / or receive information from one or more sensors associated with cells, the cell environment, or a combination thereof within one or more sections of the sandbox bioreactor. The controllers can change the cell environment in response to information from one or more sensors. Exemplary changes include liquid culture medium flow rate, liquid culture medium component concentration, pH, temperature, gas concentration, gas content, and cell density.

[0120] In some embodiments, the system includes a production bioreactor module. The production bioreactor provides an environment for the large-scale cultivation and production of cells and / or biological products from cells. The production bioreactor provides a 3-D structure comprising a plurality of micro-modules. The production bioreactor may include greater than or equal to 1, 2, 4, 6, 8, 10 or more micro-modules. The micro-modules create a series of channels and chambers for the growth and movement of cells, and for the flow of liquid culture medium, gas, and biological products. The micro-modules of the production bioreactor may include shapes such as a double helix icosahedron, a modified double helix icosahedron, or any shape that can be described as a triply periodic minimal surface (TPMS). This type of surface forms a lattice system that can grow periodically in all three axes (X, Y, Z). The TPMS can have no self-intersections and divides a given volume into two (or more) separate sub-volumes. Self-intersections can include surfaces where each point defining the surface has a single normal vector. If the surface divides the circumscribing volume in which it lies into two separate and congruent self-volumes, the surface is called a balanced surface. The description of the TPMS is in terms of a fundamental patch or asymmetric unit, and the entire surface can be accumulated from the fundamental patch or asymmetric unit by its symmetry elements. The micro-modules can be fluidly connected to each other (e.g., interconnected) such that gas, culture medium, and / or by-products can flow from one micro-module to another.

[0121] In some embodiments, the micro-module of the production bioreactor can include a double-helix icosahedron or a modified double-helix icosahedron shape. The double-helix icosahedron (DG) can include two icosahedra and can include two mutually-entwined non-overlapping regions. The modified double-helix icosahedron (DG) can include two mutually-entwined non-overlapping regions that can interface with two constant mean curvature (CMC) surfaces separated by a matrix phase. The modified double-helix icosahedron structure can include minor modifications to the junctions of the non-modified double-helix icosahedron to adapt the structure to a given macroscopic structure or function. The modifications can include: blocking the connection or intersection of parts (e.g., 'ports'), modifying the diameter of one or both of the phase channels of the structure, or completely or partially eliminating any phase channels present in the DG structure. The DG or modified DG can include a first icosahedron structure intertwined with a second icosahedron structure. The two channels can be separated by a porous membrane (matrix phase). The matrix phase can allow gas molecules to diffuse in a manner that can be at least partially based on specific pressures and gas compositions. When the microchannel radii of the liquid and gaseous components are equal, the matrix phase surface can be equal to the sum thereof. When multiple DGs are connected (e.g., assembled together), the two CMC surfaces give rise to two continuous channels. These two channels create two non-overlapping channels for the flow of liquid medium and / or gas. The porous membrane can provide a surface on which certain cell types can attach and grow. In some embodiments, one channel supplies liquid medium to the entire production bioreactor. In some embodiments, both channels supply liquid. In some embodiments, to the production bioreactor, one channel supplies liquid medium and the other channel supplies gas. In some embodiments, the diameter of the microchannels of the micro-module can be varied according to the needs of specific cell types, production requirements, and the like. In some embodiments, the micro-module can have a regular cubic wrapping structure with a side length "L". L can be related to the sweeping diameter. In some embodiments, L is equal to two-thirds of the sweeping diameter of the microchannel multiplied by the square root of two multiplied by the square root of three. The total surface and volume of the microchannels corresponding to the liquid component can be equal to the corresponding measures of the gas component, provided that the radii of the two components are the same within the micro-module. In some embodiments, the radii of the components can be different. In some embodiments, when the two radii are equal, the microchannel radius cannot be greater than 0.7 times the sweeping radius. The shortest distance between two micro-modules of two different faces is equal to the square root of two times the sweeping radius minus the sum of the radii of each component channel.

[0122] In some embodiments, the area of the first channel having a DG is equivalent to the area of the second channel within the DG, and wherein the area of the matrix phase is the sum of the area of the first channel and the area of the second channel.

[0123] The distance separating the matrix phase of the channels from the center of each channel is constant. The flow rates of the culture medium and gas flowing through the production module can depend on the selected cell type and cell density, as well as the stress conditions generated on the cells. The rate of gas diffusing through the matrix into the liquid culture medium depends on the gas composition, the pressure of the gas in the gas channels formed by the structure, the membrane thickness, and the materials selected for fabricating the channels and the surrounding area. The gas flow rate and the operating pressure can be related to the cultured cell density. In some embodiments, the gas flow rate can be equal to the volume of gaseous components per minute. In some embodiments, the gas flow rate is greater than or equal to about 2, 3, 5, or 10 times the volume of gaseous components per minute. In some embodiments, the operating pressure can vary from about 1 atmosphere (atm) to 5 atm. In some embodiments, the operating pressure is greater than or equal to 1 atm, 2 atm, 3 atm, 4 atm, 5 atm, or greater.

[0124] One advantage of the DG is reduced gravity, which in other structures can cause uneven exposure to the culture medium and gas exchange. This shape creates three-dimensional (3-D) laminar forces such that variations in the distance of any cell to the structure wall are averaged out, providing more constant and uniform exposure across the cell population. Additionally, the DG shape avoids stagnant regions of liquid or gas where flow cannot occur or may be interrupted. This allows for higher fluxes at lower velocities throughout the bioreactor and results in lower shear stress on the cells. In some embodiments, the average velocity can be greater than or equal to about 1 micron per second, 3 microns per second, 5 microns per second, 10 microns per second, 15 microns per second, 20 microns per second, 50 microns per second, 100 microns per second, 200 microns per second, or greater. The DG structure provides better culture medium and gas diffusion compared to other bioreactor systems. In some embodiments, the velocity of the liquid culture medium flowing through the channels within the DG is greater than the free settling velocity of the cells flowing through the same channels.

[0125] The DG structure provides an increased surface area relative to many shape options, and this increased surface area provides a surface area for cell growth as well as improved liquid culture medium flow, mixing, and gas exchange. When L equals L1, the surface of each component can be expressed as Y = 3258.6.XE(-1), where Y is square millimeters per microliter and X equals the radius bounded by L1.

[0126] The DG micro-modules are assembled together into a macroscopic structure or macroscopic shape, which constitutes a production bioreactor. In some embodiments, the macroscopic structure is a pyramid. In some embodiments, the macroscopic structure is a hollow pyramid, a layered pyramid, a checkerboard arrangement, or a log-like shape. The number of macroscopic structures and micro-modules within the production bioreactor can be adjusted according to the cell division rate of the cells to be cultured, as well as the rate of liquid medium, gas exchange, and cell movement through the bioreactor. Each macroscopic structure can provide different possibilities for interaction with cells, and it can be selected according to the specific process that the production bioreactor is desired to stimulate. Pyramidal and hollow pyramidal macroscopic structures can achieve an environment suitable for growth while maintaining a constant rate and cell density. More sensitive cell strains may require more intervention over time, and in such cases, the hollow pyramid can provide this ability. Layered pyramids can achieve an environment suitable for growth and development by maintaining both the rate and density constant while providing complete access to each cell at all times, enabling direct intervention and treatment. Checkerboard and log-like arrangements can also provide complete access to each cell at each point in the process while allowing control of uniform rate and density. In some embodiments, cells are input at the top of the macroscopic structure and a cell collection device is at the base of the macroscopic structure.

[0127] The arrangement of DG micro-modules into a macroscopic structure provides a mechanism for determining and optimizing the liquid medium and gas flow rates within the bioreactor. In some embodiments, the macroscopic structure includes layers or tiers of micro-modules. In some embodiments, the micro-modules are arranged in tiers or layers, and the rate of the liquid medium is substantially the same in each tier. Alternatively or additionally, the rate of the liquid medium can vary in each tier or layer. For example, the rate of the liquid medium can increase or decrease between tiers or layers. The rate of the liquid medium can vary depending on the different micro-modules, or can be substantially the same between micro-modules. In some embodiments, the production bioreactor further includes a liquid medium input device. The liquid medium device can be configured to supply the liquid medium to the micro-modules of each tier within the macroscopic structure. In some embodiments, the volume of the liquid medium supplied to each tier is such that a substantially constant cell density is maintained in each tier. The microchannel radius can be associated with the radius of the cells, the cell density, or other parameters (e.g., filamentous arrangement, chain-like arrangement, etc.). In some embodiments, the cell density can range from 1x 10 6 cells / ml to 1x 10 12cells / ml. In some embodiments, the rate of the liquid culture medium through each micro-module depends on the cell division rate such that the time required for cells to traverse a single micro-module or a layer of micro-modules is substantially the same as or proportional to the cell division rate, so that the cell division during this transport is greater than or equal to 1, 2, 3, 4, 5 times or more than 5 times. In some embodiments, the first layer has a volume of x of the liquid culture medium, so that at a given number of cells, the density is X, the second layer has a volume of 2x of the liquid culture medium, and during the duration of the transport of cells from the first layer to the second layer, the number of cells doubles (e.g., each cell divides on average once), so that the density in the second layer is still X (i.e., a constant cell density between the layers).

[0128] Additional optimization can be achieved by determining the expected number of cells at the base of the macrostructure, the termination end of the macrostructure (where the cells and / or biological products reach before leaving the structure through the output to the collection container). The expected number of cells can also be determined for different layers of the macrostructure. Based on the expected number of cells at the base and different layers, due to the increase in the number of cells due to cell division, cell movement, and cell accumulation (as they travel through the bioreactor towards the base of the structure), the flow rates of the gas and liquid culture medium can be adjusted for each layer to compensate for the increased demand for gas and liquid culture medium.

[0129] Liquid culture medium supply

[0130] In some embodiments, the system includes one or more components for supplying the liquid culture medium to one or more modules. The components can include one or more of a medium formulator, an electroporator or other sterilization device, a reservoir, a pump, a bubble sensor, and a bubble trap. The medium formulator produces the liquid culture medium for one or more modules by mixing the components of the medium and water to be suitable for cell growth in the modules. The electroporator can be interconnected with the medium generator to clean the medium and provide a sterile starting medium for supply to one or more modules to culture cells. A bubble sensor and a bubble trap can be included to detect and remove any bubbles introduced in the medium generation or cleaning in the liquid culture medium.

[0131] The system may also include one or more reservoirs for containing a reserve culture medium before supplying it to the module. In some embodiments, the system includes at least 2, 3, 4, 6, 8, 10 or more reservoirs. The reservoirs can be filled asynchronously such that one reservoir is being filled while another is already fully filled for supplying the liquid culture medium to one or more modules. Another advantage of separating the reservoirs in this way is to isolate any connection of the cell growth module of the system from the electric current. Any reservoir being filled is exposed to the electric current, which may come from an upstream component such as an electroporator. The coated reservoir is isolated from the electric current so that it cannot conduct the electric current to the downstream components and modules. In some embodiments, the volume of the reservoir can be associated with the flux of the bioreactor in the division time of the cells selected for the process. In some embodiments, multiple reservoirs can be installed in parallel and not coupled to each other. In some embodiments, multiple reservoirs can be installed in series.

[0132] The liquid culture medium supply component may also include one or more sensors. The sensors can measure parameters including the pH and temperature of the culture medium. The sensors can be in-line sensors or can be connected to a sampling device that intermittently samples the culture medium from one or more liquid culture medium supply components. The supply system can provide the liquid culture medium at a range of rates depending on the use, scale, and operation of the system. In some embodiments, the liquid culture medium supplier can provide from about 100 microliters to about 1000 liters per hour to one or more downstream modules. In some embodiments, the liquid culture medium supplier provides from about 0.5 liters to 1000 liters per hour to one or more downstream modules. In some embodiments, the liquid culture medium supplier provides from about 0.5 liters to 5 liters per hour to one or more downstream modules. In some embodiments, the liquid culture medium supplier provides from about 10 liters to 80 liters per hour to one or more downstream modules. In some embodiments, the liquid culture medium supplier provides from about 100 liters to 1000 liters per hour to one or more downstream modules.

[0133] The liquid culture medium supply component may include one or more pumps to enable the culture medium to flow from a reservoir or a culture medium formulator to downstream components such as a cell chip, a sandbox bioreactor, or a production bioreactor. The system may include greater than or equal to 1, 2, 3, 4, 6, 8, 10 or more pumps. The pumps may be of the same type or may be of different types. Exemplary pumps include syringe pumps, peristaltic pumps, and pressure pumps. The liquid culture medium supply system is configured to provide a unidirectional flow up to the downstream components. In some embodiments, the pump is a syringe pump, which is used to supply the culture medium to the cell chip. In some embodiments, the pump is a syringe pump, which is used to supply the culture medium to the sandbox bioreactor. In some embodiments, the pump is a peristaltic pump, which is used to supply the culture medium to the production bioreactor. In some embodiments, the system includes three pumps, where two syringe pumps supply the cell chip and the sandbox reactor, and one peristaltic pump supplies the production bioreactor. The pumps may work synchronously or individually. In some embodiments, all three pumps work synchronously. One or more pumps supply the culture medium to the downstream module with high precision in terms of volume and flow rate. In some embodiments, the precision is within 1, 2, 3, 4 or 5 nanoliters.

[0134] Gas supply and composition

[0135] The systems herein can be compatible with cells that require specific gas components, such as cells that require oxygen to grow and survive. The materials used to construct the bioreactor module may include glass, acrylic, collagen, polydimethylsiloxane (PDMS), poly(ethylene glycol) (PEGDA), Poly(D,L-lactide), and other biocompatible polymers that allow oxygen supply to the culture medium. In some embodiments, the system includes a controller that controls the diffusion of oxygen and other gas solutions in one or more modules. The gas solutions are formulated from pure gas components (such as from gas storage tanks or other supply agencies) to determine the mixture or pure gas solution at various concentrations and flow rates. Alternatively or additionally, the gas mixture may be provided by a purified air mixture. The gas solutions can be used to provide a ventilation environment and control the pH, as well as to provide carbon, nitrogen, phosphorus, and sulfur to the liquid phase. In some embodiments, the system has more than one gas controller or mechanism, so that different gas solutions can be provided to different modules within the system.

[0136] Sensors and environmental monitoring

[0137] In some embodiments, one or more bioreactor modules are interconnected with one or more sensors that monitor one or more characteristics of the environment. The sensors can be placed at specific points within the module or system. Measurements are taken over a specific time range and at specific time points in order for the sensors to monitor a specific group of cells. In some embodiments, one or more sensors track the characteristics of a specific group of cells as they move through the bioreactor module. The sensors can trigger specific processes in response to measurements of optimized conditions for a specific group of cells. Exemplary measured and optimized characteristics include: pH, diluted oxygen, total gas composition, dilution of other gases, temperature, cell density, sugar analysis, transcriptomics and targeted transcript monitoring, metabolomics, and proteomics. In some embodiments, the characteristics are measured at at least 1, 2, 3, 4, 6, 8, 10 or more time points. In some embodiments, the characteristics are measured for a specific group of cells and can be time-controlled to monitor the group as it moves through the modules of the bioreactor and between modules.

[0138] In some embodiments, at least one sensor measures parameters of cells, such as biological, physical, or chemical parameters, as the cells are within, traveling through, entering, and / or leaving the bioreactor module. In some embodiments, at least one sensor measures parameters of the cellular environment within the bioreactor module, such as biological, physical, or chemical parameters. Exemplary biological parameters include: cell division rate, cell growth rate, cell stress response, cell protein content, cell carbohydrate content, cell lipid content, and cell nucleic acid content. Exemplary physical parameters include: cell size, cell density, cell flow rate, liquid media flow rate, mixing rate, turbidity, temperature, and pressure. Exemplary chemical parameters include: pH, liquid media composition, concentration of individual liquid media components, gas composition, gas concentration, and dissolved gas composition.

[0139] In some embodiments, the system or module communicates with a camera device. The camera monitors the output of at least one bioreactor module. The camera can capture biochemical, physical, or chemical characteristics of the bioreactor output. In some embodiments, the camera device captures information such as cell count, cell velocity, and cell density. In some embodiments, the camera is a hyperspectral camera that captures information over the entire wavelength range. In some embodiments, the camera device captures information from the output of two or more bioreactor modules.

[0140] Method of use

[0141] In some embodiments herein, one or more modules are used to produce cells. Figure 8An exemplary method for producing cells using one or more modules is shown. Cells can be collected through the output channels of the modules. In some embodiments, the cells are collected within the module and stored in the module. For example, the cells are stored within a cell chip module. In some embodiments, one or more modules are used to produce biological products from cells, such as small molecules, proteins, antibodies, metabolites, or other products produced by the cells grown in the module. The biological products can be collected through the output channels of the module and separated from the growing cells, for example, by diffusion through a porous membrane or by filtration. In some embodiments, the biological product is inside the cell. To harvest the biological product, the cells are collected, lysed, and then the biological product can be further purified. In some embodiments, the biological product is secreted by the cells and can be collected without harvesting or lysing the cells.

[0142] The bioreactor modules and systems herein can be used to produce specific cell types and have the flexibility to culture various cell types. In some embodiments, a cell chip module is used to provide a specific type of cell to a sandbox bioreactor, a production bioreactor, or a sandbox module in series with a production module. The cell chip module allows for customized cell production and environmental optimization.

[0143] The cell chip can be adapted to produce (including autologous production and allogeneic production) stem cells and other types of cell therapies. The cell chip can be adapted to produce (including autologous production and allogeneic production) stem cells and other types of cell therapies. In some embodiments, it can perform expansion, gene delivery, or activation of T-cells for personalized chimeric antigen receptor T-cell (CAR-T) therapy. In some embodiments, the stem cells can be undifferentiated, cultured, and / or differentiated.

[0144] In some embodiments, the cells cultured in the system are prokaryotic cells, such as bacterial cells. In some embodiments, the cultured cells are eukaryotic cells, such as yeast cells, fungal cells, algal cells, plant cells, avian cells, or mammalian cells. The cells can float freely in the culture medium or can be adherent cells that attach to one or more surfaces within the bioreactor. The cells can be transformed or otherwise engineered to produce biological products such as heterologous proteins, small molecules, or metabolites.

[0145] In some embodiments, the systems, devices, and methods described herein can be used under zero-gravity or microgravity conditions to culture cells under zero-gravity or microgravity conditions.

[0146] Method for constructing a bioreactor module

[0147] The systems, components, and modules herein can be fabricated from a variety of materials and these materials can be tailored according to cell growth and the cell environment used. In some embodiments, the components and modules or parts thereof are fabricated by 3-D printing. The printing can employ commercially available resins and ultraviolet (UV) curable biocompatible polymers. In some embodiments, each micro-module shape is designed independently in a virtual environment. In some embodiments, components and modules assembled from commercially available components are combined and arranged together as described herein. In some embodiments, the biomaterials used can include a combination of three sub-components (biocompatible polymer, photoinitiator, and UV absorber).

[0148] The devices and systems of the present disclosure can be formed by 3-D printing, such as stereolithography. In some embodiments, a computer-aided manufacturing (CAM) or computer-aided design (CAD) model of the device of the present disclosure is provided to a 3-D printing system that employs stereolithography, including providing a container with a resin that contains a photoinitiator and one or more polymer precursors. An ultraviolet (UV) laser can be used to engrave a pre-programmed design or structure onto the surface of the container with the resin. The resin can be a photosensitive polymer that photochemically cures upon contact with the UV laser to form a single layer. Additional resin can be added and cured during the layer-by-layer production process. Stereolithography can be used to construct modules in top-down or bottom-up additive manufacturing methods.

[0149] Alternative methods for constructing reactor modules can include self-assembly of polymers, such as block copolymers, to form 3-D helical icosahedral structures or subtractive manufacturing methods. Subtractive manufacturing methods can include chemically or mechanically removing sacrificial materials. For example, the sacrificial material can be formed using a sintering laser for binder jetting. The sacrificial material can be immersed, impregnated, or otherwise coated with a biocompatible polymer. The sacrificial material can then be dissolved or mechanically removed to form a 3-D helical icosahedral structure from the biocompatible polymer.

[0150] Customized bioreactors and systems

[0151] The bioreactor modules and systems herein can be designed and tailored for specific cell types and specific bioproduct outputs. Specifically, production bioreactors can be designed for specific and personalized uses. The volume of the micro-modules, the channels within each micro-module, the number of micro-module tiers, and the macroscopic structure can be selected to provide a cellular environment (e.g., growth conditions) to achieve a given cell density, cell division rate, liquid shear force, to accommodate the selection of liquid medium, liquid for harvesting bioproducts (e.g., solvents for separating and / or fractionating cell components), liquid flow rate, gas composition, and gas flow rate.

[0152] In some embodiments, the system includes means for optimizing the bioreactor module and the cellular environment with one or more cells. The optimization means can include a data loop that receives information about the cellular environment (E) and cell performance (B) from one or more sensors and cameras within the system. Additionally, for cells input into the system, the genetic information (G) of the cell type can be input into the optimization means to provide relationships such as G*E = B.

[0153] In some embodiments, the cellular environment varies in one or more parameters (e.g., pH, liquid medium components, gas composition, flow rate), and information about the cellular environment is collected for each variation (E1 - E n ) and the resulting cell performance (B1 - B n ) under each environmental variation. In some embodiments, the input cells vary (e.g., different genetics, e.g., cells that differ in division rate, nutrient consumption, morphology, stress tolerance), and information about each cell type is collected for each cell variation (G1 - G n ) and the resulting cell performance (B1 - B n ) in the environment within the bioreactor. In some embodiments, variations are made to the cellular environment and cell genetics, and performance is measured such that for more each cell variation (G1 - G n ), the optimization means measures the performance (B1 - B n ) in more than one cellular environment (E1 - E n ). The optimization means rates, indicates, or otherwise compares the G*E = B relationship across the entire cell genetics and cellular environment, thereby providing information about the compatibility, optimization, and rating of different cell genetics in various tested cellular environments. The optimization means can thereby optimize production based on cell genetics, cellular environment, or both. The optimization means can thereby provide parameters to optimize the production bioreactor, which includes the design of customized bioreactors and customized cellular environment conditions for the cell type and production output.

[0154] In some embodiments, the optimization mechanism is a computer-controlled device. In some embodiments, the optimization mechanism includes a machine learning algorithm that uses data from one change or a set of changes to create the next change or the next set of changes for testing. In some embodiments, the systems, devices, and methods described herein can be used under zero-gravity or microgravity conditions to culture cells under zero-gravity or microgravity conditions.

[0155] Exemplary bioreactor modules and systems

[0156] The bioreactor modules provided herein can be used individually or in combination to construct systems for the production, optimization, and (in some cases) storage of cells. In some embodiments, the systems, devices, and methods described herein can be used under zero-gravity or microgravity conditions to culture cells under zero-gravity or microgravity conditions. An exemplary embodiment of the 3-module system is shown in Figure 1 . In this exemplary embodiment, three bioreactors are interconnected with the system: the cell chip 200, the sandbox bioreactor 400, and the production bioreactor 800. The liquid medium is mixed in the medium formulator 101 and transferred to the electroporator 20 for cleaning. The electroporator may include a cooler 21 to bring the temperature of the liquid medium to the operating temperature of the system. The pump 40 moves the liquid medium from the formulator 101 to the electroporator 20. The pump 50 causes the medium to flow through the bubble trap 60 to remove the bubbles generated by the electroporation process, and the bubble sensor 70 monitors the flowing medium to achieve an appropriate bubble-free state. The medium then reaches the reservoir 30. In some embodiments, the reservoir 30 consists of two or more reservoirs such that while the filled first reservoir 31 supplies the liquid medium to the bioreactor module, the second reservoir 32 is being filled. When using pumps 100, 110, and 120 respectively, each bioreactor module 200, 400, and 800 receives the liquid medium from the reservoir. Using the gas supplier 500, gas is supplied to the bioreactor, and the gas supplier may include one or more gas storage devices for each gas component. The gas supplier may include control devices to mix and regulate the gas composition and gas flow rate flowing to the bioreactor, including different mixtures and different flow rates flowing to each bioreactor module respectively.

[0157] The system and bioreactor may also include certain output containers and devices. Each bioreactor including an input and flowing liquid culture medium may also include fluid handling means. A common liquid handler 600 may be used to collect the spent liquid culture medium from all bioreactor modules. Similarly, for each bioreactor including an input and flowing gas, a gas handler may collect the spent and cell-excreted gas components. A common gas handler 550 may be used to collect the output gas from all bioreactor modules.

[0158] One or more sensors may be included, which are used with the bioreactor or in a system for multiple bioreactors, to monitor one or more parameters, including physical parameters, biological parameters, chemical parameters, and combinations thereof. Figure 1 Exemplary sensors shown include sensor 80 (for monitoring the media preparator), sensor 81 (for monitoring the electroporator), and sensor 82 (for monitoring one or more reservoirs). Additional sensors 83 monitor the output from the cell chip module. Sensors 84 and 85 monitor the liquid and gas outputs from the sandbox module, respectively. Similarly, sensors 86 and 87 monitor the liquid and gas outputs from the production bioreactor module, respectively.

[0159] A system of one or more bioreactors may further include interconnections between the bioreactor modules. As Figure 1 shown in the exemplary system, the cell chip is interconnected with the sandbox module via connector 280 and can be adjusted to allow cells to move from the output end of the cell chip into the input end of the sandbox module. Connector 380 allows cells to flow from the output end of the sandbox module to the input end of the production bioreactor module. Connector 480 is the interconnection between the output end of the production bioreactor and the collection container or device collector 700. Collector 700 may collect cells, biological products, or combinations thereof from the production bioreactor 800. In some embodiments, collector 700 may include filters, membranes, or other units and / or modules for separation, such as separating cells from the liquid culture medium, separating biological products from cells, and separating between cell components.

[0160] Figure 2A An exemplary cell environment 205 in chip module 201 is shown. In this embodiment, the cell chip includes: a gated trap 220, a plurality of suction traps 240, and a plurality of overflow traps 260. At the input end is a media input port 202 for supplying media to the cell environment. At the opposite end (in the direction of liquid media flow) is a harvest port 210 for harvesting the cells cultured in the cell chip module. Figure 2BDetails of an exemplary culture medium circuit 230 of the cell chip 201 are shown. The culture medium circuit may include an input culture medium port 231, an output culture medium port 233, and a culture medium feed channel 235. Figure 2B Details of an exemplary gas circuit 250 of the cell chip 201 are shown. The gas circuit may include an input gas circuit 251, an output gas circuit 253, and a gas distribution channel 255. Figure 2C A side view of the cell chip 201, and an example of the arrangement of the culture medium circuit 230, the gas circuit 250, and the cell environment 205 are shown.

[0161] Figure 3A-3F An exemplary trap used with the cell chip module is shown. The exemplary gate trap 221 is illustrated in Figure 3A (top view) and 3B (side view), and is shown near the culture medium input port 202. The gate trap is constructed by a flow rate protector 225 surrounding the internal space, which has an opening 227 therebetween that restricts the outflow of cells 203 from the internal space of the trap. In some embodiments, the opening 227 between the protectors 225 is sized to be twice the diameter of the cells grown on the cell chip. In some embodiments, the opening 227 is sized to be greater than the diameter of the cells grown on the cell chip. Cells can be inoculated onto the chip and into the gate trap 221 using the inoculation port 223.

[0162] Figure 3C and 3D From a top view ( Figure 3C ) and a side view ( Figure 3D ) an exemplary overflow trap 240 arrangement is shown. The trap is constructed by an arrangement of perforated box walls 245 that are arranged to surround the internal space on three sides and are arranged to create an opening 247 between the wall partitions 246. The size of the opening 247 can be adjusted according to the cells grown on the chip. In some embodiments, the diameter of the opening 247 is smaller than that of live cells but larger than that of dead cells, such that live cells 203 are still trapped in the internal space of the perforated box walls 245 until they reach a sufficient number to overflow the box arrangement, while dead cells pass through the opening 247 and are not retained in the internal space of the perforated box walls 245.

[0163] An exemplary suction trap 260 is shown in Figure 3E and 3F in the top view and side view. The trap has a porous domain 262 and an environmental wall 264. The porous domain is designed such that the size of the cells exceeds the size of the pores.

[0164] Figure 4A-4E An exemplary sandbox module and its components are shown. Figure 4AShows exemplary sandbox section 405. This exemplary section includes channel 445, in which cells and the associated cellular environment flow into mixing module 420, in which liquid medium from channel 443 is mixed with the cells, and then the mixture flows into cell chamber 410 for cell growth and optionally for measurement by one or more sensors or cameras. Figure 4B Shows exemplary sandbox module cellular environment 401, which includes multiple sections in series and parallel, cell input 440 and cell output 460 from the sandbox module. Each exemplary section includes a mixing module (e.g., 421, 422, 423) and a cell chamber (e.g., 411, 412, 413). Figure 4C Shows an example of gas circuit 470 (top) such as sandbox cellular environment 401 (bottom). Figure 4D Shows an example of liquid medium circuit 480 with a medium input (top), and an example of cell input channel 490 (bottom). Figure 4E Shows an example of liquid medium circuit 480, gas circuit 470, and cell input channel 490 superimposed on exemplary sandbox cellular environment 401 to construct exemplary sandbox module 400, shown in a top view and a side view.

[0165] Figure 5 Shows exemplary production bioreactor 800, which includes inoculation port 802 for cells to enter the bioreactor, and cell distribution structure 804, in which cells flow into the bioreactor and are mixed with liquid medium and other cellular environment components. Liquid medium input 820 allows liquid medium to be introduced into the production bioreactor, and the liquid medium is then distributed through medium feed system 822 by using a medium connection (e.g., 824), and the unused medium is collected 826. Gas enters at gas input end 810 and is distributed throughout the bioreactor through gas phase channel 814, and is output through gas output end 818. Cells and / or products produced by the cells can be harvested through port 870.

[0166] Figure 6A-6C Shows an example of a macroscopic structure used with the production bioreactor described herein. Figure 6A Shows exemplary pyramid macroscopic structure 881 and pyramid macroscopic structure 883 with a superimposed medium distribution system. Figure 6B Shows exemplary hollow pyramid macroscopic structures 885 and 887. Figure 6A Shows exemplary log macroscopic structure 889.

[0167] In some embodiments, the macroscopic structure of the production bioreactor is composed of micro-modules, each of which can have a given shape, such as a double-helix icosahedron or a modified double-helix icosahedron. Figure 7A An exemplary embodiment of a modified double-helix icosahedron shape 841 for constructing one or more micro-modules for a production bioreactor is shown. As shown at the bottom of this figure, the double-helix icosahedron micro-module is schematically illustrated as box 841A, as Figure 6A-6C shown in the macroscopic structure of. The modified double-helix icosahedron shape 841 can include phases 842 and 844, which, when assembled, as Figure 7B shown in, can be assembled into stacked channels 843 and 845. In addition, there is a membrane gap 846 between phases 842 and 844. Figure 7C An exemplary arc of a portion of an exemplary channel 842 is shown, which shows a constant diameter throughout the channel.

[0168] Micro-modules assembled into a macroscopic structure

[0169] The micro-modules described herein can be assembled into a macroscopic structure to provide targeted control of the medium and gas flow rates and distributions. In some embodiments, the micro-module is a modified double-helix icosahedron (DG), which is assembled into a macroscopic structure to produce a production bioreactor. Figure 9A-9F An assembly starting from a first micro-module (e.g., DG) and assembling additional micro-modules is shown, such that the geometry repeats to form a three-dimensional (3-D) matrix, the growth of which is restricted to two of the three possible dimensions. The connection point of one micro-module to another is called a "port". This first assembly of interconnected micro-modules with the same orientation is called a "layer". The layer can be arranged, for example, in a long rhombus, such that in some embodiments, if the same number of modules are connected in a selected direction, the resulting growth is disproportionate, and thus, the growth of the layers is irregular relative to each other. In some embodiments, the layer is arranged in a square, such that the resulting growth is proportional. Figure 9D-9F An exemplary embodiment of layer assembly and growth is illustrated.

[0170] At the edges of each layer, the unconnected ports of the micro-modules can be used to connect the layer to other functional components, such as for the input of the medium flow or for the input of gas, as well as the output of the used medium, the output of the used gas, and the output of cells or biological products produced by the cells.

[0171] In some embodiments, an assembly of layers of a micro-module (“first matrix”) can be juxtaposed with a second assembly of layers of the micro-module (“second matrix”), whereby the second matrix occupies the free space remaining from the first matrix, and whereby the matrices occupying the same volume have no points of contact and maintain a constant minimum distance. Exemplary assemblies of the two matrices are shown in Figure 10A-10F In. Figure 10A An example of a portion of a double helix icosahedron inscribed in a cube is shown. Figure 10B Shows Figure 10A Orthogonal and cross-sectional views of the structure of. Figure 10C And 10D Shows an example of the direction of growth of a second layer relative to a first layer. Figure 10E Shows an example of a volume subtracted from a pyramid and growing counterclockwise. Figure 10F Shows an example of the macroscopic structure growing clockwise along the vertical axis of a hollow pyramid.

[0172] In some embodiments, the micro-modules are assembled into a hollow pyramid macroscopic structure. The hollow pyramid has a hollow center and a volume with an increasing cross-section. With the hollow pyramid macroscopic structure, the feed circuit can serve as both the outer and inner perimeters. To construct the hollow pyramid, the matrix has an initial layer connected to a dispenser and subsequent layers connected to a collector. Starting from the lower orifice of the layer connected to the collector, the number of upper orifices of the initial layer can belong to the set M = 2 n . In this way, it is ensured that the connecting channels or tree can branch in pairs in a balanced manner. The tree can be the distribution (input) structure and collection (output) of a bubble-free bioreactor. In both the input and output, the channels can transition or branch from a channel to 2 n channels. The increment in volume between layers (which is the number of micro-modules added between one layer and the next layer in the flow direction) depends on the bioreactor and is organized according to: (i) the manner of alternating growth between the edges of its outer perimeter; and (ii) the increase in its inner perimeter (i.e., the perimeter of the internal hollow center). For example, if N is the number of modules in one of the edges of the outer perimeter of the hollow pyramid and n is the number of micro-modules contained in one of the edges of the inner perimeter of the hollow pyramid, then if N = (8; 8) at one layer, then n = (4; 4) (see Figure 12 ). This logic is repeated alternately between the outer edges of the pyramid in a clockwise manner (considering the flow direction) in each layer. The result is a stepped pyramid, where its steps form a faceted spiral icosahedron. The inner perimeter also has a spiral icosahedron growth, but with a lower frequency than the outer perimeter, and the growth direction of the inner perimeter is opposite to the growth direction of the outer perimeter (see Figure 13A and 13B)。The interaction between the inner and outer spirals and the flow direction result in a vortex-type movement of the culture medium flowing within the hollow pyramid structure.

[0173] In some embodiments, the feed system is connected to the bioreactor through one or more sub-channels. The sub-channels of the feed loop can be equidistantly located around the perimeter of one or more layers of the bioreactor on each side of a given layer. The sub-channels can be connected at one or more ports of the micro-module as the edges of the layer. Exemplary connection groups are shown in Figure 11A –11F.

[0174] In some embodiments, the feed loop is connected to the bioreactor at 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 points. In one embodiment, the feed loop serves as a hollow pyramid macroscopic structure bioreactor and the feed loop has a portion with 5 sub-channels. One of these sub-channels extends to the inner side of the inner channel of the pyramid, and the remaining feed sub-channels are parallel to the edges of each layer (outer channels). The pressure and flow rate balance of the feed loop is maintained by the ratio of the outer and inner channels of the feed system. An exemplary feed loop in the shape of a hollow pyramid is shown in Figure 14 Figure.

[0175] In some embodiments, the macroscopic structure of the bioreactor is layered. In some embodiments, the feed system is connected to the bioreactor through one or more sub-channels. The sub-channels of the feed loop can be equidistantly located around the perimeter of one or more layers of the bioreactor on each side of a given layer. The sub-channels can be connected at one or more ports of the micro-module as the edges of the layer. Exemplary connection groups are shown in Figure 11A –11F.

[0176] In some embodiments, the bioreactor employs a layered macroscopic structure composed of micro-modules such as DG. The layered macroscopic structure has sheets with a constant thickness and an increasing cross-section including the micro-modules. The constant thickness of the sheets allows for uniform access to substances from the feed loop. The increment in volume between layers (which is the number of modules added between one layer and the next layer in the flow direction) depends on the bioreactor and is organized according to an alternating pattern of growth between the shortest edges of the sheets (see, for example, Figure 15 ). In the layered macroscopic structure, there can be one or more than one sheet arranged in parallel, such as 2, 3, 4, 5, 6, 7, 8 or more than 8 sheets. The space between each sheet can be used to accommodate the feed loop or the portion of it that feeds the modules in the sheet (see, for example, Figure 16 ).

[0177] In some embodiments, the micro - modules are assembled into a tree - checkerboard macrostructure having at least one hollow column with a constant cross - section that longitudinally traverses the micro - module layer. In some embodiments, the tree - checkerboard macrostructure has 1, 2, 3, 4, or more than 4 such columns. The column can be used to provide areas for transporting liquid culture media and other substances through channels along the longitudinal column. Collection of used media, gases, cells, and biological products can occur on one or more or all of the external faces of the structure, driven by the pressure difference between the center of the column and the face. An exemplary tree - checkerboard macrostructure is shown in Figure 17 and an exemplary feeding and collection arrangement is shown in Figure 18 .

[0178] Bioreactor connection system

[0179] The modules can be connected, coupled, or in fluid communication through one or more connection systems. Figure 19A and 19B show exemplary connection systems that include connectors between the cell - chip module and a fluid source or fluid collection module. The connector includes a support and a set of hollow needles that allow the entry and exit of fluids and / or fluids containing cells. In some embodiments, the connector is connected to a first module, such as a cell - chip module, through the needles. The needles can be arranged in groups such that each group of needles can include a needle for inputting fluid and another needle for outputting fluid from the cell - chip module. One end of the needle is for entering a chamber or channel of the cell - chip module, and the other end of the needle can be connected to a fluid source, a collection device, or another module.

[0180] In some embodiments, the set of needles includes at least one input needle and one output needle. In some embodiments, there are multiple sets of needles. Each set of needles can be oriented to a separate chamber and / or separate channel to direct the input of fluid thereto or the removal of fluid therefrom.

[0181] In some embodiments, the connector can connect the cell - chip module to one or more fluid sources (e.g., culture media, nutritional supplements, chemical inputs, trypsin, wash / buffer solutions) that can be used to supply fluid to the cell - chip module and optionally remove used fluid. In some embodiments, the connector can connect the cell - chip module to a second module, such as a sandbox bioreactor or a production bioreactor, such that, for example, cells can be transferred from one module to another.

[0182] In one embodiment, the connection system includes a clean room so that the needles are cleaned and / or sterilized before entering a module such as a cell chip module. In one embodiment, the clean room is one or more separate rooms at one end of the cell chip module. The clean room(s) abuts a partition at the first end, which isolates the clean room from the environment, and the needles can penetrate the partition from one end to enter the clean room. The clean room can abut a safety membrane or other boundary at the second end, which can confine the cleaning or sterilizing fluid (or gas) within the clean room. In such an embodiment, the connector is connected to a fluid source (e.g., containing a cleaning agent or disinfectant and a washing solution(s)) at the other end of the needle.

[0183] On the other side of the safety membrane or boundary is a channel. Once cleaned and sterilized, the needles can penetrate the safety membrane or boundary to enter the channel. The channel can be a culture medium channel that allows the culture medium to flow from the needles to other locations in the cell chip. The channel can be a cell harvest channel, and the cells present in the chip (e.g., cells grown and proliferated in the chip) can be directed to the channel and then through the needles to a separate module or harvest component. The channel can be a waste channel through which the used culture medium can be directed and removed from the chip.

[0184] Figure 19C Exemplary embodiments of the connection of components including a culture medium, a disinfectant, and a waste collection and sandbox module by a connector system are shown. Connecting tubes or channels are connected from the connector system and valves are used to direct the fluid from the connector to the appropriate source, collector, or module.

[0185] Figure 19D Exemplary embodiments of the connection system are shown where a cell chip module's first chamber is pierced with needles for, e.g., cleaning and sterilizing. Exemplary embodiments of the connection system during the cleaning process are illustrated, causing fluid to flow from a component containing a disinfecting fluid to a disinfecting chamber of the cell chip and causing one of each set of needles to remove the used disinfecting fluid.

[0186] Figure 19E Exemplary embodiments of the connection system are shown where the needles pierce into the second chamber after cleaning / sterilization. The first set of needles (left side) is positioned so that the input needles enter the culture medium channel / chamber and allow fresh culture medium to flow into the cell chip module. The middle set of needles is positioned so that one needle is positioned to output used culture medium and culture medium waste from the channel in the cell chip module. The third set of needles (right side) is positioned so that only the output needles enter the chamber / channel and are positioned to output culture medium and cells from the cell chip module.

[0187] Module for adherent cell culture

[0188] In another aspect, the present disclosure provides a method of culturing cells. The method can include providing a plurality of cells to an adherent bioreactor. The adherent bioreactor can include at least one channel and a microporous membrane. The adherent bioreactor can be part of a cell chip module or a sandbox bioreactor. At least a portion of the cells can be allowed to attach to the surface of at least one channel such that at least the portion of the plurality of cells grow and / or replicate on the surface of the channel to produce adherent cells. A liquid culture medium can flow from at least one channel across the microporous membrane. The flowing of the liquid culture medium from the channel across the microporous membrane can wash the adherent cells (e.g., with fresh culture medium or a sterile wash buffer), or can be used to detach the cells from the surface of the channel, or can wash the detached cells. The detached cells (e.g., suspended cells) can be collected by flowing another liquid culture medium containing the cells through the channel. The adherent bioreactor can be fluidly coupled to a cell chip module, a sandbox bioreactor, a bioreactor (e.g., a production bioreactor), or any combination thereof. The cell chip module can provide cells to the adherent bioreactor. The adherent bioreactor can provide cells to a bioreactor (e.g., a production bioreactor).

[0189] Cells can be cultured or grown under controlled conditions on a surface or an artificial substrate. For example, cells can be cultured in a monolayer or other surface growth configuration. Figure 20 An exemplary layered module for adherent cell culture is shown. Figure 21A An embodiment of a module suitable for adherent cell culture is shown. Cells can attach to a material suitable for cell attachment such as PDMS. At a selected time point or a selected cell density, the adherent cells can be removed from the adherent material and released into the surrounding culture medium, for example, by adding an enzyme such as trypsin to the liquid flowing over the adherent (e.g., attached) cells, such that the cells or portions thereof can be harvested from the module.

[0190] In some embodiments, the adherent material abuts one or more open channels such that the culture medium flows over the adherent cells. The module can also include a boundary such as a microporous membrane that allows the culture medium or other fluid to pass through, but the cells cannot pass through the microporous membrane due to the size of the micropores.

[0191] In some embodiments, the module for adherent cells can have one or more chambers. Exemplary embodiments are shown in Figure 20 which has an inoculation chamber for inputting cells into the module, a filtration chamber, and an attachment chamber. The attachment chamber can have an adherent material such that cells attach to their surface.

[0192] Cells can be removed from an adherent location by, for example, including trypsin in a flowing culture medium or other fluid or incubating with adherent cells. Once the cells (e.g., from a PDMS material) are detached, the trypsin can be removed by washing with a culture medium or other fluid, and then this culture medium or fluid is passed through a microporous membrane, leaving the cells. Such cells can then be resuspended in fresh culture medium or other liquid, and the cells or portions thereof can be moved to a harvest chamber or other output channel for collection by utilizing the fluid flowing through the channels.

[0193] Figure 21B-21E An exemplary sequence showing cell attachment, trypsin detachment of cells, and washing and harvesting is shown. In some embodiments, the adherent cell module is a cell chip module or a portion thereof. In some embodiments, the adherent cell module is a sandbox module or a portion thereof. In some embodiments, the adherent cell module is a part of a sandbox module or a portion thereof, such as one or more micro-modules.

[0194] Computer system

[0195] The present disclosure provides computer systems programmed to implement the methods of the present disclosure. Figure 34 A computer system 3401 is shown that is programmed or otherwise configured to control all internal processes of the system according to a program, such as culture medium formulation and sterilization, flow rate control, gas flow rate, pressure and formulation, obtaining data through embedded sensors (e.g., physical, chemical, and biological data), sensor data fusion and command control loops, image processing, and creating data sets associated with each process. The computer system 3401 can regulate various aspects of the microenvironment conditions that cells experience within the systems of the present disclosure, such as, for example, culture medium flow rate and sub-component concentration, mixing time, reservoir filling; pulse number, pulse voltage, duty cycle applied to the culture medium by an electroporator, gas flow rate and pressure, differential gas solution formulation, differential gas flow rate of some or all components, differential culture medium flow rate of some or all components; differential pH value of some or all components; differential dissolved gas of some or all components, differential sugar analysis of some or all components, differential temperature of some or all components, sampling time of some or all components, analysis of physical, chemical, biological parameters of some or all components such as temperature, pH, amount and type of dissolved gas, flow rate, cell density, sugar analysis, and biochemical analysis such as proteomics, metabolomics, transcriptomics, and the like. The computer system 3401 can be an electronic device of a user or a computer system (remote with respect to the electronic device). The electronic device can be a mobile electronic device.

[0196] The computer system 3401 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 3405, which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 3401 also includes: a memory or storage location 3410 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 3415 (e.g., hard disk), a communication interface 3420 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 3425 such as cache memory, other memories, data storage, and / or an electronic display adapter. The memory 3410, storage unit 3415, interface 3420, and peripheral devices 3425 communicate with the CPU 3405 via a communication bus (solid line), such as a motherboard. The storage unit 3415 can be a data storage unit (or data repository) for storing data. The computer system 3401 can be operably coupled to a computer network ("network") 3430 by means of the communication interface 3420. The network 3430 can be the Internet, an intranet, and / or an extranet, or an intranet and / or an extranet connected to the Internet. The network 3430 is in some cases a telecommunications and / or data network. The network 3430 can include one or more computer servers, which can implement distributed computing such as cloud computing. The network 3430 can, in some cases, implement a peer-to-peer network by means of the computer system 3401, which can enable devices coupled to the computer system 3401 to act as clients or servers.

[0197] The CPU 3405 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location, such as the memory 3410. The instructions can be directed to the CPU 3405, which can then program or otherwise configure the CPU 3405 to implement the methods of the present disclosure. Examples of operations performed by the CPU 3405 can include fetching, decoding, executing, and writing back.

[0198] The CPU 3405 can be part of a circuit, such as an integrated circuit. One or more other components of the system 3401 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0199] The storage unit 3415 can store files such as drives, libraries, and saved programs. The storage unit 3415 can store user data, e.g., user preferences and user programs. The computer system 3401 can, in some cases, include one or more additional data storage units, which are external to the computer system 3401, e.g., located on a remote server and communicating with the computer system 3401 via an intranet or the Internet.

[0200] The computer system 3401 can communicate with one or more remote computer systems via a network 3430. For example, the computer system 3401 can communicate with a user's remote computer system (e.g., virtual private network, computer hosting service such as Amazon Web Services (AWS), satellite communication). Examples of remote computer systems include personal computers (e.g., portable PCs), touchscreen tablets or tablet PCs (e.g., iPad, Galaxy Tab), telephones, smartphones (e.g., iPhone, Android-enabled devices, ) or personal digital assistants. The user can access the computer system 3401 via the network 3430.

[0201] The methods described herein can be implemented by machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 3401 (e.g., on the memory 3410 or the electronic storage unit 3415). The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 3405. In some cases, the code can be retrieved from the storage unit 3415 and stored on the memory 3410 for access by the processor 3405. In some cases, the electronic storage unit 3415 can be excluded and the machine executable instructions are stored on the memory 3410.

[0202] The code can be pre-compiled and configured to be used with a machine having a processor adapted to execute the code, or can be compiled during run-time. The code can be provided in a programming language that can be selected to enable the code to be executed in a pre-compiled or just-in-time compiled manner.

[0203] Aspects of the systems and methods provided herein, such as computer system 3401, may be embodied in programming. Various aspects of the technology may be considered a "product" or "article of manufacture" in the form of machine (or processor) executable code and / or associated data that is loaded or embodied in a type of machine-readable medium. The machine executable code may be stored in an electronic storage unit such as a memory (e.g., random access memory, read only memory, flash memory) or a hard disk. A "storage" type medium may include any one or all of the tangible memory of a computer, a processor, or the like, or associated modules (e.g., various semiconductor memories, tape drives, disk drives, etc.), which may provide non-transitory storage for a software program at any time. All or part of such software may sometimes be communicated through the Internet or various other telecommunications networks. Such communication may, for example, enable software to be loaded from one computer or processor to another, e.g., from a management server or host computer to an application server computer platform. Thus, another type of medium that may carry software elements includes light waves, radio waves, and electromagnetic waves, such as used via a physical interface between local devices, via wired and optical fiber fixed networks, and via various air-links. Physical elements carrying such waves, such as wired or wireless links, optical links, or the like, may also be considered a medium carrying software. Unless restricted to non-transitory, tangible "storage" media, the term computer or machine "readable medium" as used herein refers to any medium that participates in providing instructions to a processor for execution.

[0204] Thus, machine-readable media such as computer-executable code can take many forms, including but not limited to, tangible storage media, carrier media, or physical transmission media. Non-volatile storage media includes, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, which can be used to implement databases, etc., as shown in the figures. Volatile storage media includes dynamic memory, such as the main memory of such a computer platform. Tangible transmission media includes coaxial cables; copper wire and fiber optics, including the wires within a computer system (including the bus). Carrier transmission media can take the form of electrical or electromagnetic signals or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, the general forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched cards, paper tapes, any other physical storage media with hole patterns, RAMs, ROMs, PROMs, and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves transmitting data or instructions, cables or links transmitting such carrier waves, or any other medium from which a computer can read program code and / or data. Many of these forms of computer-readable media can participate in transmitting one or more sequences of one or more instructions to a processor for execution.

[0205] The computer system 3401 can include or communicate with an electronic display 3435, which includes a user interface (UI) 3440 to provide, for example, settings, a list of biological process reports of variables measured in real time at each stage of the system, the ability to export and import files (e.g., configuration files, updates), calibration, alerts (e.g., errors, maintenance, consumable replacement). Non-limiting examples of the UI include a graphical user interface (GUI) and a web-based user interface.

[0206] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented by software when executed by a central processing unit 3405. The algorithms can, for example, detect problems in a process by using feedback loops to adjust variables of a control system, by image recognition and pattern analysis, fuzzy logic, and hard and soft threshold execution, and associate specific and non-specific data through machine learning (e.g., supervised, unsupervised, and / or reinforcement) to optimize process conditions, process results, modeling performance, and simulations within the system.

[0207] Embodiments

[0208] Embodiment 1: Simulation of Fluid Flow and Mixing in Layers of a Micro Module

[0209] Given that the flow is laminar, the simulation can be solved in two scenarios; on the one hand, the velocity field, and on the other hand, the convective-diffusive transport of the microorganisms to be analyzed. In this case, two species (S1 and S2) are decided to be used, which enter each branch of the inlet of the module at a certain concentration, or enter the branches of the inlets of different coupled modules. The diffusion values are determined based on the diffusion coefficient of fluorescein in water for S1 and a diffusion coefficient two orders of magnitude lower for S2.

[0210] The process between the flows and the mixing process are simulated considering an average inflow velocity of a constant 5 micrometers per second (μm / sec), and the species concentration is tracked through the entire section from the outlet. An inflow concentration value of 50% can be expected for good mixing, assuming equal flow rates for the two inflow branches.

[0211] In Figure 22A the cross-sectional view of a single micro-module shows a case of poor or low mixing due to the low diffusion coefficient of the species, while in Figure 22B the diffusion coefficient is higher and successful mixing can be understood. Figure 23A-23B The results of assembling ten micro-modules through the simulation process are shown. The scalar distribution (representing cells) and the concentration of the culture medium (color) are shown. It can be observed that, on the one hand, this specific distribution of the micro-modules, where the cells enter from the top and the culture medium enters laterally, the cells do not leave the lower outlet at all, and the culture medium is not evenly distributed (due to the short flow path in the simulation). Figure 25A-25C Another arrangement of the micro-modules is shown, and the simulation is carried out in a module with 6 levels. In this arrangement, the first level starts with a larger number of modules, so that the growth percentage of the modules in each level is smaller (increasing by 1 module column per row / level), and a different culture medium inflow array with fewer ports is used. In this arrangement, both the distribution of the cells and the distribution of the culture medium are appropriately uniform.

[0212] Example 2: Construction of the macrostructure of the micro-module

[0213] The bioreactor is designed by using the Figure 24 macrostructure shown in Figure 23A and 23B which includes layers of DG micro-modules and has the shown feed circuit. An SLA 3D printer (Peopoly Moai) with commercial resin is used for 3D printing, including all the systems and connections. The printed bioreactor is shown in cross-section in

[0214] Another bioreactor is printed with an SLA printer and a photocurable resin. The design includes two phases, the "positive form" (the volume of the two intertwined phases without an intermediate membrane). Figure 25AAn isometric view of the test file used is shown, including a double helix icosahedron with several layers (diameter: 600 micrometers (μm)) and a solid base for better operation. Note that there is no feeding system for each phase. Figure 25B A test file successfully printed on a PEGDA photocurable resin is shown, Figure 25C A test file successfully printed with a commercial resin.

[0215] Example 3: Demonstration of fluid flowing through a micro module

[0216] A Direct Light Projection type 3-D printer with a commercial resin is used to print a substrate, which includes 4 layers with input / output connections and a 4x2 module row, where the diameter of the helical icosahedron is 500 μm). The printed bioreactor is injected with red dye at one input connection. Figure 26A A loop saturated with red dye is shown. The second print includes a first substrate and a second substrate to form a double helix icosahedron, both of the helical icosahedrons having a diameter of 500 μm. One substrate is injected with red dye at the input connection, and the other substrate is injected with blue dye. Figure 26B Two substrates differentiated by color are shown.

[0217] Example 4: Cell lines on a chip

[0218] Exemplary cell lines on a chip implementation are constructed by using Figure 27 the design shown in Figure 28 The progression of images obtained over time in an assay is shown. To simulate microorganisms, glass particles with an approximate average diameter of 50 micrometers are introduced into the following structure.

[0219] With port 2 closed, distilled water flow is applied from port 3 to 1. In the initial stage, the loop is saturated with freeze-dried water for simulation; for inoculating microorganisms, the loop can first be saturated with a culture medium. The harvest output (2) is closed, and the chip is "inoculated" with glass particles to simulate microorganisms. The flow behaves to transport the particles to the chamber, and once port 2 is opened, the suction effect through the porous membrane keeps most of the particles in place.

[0220] The loop has input ports 3 - 4 and an output end for the culture medium (1). With this configuration, the main loop is inoculated through port 3, and liquid culture medium (here water) is introduced into port 4. The balance of pressure forces the culture medium to occupy the second loop and leave the loop through the output port 1. The porous membrane acts as a filter, and the simulated microorganisms are trapped in the main loop chamber.

[0221] Once the chamber is saturated, the inoculation port is closed and the harvest outlet is opened. Due to the dynamics of the flow through the chamber, the velocity and the effect on the particles in the chamber decrease sharply as they move away from the central axis (see Figure 28 . Together with the suction effect of the second circuit, this causes the microbiota to settle and proliferate in the chamber (when using live microorganisms instead of the simulation used in this example). As their number increases, some microorganisms shift to a point near the central axis of the flow coming from port 4 and are pulled towards port 2 (see Figure 28 ). Figure 29 Shows the movement of particles over time.

[0222] Example 5: Exemplary sandbox bioreactor unit

[0223] Figure 30A Shows the design of an exemplary sandbox unit with a mixing module, a single growth chamber, and a harvest port. Figure 30B –30D shows the construction unit made of PDMS. In Figure 30B , water (colorless) was inoculated into the central channel at a flow rate of 985 microliters (μL) / hour, and water containing blue dye was inoculated into each side channel at a flow rate of 985 μL / hour. In Figure 30C , water (colorless) was inoculated into the central channel at a flow rate of 984 μL / hour, and water containing blue dye was inoculated into each side channel at a flow rate of 3335 μL / hour. Figure 30C Shows the module inoculated with an unbalanced flow rate between the side channels.

[0224] Example 6: Constructing a sandbox bioreactor with multiple interconnected units

[0225] Figure 31 Shows the design of the culture medium circuit and the gas circuit for the exemplary sandbox bioreactor module. The three - layer module shares a series of through - holes, where screws, washers, and nuts are adjusted to prevent leakage.

[0226] In this module, the culture medium distribution layer is a composite distribution system with hoses. The hoses can have a diameter between 50 and 500 microns and can be formed from biocompatible materials. The length of the hoses is adjusted by calculating the loading loss of the culture medium and comparing the pressures in each mixing module. The PDMS layer separates the gas layer and the culture medium layer. Figure 32 Shows the printed culture medium layer of the sandbox. Figure 33 Shows the assembled layers. Water containing blue dye was used to inoculate the sandbox from the input port to show the flow through the sandbox module.

[0227] While the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The present invention is not intended to be limited to the specific embodiments provided within this specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Many variations, changes, and alternatives will occur to those skilled in the art without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific formulations, configurations, or relative proportions set forth herein that depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. Accordingly, it should be regarded that the present invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the present invention and are intended to thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A bioreactor, comprising: An inlet configured to receive a plurality of cells; A macrostructure including a plurality of micro-modules in fluid communication with the inlet and configured to flow the plurality of cells, wherein a micro-module in the plurality of micro-modules (i) includes a double-helix icosahedron structure, and (ii) is fluidly connected to another micro-module via one or more connections, and wherein the plurality of micro-modules are arranged in layers within the macrostructure and are fluidly interconnected to provide at least two micro-channels; And An outlet in fluid communication with at least one of the at least two micro-channels of the macrostructure, the outlet being configured to direct the plurality of cells or their derivatives out of the at least one micro-channel.

2. The bioreactor according to claim 1, wherein the at least two micro-channels include at least two non-overlapping micro-channels each having a constant mean curvature.

3. The bioreactor according to claim 2, wherein a first micro-channel of the at least two non-overlapping micro-channels is configured to flow a liquid culture medium, and wherein a second micro-channel of the at least two non-overlapping micro-channels is configured to flow a gas component.

4. The bioreactor according to claim 2, wherein the at least two non-overlapping micro-channels provide a liquid.

5. The bioreactor according to claim 2, wherein the first micro-channel of the at least two non-overlapping micro-channels and the second micro-channel of the at least two non-overlapping micro-channels are separated by a porous membrane.

6. The bioreactor according to claim 5, wherein the area of the first micro-channel is equal to the area of the second micro-channel, and wherein the area of the porous membrane is the sum of the areas of the first micro-channel and the second micro-channel.

7. The bioreactor according to claim 1, wherein the macrostructure is selected from the group consisting of: a pyramid, a hollow pyramid, a layered pyramid, a layered structure, a checkerboard arrangement, and a log-like structure.

8. The bioreactor according to claim 7, wherein the layer is configured such that the velocity of the liquid culture medium is substantially the same in each layer.

9. The bioreactor according to claim 1, wherein the velocity of the liquid culture medium flowing through one of the at least two micro-channels is greater than the free sedimentation velocity of the cells flowing through the micro-channel.

10. The bioreactor according to claim 1, further comprising a gas input end and a gas output end.

11. The bioreactor according to claim 1, further comprising a cell input end at the top of the macrostructure and a cell collection device at the base of the macrostructure, the cell input end being configured to provide the plurality of cells, and the cell collection device being configured to harvest the plurality of cells.

12. The bioreactor according to claim 1, further comprising a liquid culture medium input device configured to cause the liquid culture medium to flow into each layer of the plurality of micro-modules.

13. The bioreactor according to claim 12, wherein the volume of the liquid culture medium provided to each layer by the liquid culture medium device maintains a substantially constant cell density in each of the layers.

14. The bioreactor according to claim 12, wherein the velocity of the liquid culture medium through each micro-module depends on the cell division rate such that the time required for the cells to traverse a single micro-module or a layer of micro-modules is substantially the same as the cell division rate.

15. The bioreactor according to claim 1, wherein the bioreactor is interconnected with a second module.

16. The bioreactor according to claim 1, wherein the bioreactor is interconnected with a cell chip module.

17. The bioreactor according to claim 1, further comprising a connector configured to provide fluid communication between the bioreactor and the second module.

18. The bioreactor according to claim 17, wherein the second module is a cleaning module, a culture medium reservoir, a sandbox bioreactor or a cell chip module.

19. The bioreactor according to claim 17, wherein the connector comprises: At least one needle or channel configured to allow a liquid or the plurality of cells to flow into the bioreactor, and at least one needle or channel configured to allow a liquid or the plurality of cells to flow out of the bioreactor.

20. A method of processing a plurality of cells, the method comprising: (a) providing a bioreactor comprising: (i) an inlet for receiving a plurality of cells; (ii) a macroscopic structure comprising a plurality of micro-modules in fluid communication with the inlet and configured to allow the plurality of cells to flow; wherein the micro-modules among the plurality of micro-modules comprise a double helix icosahedron structure and are fluidly connected to another micro-module via one or more connections, wherein the plurality of micro-modules are arranged in layers within the macroscopic structure and are fluidly interconnected to provide at least two micro-channels; and (iii) an outlet in fluid communication with at least one of the at least two micro-channels of the macroscopic structure; and (b) guiding the plurality of cells to the inlet, and the plurality of cells or their derivatives are guided from the inlet through the at least one micro-channel to the outlet.

21. The method according to claim 20, wherein a first micro-channel of the at least two micro-channels allows a liquid culture medium to flow.

22. The method according to claim 21, wherein a second micro-channel of the at least two micro-channels allows a gas component to flow.

23. The method according to claim 20, wherein the at least two micro-channels are separated by a porous membrane.

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