Systems, devices, and methods for production of bioproducts including high-density cell respirators for enhanced production of adeno-associated virus

Abstract

To provide a cell culture apparatus for culturing microorganisms and proliferating cells in high density.SOLUTION: The apparatus includes a membrane including a plurality of surface features on a first side of the membrane for cell placement. The surface features include one or more compartments in which cells can be disposed. The membrane comprises a material that is at least partially permeable to gas. The second side of the membrane defines a gas region. The second side of the membrane is separated from the first side of the membrane by the membrane. The device further includes a media region for receiving media. The compartment is configured to at least partially reduce media flow shear forces on the one or more cells within the compartment. The surface features may be bumps, protrusions, fins, wells, and / or posts.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 936,308, entitled "HIGH DENSITY CELL RESPIRATOR FOR PRODUCTION OF BIOPRODUCT," filed November 15, 2019, which is incorporated herein by reference in its entirety.

[0002] Embodiments of the disclosed technology generally relate to systems, devices, and methods, including a respiratory apparatus, for producing, culturing, and / or growing biological products, including microorganisms, tissues, and cells. In some exemplary embodiments, the respiratory apparatus is a high-density cell respiratory apparatus (HDCR). The HDCR can be a bioreactor. The disclosed systems, devices, and methods, including HDCR bioreactors, are effective platforms for enhanced production of a wide variety of biological materials, including adeno-associated viruses (AAV), oncolytic viruses, and other forms of cell-based production. The HDCR systems, devices, and methods lower the sometimes prohibitively high cost of goods associated with therapies such as AAV, making such therapies more widely available to more individuals in need without excessive cash outlay. [Background technology]

[0003] In gene therapy, inefficient cell factories and stirred tank reactors (STRs) have been employed to scale up the production of bioproducts, but as repeatedly noted in the literature, the demand for AAV for systemic therapy far exceeds the production capacity of STRs.

[0004] As the genetic basis of many diseases is elucidated, the possibility of treatment through gene transfer and gene editing is becoming a clinical reality. Many of these new therapies rely on viral vectors. Cell-based vector production has been developed that relies on traditional tissue culture techniques that have reached their production capacity limit. The large quantities of viral particles required for these applications and the inefficiencies of current manufacturing methods have led to an astoundingly high cost for these medicines.

[0005] Cost and production time are among the biggest barriers to clinical studies of promising approaches in Phase I and II. High costs are due to the fact that the amount of viral vectors required for clinical trials is on the trillion cell scale (10 12 This is because it requires cell culture at the individual cell scale. With existing development technologies, it can take 9-12 months to produce enough vectors for early-stage clinical trials, and existing manufacturing facilities are already near maximum capacity. The wait time from vector production to clinical trials is 2-3 years, delaying the testing and clinical application of revolutionary therapies.

[0006] High costs and long production wait times are delaying the realization of potentially disruptive therapies that could alleviate suffering and preserve lives, and some promising approaches may be abandoned entirely due to significant financial obstacles.

[0007] Even in the R&D and preclinical stages, vector development requires the use of billion-cell scale (10 9 Currently, managing dozens of flasks or Petri dishes is tedious, occupying valuable operator time and limited incubator space.

[0008] To support more parallel efforts, new culture systems that are medium-efficient, compact, and robust for virus production are needed. Because hundreds of virus candidates are often screened before a viable candidate is identified, new technologies are needed to facilitate screening, expand laboratory capacity, and accelerate the development of therapeutics for humanity.

[0009] Vaccine production has been a major advancement in human public health. Routine vaccination against the most important and common human pathogens has led to the control of many diseases, including influenza, polio, and measles, and the eradication of some devastating diseases, including smallpox. Vaccination against seasonal and mutable viruses, such as influenza, requires the production of millions of doses of vaccine each year. The production and distribution timeline for the U.S. influenza vaccine supply takes nearly a year. Therefore, the most prevalent serotypes for the next influenza season must be predicted almost a year in advance. This means that, depending on the season, the vaccine administered may not protect against the prevalent strain. Compressing the production timeline would allow for better determination of the vaccine strains produced and could allow for "last-minute" changes in the event of a newly emerging strain. With current technology, it could take 5–10 years to produce enough vaccines to vaccinate a global population of over 7 billion in the event of a pandemic or, worse, the release of a viral bioterrorism weapon. Reducing production time by 2–10 times would save many lives.

[0010] Improved understanding of the genetic basis of many diseases makes it possible to treat many diseases through gene transfer and gene editing. The development and production of these new treatments relies on trillion-cell-scale production of viral vectors and other bioproducts. Existing tissue culture and culturing technologies are inefficient and costly, often resulting in high costs and long delays in the production of viral vectors, drugs, vaccines, and other bioproducts.

[0011] Regarding cell-specific AAV production, the literature suggests that the cell density at the time of transfection is approximately 10 5 ~10 7 cells / mL, specific productivity of 10 3 ~10 6 Only the systems, devices and methods developed for vg / cells are discussed.

[0012] The embodiments described herein generally relate to improving cell and tissue growth production apparatus and methods. Disclosed herein are systems, devices, and methods that reduce the cost and time of viral vector production, facilitating the testing and clinical use of promising therapies. The disclosed systems, devices, and methods also provide significant cost savings, enabling new therapies and global access. The systems, devices, and methods advance production beyond the capabilities of existing development technologies, including STR. Summary of the Invention

[0013] Large- and small-scale high-density cell and tissue culture is important for many biotechnology applications in which cells are used to produce specific molecules, proteins, viruses, or other products. Increasing cell density allows for greater production per unit volume, saving space and reducing costs through higher product concentrations.

[0014] One challenge of high-density cell growth arises from mass transport limitations, particularly for oxygen, nutrients, and waste products. In low-density cell culture systems, passive diffusion of metabolites may be sufficient to meet the metabolic demands of cells. However, in high-density cell culture systems, the metabolic demands of cells exceed the supply provided by diffusion alone, necessitating the addition of mass transport mechanisms such as convection.

[0015] A further requirement for high-density cell culture, especially for adherent cell populations, is a high surface area to volume ratio, since many cells grow in monolayers and are inhibited in growth when they become confluent.

[0016] Several technologies have been developed to increase cell density, including cell factory systems, wave / agitated bioreactors using microcarriers, and perfusion dialysis membrane systems. The present inventors have determined that the existing technologies have numerous drawbacks.

[0017] Cell factory systems are most similar to traditional flask culture systems, except that cell factory systems contain multiple layers of growth substrate within a single flask. The large spacing between layers results in a low surface-to-volume ratio, and cell densities that can be achieved are limited due to the reliance on diffusive transport of all metabolites.

[0018] Wave and agitator bioreactor systems add convection and enhance mass transport by gently mixing cell microcarriers, small, neutrally buoyant particles with surface chemistry favorable for cell attachment and growth, within a medium vessel. The combination of the high surface area provided by microcarriers and convective mixing allows for higher cell densities to be achieved compared to cell factory systems. However, convective mixing induces undesirable shear forces on the cells, which can induce cell death, thereby limiting the degree of mixing and mass transport to the cells that can be achieved. Such systems are often shear-limited, not by surface area but by the need to enhance mass transfer through mixing.

[0019] Perfusion dialysis membrane systems overcome the shear force problem by perfusing a gas or oxygenated medium through tightly packed, semipermeable dialysis tubing, delivering oxygen to cells by diffusion. However, the geometry of the dialysis tubing precludes a very high surface area-to-volume ratio. Additionally, challenges exist regarding cell removal from the highly porous membrane on which the cells grow.

[0020] Thus, some of the challenges of high-density cell growth include 1) achieving a high growth surface area to volume ratio, 2) maintaining adequate metabolite transport within the system, and 3) keeping shear forces experienced by the cells below growth-inhibitory or lethal levels. While existing technologies attempt to overcome these challenges, there is significant room for technological improvement. Described herein are various improved systems, devices, and methods for cell, tissue, and microbial culture.

[0021] One feature is a cell culture device. The device includes a membrane. The membrane includes surface features on a first side of the membrane. The surface features include one or more compartments capable of trapping one or more cells. The membrane includes a material that is at least partially permeable to gas. A second side of the membrane defines one boundary of a gas region. The membrane is configured to allow gas to pass through the second side of the membrane to the first side of the membrane. The device further includes a medium region having a boundary on the first side of the membrane. The medium region is configured to allow medium to pass over the first side of the membrane. The one or more compartments are configured to at least partially reduce medium flow shear forces within the compartment.

[0022] Another feature is a Petri dish. The Petri dish includes a bottom surface and a sidewall forming the dish. The bottom surface includes an at least partially gas-permeable material. The bottom surface includes a first side and a second side. The first side includes a plurality of structures extending from a base of the first side, forming one or more cell niche regions (compartments) below an upper surface of the plurality of structures. The sidewall is connected to the bottom surface. The sidewall forms a continuous and substantially vertical wall around the periphery of the bottom surface.

[0023] Another feature is a multi-well cell growth device. The device includes a plurality of wells. The wells include a bottom surface including a first side that contacts the interior of the wells and a second side that contacts the exterior of the multi-well cell growth device. The first side includes a topography that provides a plurality of cell growth compartments. The bottom surface includes a material that is at least partially permeable to gas. The bottom surface is configured to allow gas from outside the multi-well growth device to pass from the exterior to the interior of the wells and contact one or more of the plurality of cell growth compartments, and / or allow gas from within one or more cell growth compartments to pass from the first side to the second side.

[0024] Another feature is a cell culture device. The device includes a membrane. The membrane includes a plurality of fin structures extending substantially parallel to one another. The substantially parallel fin structures define grooves between adjacent fin structures. At least one groove on a first side of the membrane provides a compartment for at least cell placement. The membrane includes a material that is at least partially permeable to gas. A second side of the membrane defines a gas region. The second side of the membrane is separated from the first side of the membrane by the membrane. Gas can pass through the membrane. A medium region on the first side of the membrane is configured to receive medium containing one or more cells that can be deposited within the compartment.

[0025] Another feature is a cellular respirator device. The device includes one or more membranes. At least one of the one or more membranes has a plurality of fins, including at least a first fin and a second fin, protruding from a first surface of the membrane to form a first of a plurality of compartments configured to hold a plurality of cells. The membrane is formed from a gas-permeable and air-permeable material to facilitate delivery of gas to the plurality of compartments through one or more channels formed below the first surface of the membrane below the fins. At least a first channel of the one or more channels is configured to deliver gas directly to the first compartment.

[0026] Another feature is a method of culturing biological cells. The method includes providing a device according to any of the above features. The method further includes introducing biological cells into the device. The method further includes providing a cell culture medium on a first side of the device such that the cell culture medium contacts the biological cells.

[0027] The cell culture device may include a membrane that may include a plurality of surface features on a first side of the membrane, the surface features may include one or more compartments capable of entrapping one or more cells, and the membrane may include a material that is at least partially permeable to gas; a second side of the membrane that defines a boundary of a gas region, the second side being configured to allow gas to pass through the second side of the membrane to the first side of the membrane; and a medium region having a boundary on the first side of the membrane and configured to pass medium across the first side of the membrane, the one or more compartments being configured to at least partially reduce medium flow shear forces within the compartments.

[0028] The device may include one or more medium inlets and one or more medium outlets fluidly connected to the medium region, the one or more medium inlets configured to facilitate introduction of medium into the medium region on the first side of the membrane, and the one or more medium outlets configured to facilitate removal of medium from the medium region.

[0029] The device, wherein at least one of the one or more medium inlets is fluidly connected to at least one of the medium outlets to facilitate reintroduction of medium into the medium region on the first side of the membrane.

[0030] The device is configured to minimize medium flow shear forces in one or more compartments upon introduction or removal of medium from the medium region.

[0031] The apparatus may further include one or more gas inlets and one or more gas outlets in fluid contact with the gas region, the one or more gas inlets configured to introduce gas into the gas region and the one or more gas outlets configured for removal of gas from the gas region.

[0032] The device may include one or more channels formed in the second side of the membrane, at least a first of the one or more channels configured to deliver gas through the membrane to at least one of the compartments by permeation and diffusion through the membrane.

[0033] The device can include an enclosure disposed above the first side of the membrane.

[0034] The device, wherein the enclosure defines a second boundary of the medium region.

[0035] The device is configured to be stacked with at least a second cell culture device.

[0036] A device wherein one or more of the medium inlet or gas inlet or medium outlet or gas outlet is configured such that when stacked, the one or more of the medium inlet or gas inlet or medium outlet or gas outlet is in fluid communication with the inlet or outlet of an immediately adjacent device.

[0037] A device wherein one or more of the medium inlets or gas inlets are configured such that, when stacked, medium or gas introduced into the device is combined and enters a combined inlet line for delivery to multiple membranes.

[0038] A device wherein one or more of the medium outlets or gas outlets are configured such that when stacked, medium or gas removed from the device is combined and enters a combined outlet line for delivery from the multiple membranes.

[0039] The device may include a liquid-tight container for holding the device.

[0040] A device wherein the membrane comprises pores having a size that allows permeation and diffusion of oxygen through the membrane.

[0041] A device that supports tissue-level cell density such that one or more compartments have a volume of cells that occupy at least about 10% of the volume of the one or more compartments that can confine one or more cells.

[0042] The device, wherein the negative pressure deforms a bottom surface of at least one of the one or more compartments downward, drawing liquid medium into the at least one compartment through an opening in the top of the at least one cell niche, and / or the positive pressure deforms a bottom surface of at least one of the one or more cell niches upward, pushing liquid medium out of the at least one compartment through an opening in the top of the at least one compartment.

[0043] The device, wherein mechanical stretching of the membrane expands at least one of the one or more compartments to facilitate release of one or more cells from the at least one cell niche through an opening in the top of the at least one compartment.

[0044] The device can include a plurality of microdiffusers configured to rectify the pulsating pressure into a net flow normal to the first surface of the membrane.

[0045] The device may include at least one porous wick configured to transport a fluid medium to one or more compartments.

[0046] A device in which the membrane comprises a micropatterned architecture having multiple compartments designed to provide a significantly high gas exchange area to volume ratio to maximize the oxygen permeation rate into the compartments formed in the membrane.

[0047] A device in which the membrane is configured to expand or contract in response to changes in pressure or flow associated with the gas.

[0048] The device, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with the liquid medium.

[0049] The device, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with at least one of the gas and liquid medium to promote uniform distribution of reagents or cells in one or more compartments.

[0050] The device, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with at least one of a gas and a liquid medium to facilitate efficient cultivation or harvesting of cells located within one or more compartments within the membrane.

[0051] The device, wherein the fluid path for the medium is gravitationally supported by one or more flow regulators that regulate the flow of liquid medium across the first side of the membrane.

[0052] The device may include a plurality of microcarriers to which cells can attach.

[0053] The device, wherein the plurality of surface features include a plurality of fin structures extending substantially parallel to one another, the substantially parallel fin structures defining grooves between adjacent fin structures, at least one groove on a first side of the membrane providing at least one of one or more compartments for cell placement, and a longitudinal direction of the at least one groove corresponding to a longitudinal direction of the one or more compartments.

[0054] The device, wherein one or more medium inlets are configured to introduce medium into the medium region to create a medium flow that is not parallel to the longitudinal direction of the channel.

[0055] The device, wherein one or more medium inlets are configured to introduce medium into the medium region to create a medium flow substantially perpendicular to the grooves.

[0056] The device, wherein one or more medium inlets are configured to introduce medium into the medium region to create a medium flow substantially aligned with the grooves.

[0057] The device, wherein an upper opening of one or more of the one or more cell niches is narrower than the width of the lower opening.

[0058] The device, wherein a plurality of fins protrude from the base of the membrane to retain and protect one or more of the cells within the one or more cell niches from media flow shear forces generated by media delivery.

[0059] The device, wherein the membrane has a multi-layer monolithic structure such that one or more channels for delivering gas are formed in a first layer of the membrane and compartments are formed between a plurality of fins in a second layer of the membrane above the first layer.

[0060] A device in which a dedicated space is provided above a plurality of compartments formed between the plurality of fins to support a fluid path for a liquid medium that flows above the plurality of fins and is substantially perpendicular to the plurality of fins.

[0061] A device in which a dedicated space is provided above a plurality of compartments formed between the plurality of fins to support a fluid path for a liquid medium to flow above the plurality of fins and substantially aligned with the plurality of fins.

[0062] A device wherein the fluid path has relatively low resistance to liquid medium flowing over the plurality of fins, thereby eliminating or reducing the need for the use of pumps to regulate liquid medium flow.

[0063] A device wherein the surface features include a plurality of well structures, at least one of the plurality of well structures providing at least one of the one or more compartments for cell placement.

[0064] A device wherein an upper opening of at least one of the well structures includes a diametric width that is narrower than the diametric width of the well structure below the opening.

[0065] The device, wherein one or more of the well structures includes an opening that is circular, oval, square, rectangular, hexagonal, or octagonal.

[0066] A device wherein the well structure protects one or more cells within one or more cell niches of the well structure from shear forces caused by media delivery.

[0067] A device wherein the membrane has a multi-layer monolithic structure such that one or more channels for delivering gas are formed in a first layer of the membrane and compartments are formed within well structures in a second layer of the membrane above the first layer.

[0068] An apparatus in which a dedicated space is provided above the plurality of well structures to support a fluid path for liquid medium to flow above the plurality of well structures.

[0069] A device in which the fluid pathway has relatively low resistance to liquid medium flowing over the multiple well structure, thereby eliminating or reducing the need for the use of pumps to regulate liquid medium flow.

[0070] An apparatus in which a dedicated space is provided above the plurality of well structures to support a fluid path for liquid medium to flow above the plurality of well structures.

[0071] A device wherein at least one well structure has a circular opening.

[0072] A device wherein at least one well structure has a polygonal opening.

[0073] A device wherein at least one well structure has a curved opening.

[0074] A device wherein the surface structure includes a plurality of post structures, at least one post structure providing at least one of a plurality of compartments for cell placement.

[0075] A device in which cells are attached to at least one post structure.

[0076] The device, wherein the membrane has a multi-layer monolithic structure such that one or more channels for delivering gas are formed in a first layer of the membrane and a cellular niche is formed proximal to the post structure in a second layer of the membrane above the first layer.

[0077] An apparatus in which a dedicated space is provided above the plurality of post structures to support a fluid path for a liquid medium to flow above the plurality of post structures.

[0078] An apparatus wherein the fluid path has relatively low resistance to liquid medium flowing over the plurality of post structures, thereby eliminating or reducing the need for the use of pumps to regulate liquid medium flow.

[0079] An apparatus in which a dedicated space is provided above the plurality of post structures to support a fluid path for a liquid medium to flow above the plurality of post structures.

[0080] The apparatus, wherein at least one post structure has a circular cross-section.

[0081] The device, wherein at least one well post has a polygonal cross-section.

[0082] A device wherein at least one post structure has a curved, angled, crescent-shaped, or U-shaped cross-section.

[0083] The apparatus may include a Petri dish configured to receive the membrane.

[0084] A device in which the membrane forms the bottom of a Petri dish.

[0085] The device may include a tension ring configured to keep the membrane in tension and provide additional rigidity.

[0086] The device may include spacing pillars protruding from a second surface of the membrane opposite the first surface, the spacing pillars configured to allow gas exchange through the membrane to the cell niche.

[0087] A Petri dish may include a bottom surface and sidewalls forming a dish, the bottom surface may comprise at least a partially gas permeable material, the bottom surface may include a first side and a second side, the first side may include a plurality of structures extending from a base of the first side, forming one or more cell niche regions below an upper surface of the plurality of structures, and the sidewalls connected to the bottom surface and forming a continuous and substantially vertical wall around the periphery of the bottom surface.

[0088] A Petri dish in which the bottom and side walls contain the same material.

[0089] A Petri dish, the bottom and sidewalls of which at least partially comprise a gas-permeable material.

[0090] A Petri dish in which the sidewalls comprise at least one material different from the bottom surface.

[0091] The Petri dish may include one or more retaining elements that provide support to the sidewalls.

[0092] A Petri dish, wherein a bottom surface of the Petri dish exposed to the external environment comprises at least a partially gas-permeable material such that gas from the external environment can pass at least partially through the gas-permeable membrane to the first side, and / or gas from the first side can pass from the first side to the external environment at least partially through the gas-permeable membrane.

[0093] A Petri dish in which the bottom of the dish is placed on a mesh, cloth, or other open-pore material to allow gases from the external environment to exchange with the membrane.

[0094] A Petri dish, wherein the bottom surface of the Petri dish exposed to the external environment includes one or more variations in its shape that allow gas to pass at least partially underneath the bottom surface when the dish is placed on a flat surface.

[0095] A Petri dish whose shape includes pillars, channels, grooves, bumps, protrusions, or legs.

[0096] A Petri dish whose shape includes one or more spacing pillars.

[0097] The petri dish may include a top surface, which may include a sealing membrane.

[0098] A Petri dish whose top surface comprises a silicone-based membrane.

[0099] A Petri dish that may include a sealable port for transferring media to or from the Petri dish.

[0100] A multi-well cell growth device that may include a plurality of wells, each well may include a bottom surface including a first side in contact with the interior of the well and a second side in contact with the exterior of the multi-well cell growth device, the first side including a topography that provides a plurality of cell growth compartments, the bottom surface including a material that is at least partially permeable to gas, and the bottom surface configured such that gas from outside the multi-well growth device can pass from the exterior to the interior of the well to contact one or more of the plurality of cell growth compartments, and / or gas from within one or more cell growth compartments can pass from the first side to the second side.

[0101] A multi-well cell growth device, wherein the topography of the first side includes one or more of fins, subwells, and pillars, the fins, subwells, and pillars at least partially defining a plurality of cell growth compartments.

[0102] A multi-well cell growth device, wherein the sidewalls of the plurality of wells comprise, at least in part, a gas permeable material.

[0103] A multi-well cell growth device, wherein the second side includes one or more variations in its shape that allow gas to pass at least partially beneath the bottom surface when the second side is on a flat surface.

[0104] A multi-well cell growth device, the shape of which includes pillars, channels, grooves, bumps, protrusions, or legs.

[0105] A multi-well cell growth device, the geometry of which includes one or more spaced pillars.

[0106] a second side of the membrane defining a gas region, the second side being separated from the first side by the membrane, the second side allowing gas to pass through the membrane; and a medium region on the first side of the membrane configured to receive medium containing one or more cells that can be deposited within the compartment.

[0107] A cellular respirator device may include one or more membranes and at least one membrane of the one or more membranes having a plurality of fins including at least a first fin and a second fin protruding from a first surface of the membrane to form a first compartment of the plurality of compartments configured to hold a plurality of cells, wherein the membrane is formed from a material that is gas permeable and air permeable to facilitate delivery of gas to the plurality of compartments through one or more channels formed below the first surface of the membrane below the plurality of fins, and at least a first channel of the one or more channels is configured to deliver gas directly to the first compartment.

[0108] A method of culturing biological cells may include providing any of the devices described above, introducing biological cells into the device, and providing a cell culture medium on a first side of the device such that the cell culture medium contacts the biological cells.

[0109] The method can include providing a gas to a second side of the membrane.

[0110] The method may include flowing a medium over one or more cell compartments.

[0111] The method, wherein flowing the medium comprises introducing the medium through an inlet and removing the medium through an outlet.

[0112] The method may include flowing a gas onto a second side of the membrane.

[0113] The method, wherein flowing the gas includes introducing the gas through an inlet and removing the gas through an outlet.

[0114] The method may include causing media, biological cells, or another substance to flow into one or more cell compartments by creating negative pressure in the one or more compartments.

[0115] The method may include maintaining the cells in the one or more compartments by creating negative pressure in the one or more compartments.

[0116] The method may include causing medium, biological cells, or another substance to flow out of one or more cell compartments by creating a positive pressure within the one or more compartments.

[0117] The method can include directing cells out of one or more compartments by creating a positive pressure within the one or more compartments.

[0118] The method may include mixing one or more of the biological cells in the one or more compartments by creating positive and / or negative pressure in the one or more compartments.

[0119] A method in which positive and / or negative pressure is generated by flowing gas into a gas region, flowing medium into a medium region, flowing both gas and medium into their respective regions, preventing the flow of gas into the gas region and / or preventing the flow of medium into the medium region, thereby generating a pressure differential between the two regions.

[0120] Live cells, non-live mammals, insects, bacteria, fungi, yeast, high-density leukocytes, produceor cells, induced produceor cells, CAR-T, 3T3-L1, 4T1, 9L, A20, A172, A253, A431, A549, A2780, A2780ADR, A2780cis, AB 9. AHL-1, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BOSC23, BT-20, BxPC-3, C2C12, C3H-10T1 / 2, C6, C6 / 36, Caco-2, Cal-27, Calu-3, CAP, CGR8, CHO, CML T1, CMT12, COR-L23, COR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23-, COS-7, COV-434, CT26, D17, DAOY, DH82, DU145, DuC aP, E14Tg2a, vEL4, EM-2, EM-3, vEMT6 / AR1, EMT6 / AR10.0, FM3, GL261, H1299, HaCaT, HCA2, HEK293, HEK293T, HeLa, Hep G2, Hepa1c1c7, High Five, HL-60, HT-29, HT-1080, J558L, Jurkat, JY, K562, KBM-7, KCL-22, KG1, Ku812, KYO-1, L243, L1210, LNCa P, MA-104, Ma-Mel, MA2.1, MC-38, MCF-7, MCF-10A, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, MDCK II, MG63, MIA PaCa-2, Mono-Mac-6, MOR / 0.2R, MRC-5, MTD-1A, MyEnd, NALM-1, NCI-H69, NCI-H69 / CPR, NCI-H69 / LX4, NCI-H69 / LX10, NCI-H69 / LX20, Neuro-2a, Neuro2a, NIH-3T3, NK-92, NTERA-2, NW-145, OK, OPCN / OPCT cell line, P3X63Ag8, PANC-1, PC-3, PC12, Peer, PNT1A, PNT2, Pt The method may be performed using any one or more of the following cells: K2, Raji, RBL-1, RenCa, RIN-5F, RMA-S, S2, SaOS-2, Sf9, Sf21, SH-SY5Y, SiHa, SK-BR-3, SK-N-SH, SK-OV-3, T-47D, T2, T84, T98G, THP-1, U2OS, U87, U373, U937, VCaP, Vero, VG-1, WM39, WT-49, YAC-1, and YAR cells.

[0121] The method wherein the gas is selected from the group consisting of oxygen, carbon dioxide, nitrogen, carbon monoxide, nitrous oxide, hydrogen sulfide, ethylene oxide, ozone, chlorine dioxide, and nitrogen dioxide.

[0122] The method, wherein the culture medium is selected from the group consisting of DMEM, FCS, 293SFM II, AEM, CDM4HEK293, SFM4HEK293, Ex-Cell293, SFM4Transfx-293, Freestyle293, ESF SFM, CDM4CHO, CHO medium, MEM, MEM Alpha, RPMI, F-10, F-12, IMDM, Medium 199, Leibovitz L-15, McCoy's 5A, MCDB medium, William's medium, CMRL medium, OptiMEM, OptiPro, AIM V, OptiPEAK T lymphocyte, ExCellerate human T cell growth medium, StemXVivo serum-free human T cell base medium, ExpiSf CD medium, Sf-900 II / III SFM, and TC-100 insect medium.

[0123] A method wherein the other substances are selected from the group consisting of waste materials, substances secreted by cells, substances inside cells, cell debris, and gases.

[0124] The method may include introducing a cryogen into the gas region to freeze cells and cellular components in the multiple compartments, wherein the cryogen has a temperature below 0°C and the cryogen is in a gaseous or liquid state.

[0125] The device, wherein at least one of the surface features has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

[0126] The device, wherein at least one of the surface features has a height of about 700 μm to about 1000 μm.

[0127] The device, wherein at least one of the surface features has a height of about 700 μm.

[0128] The device, wherein at least one of the surface features has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

[0129] The device has a height to width to pitch ratio of about 7-10 to about 3-5 to about 2-5.

[0130] A device having a height to width to pitch ratio of about 7 to about 3-5 to about 2-5.

[0131] A Petri dish, wherein at least one of the plurality of structures has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

[0132] A Petri dish, wherein at least one of the plurality of structures has a height of about 700 μm to about 1000 μm.

[0133] A Petri dish, wherein at least one of the plurality of structures has a height of about 700 μm.

[0134] A Petri dish, wherein at least one of the plurality of structures has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

[0135] A Petri dish having a height to width to pitch ratio of about 7-10 to about 3-5 to about 2-5.

[0136] A Petri dish having a height to width to pitch ratio of about 7 to about 3-5 to about 2-5.

[0137] A multi-well cell growth device, wherein at least one of the plurality of cell growth compartments has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

[0138] A multi-well cell growth device, wherein at least one of the plurality of cell growth compartments has a height of about 700 μm to about 1000 μm.

[0139] A multi-well cell growth device, wherein at least one of the plurality of cell growth compartments has a height of about 700 μm.

[0140] A multi-well cell growth device, wherein at least one of the plurality of cell growth compartments has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

[0141] A multi-well cell growth device having a height to width to pitch ratio of about 7-10 to about 3-5 to about 2-5.

[0142] A multi-well cell growth device having a height to width to pitch ratio of about 7 to about 3-5 to about 2-5.

[0143] A cell culture device, wherein at least one of the plurality of fin structures has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

[0144] A cell culture device, wherein at least one of the plurality of fin structures has a height of about 700 μm to about 1000 μm.

[0145] A cell culture device, wherein at least one of the plurality of fin structures has a height of about 700 μm.

[0146] A cell culture device, wherein at least one of the plurality of fin structures has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

[0147] A cell culture device having a height to width to pitch ratio of about 7-10 to about 3-5 to about 2-5.

[0148] A cell culture device having a height to width to pitch ratio of about 7 to about 3-5 to about 2-5.

[0149] A cellular respiratory device, wherein at least one of the plurality of fins has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

[0150] A cellular respiratory apparatus, wherein at least one of the plurality of fins has a height of about 700 μm to about 1000 μm.

[0151] A cellular respiratory apparatus, wherein at least one of the plurality of fins has a height of about 700 μm.

[0152] A cellular respiratory apparatus, wherein at least one of the plurality of fins has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

[0153] A cellular respiratory apparatus having a height to width to pitch ratio of about 7-10 to about 3-5 to about 2-5.

[0154] A cellular respiratory apparatus having a height to width to pitch ratio of about 7 to about 3-5 to about 2-5.

[0155] The method, wherein the device comprises at least one surface feature having a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

[0156] The method, wherein the device comprises at least one surface feature having a height of about 700 μm to about 1000 μm.

[0157] The method, wherein the device comprises at least one surface feature having a height of about 700 μm.

[0158] The method, wherein the device comprises at least one surface feature having a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

[0159] The method wherein the ratio of height to width to pitch is about 7-10 to about 3-5 to about 2-5.

[0160] The method wherein the ratio of height to width to pitch is about 7 to about 3-5 to about 2-5.

[0161] A bioreactor is provided that may include one or more of a gas inlet, a gas outlet, a gas pathway connected between the gas inlet and the gas outlet, a medium inlet, a medium outlet, a medium pathway connected between the medium inlet and the medium outlet, and a membrane separating the gas supply pathway and the medium supply pathway, the membrane including a cell protection region, the cell protection region in liquid communication with the medium pathway, the cell protection region being fluidically distinct from the medium pathway, the cell protection region in gas communication with the gas pathway across the membrane, and the cell protection region configured to reduce an effect of shear force of medium flowing through the medium pathway on cells produced in the cell protection region.

[0162] The bioreactor is approximately 10 7 cells / milliliter (cells / mL) ~ approximately 10 9 The cell density at the time of transfection in cells / mL, and approximately 10 15 Adeno-associated virus genomes / liter (AAV vg / L) ~ approximately 10 16A bioreactor configured to produce a productivity of AAV vg / L.

[0163] A bioreactor, wherein the velocity of the medium at the medium inlet is about 1 μm / sec to about 1 m / sec.

[0164] The velocity of the medium at the medium inlet is about 100 μm / sec, the bioreactor has a length of about 40 cm, and the 8 Bioreactor producing cells / mL.

[0165] The velocity of the medium at the medium inlet is about 1 μm / sec, and the bioreactor is about 10 6 139. The bioreactor according to any one of claims 135 to 138, which produces cells / mL.

[0166] The device is about 10 7 cells / milliliter (cells / mL) ~ approximately 10 9 A cell density at the time of transfection of 10 cells / mL, and approximately 10 15 Adeno-associated virus genomes / liter (AAV vg / L) ~ approximately 10 16 Any of the apparatus, Petri dish, multi-well cell growth device, cell culture apparatus, cellular respiration apparatus, and methods configured to produce AAV vg / L productivity.

[0167] Any of the device, cell culture device, and cell respiration device, and method, wherein the velocity of the medium at the medium inlet is about 1 μm / sec to about 1 m / sec.

[0168] The velocity of the medium at the medium inlet is about 100 μm / sec, the device has a length of about 40 cm, and 8 Any of the devices, cell culture devices, cell respiration devices, and methods that produce cells / mL.

[0169] The velocity of the medium at the medium inlet is about 1 μm / sec, and the device is 6 Any of the devices, cell culture devices, cell respiration devices, and methods that produce cells / mL.

[0170] Either the Petri dish or the multi-well cell growth device has a length of approximately 40 cm and contains approximately 10 6 cells / mL ~ approx. 10 8 It is configured to produce cells / mL.

[0171] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0172] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain features of the subject matter disclosed herein and, together with the description, serve to explain some of the principles associated with the disclosed implementations.

[0173] [Figure 1] 1 is a functional overview of a high-density cell respiratory device, including, for example, fin-like structures, according to various exemplary embodiments. [Figure 2] Figure 1 is a model of the high-density cell respiratory device. [Figure 3] 2 illustrates the deformation of the membrane of FIG. 1 in the presence of pressure fluctuations, resulting in the flow of medium into and out of the compartments of the high-density cell respirator device of FIG. 1. [Figure 4] 10A-10C illustrate the use of pulsating flow to enhance mixing and nutrient transport in accordance with various exemplary embodiments of the disclosed technology. [Figure 5] Shown is an open membrane architecture in which cells are sedimented and held within grooves forming compartments, medium flows over the cells, and the flow of medium draws air into the compartments, and in various exemplary embodiments, a porous and / or fibrous membrane that carries medium over the compartments. [Figure 6]FIG. 6 shows a membrane having fins with a keystone shape that form grooves with openings at the top of the grooves that are narrower than at least some points closer to the surface of the membrane, according to an exemplary embodiment, and further illustrates removal of material from the keystone-shaped grooves by stretching or inverting the membrane. [Figure 7] 10A-10C illustrate membranes with no surface features, with fins and grooves, with wells, and with posts, according to example embodiments. [Figure 8A] 1 illustrates the construction and formation of a high density cellular respiratory device formed with a media layer bonded to a gas layer, according to an exemplary embodiment. [Figure 8B] 8B shows spacing pillars defining space for medium perfusion above the cell niches (compartments) formed by the surface features of the membrane of FIG. 8A. [Figure 8C] 8A and 8B. A plurality of stacked membranes are shown with gas manifolds at the corners of the stacked membranes, each corresponding to the membrane of FIGS. 8A and 8B. [Figure 8D] 8D shows an enclosure for housing the stacked membranes of FIG. 8C with gas and medium inlets and outlets to form a high-density cell respirator cartridge. [Figure 8E] FIG. 8D shows an incubator into which the high density cell respirator cartridge of FIG. 8D can be enclosed for cell incubation. [Figure 8F] 1 illustrates an example of a high density cell respirator cartridge according to an exemplary embodiment. [Figure 9] 1 shows an example of an open membrane high-volume cellular respirator device having 10 stacked membranes according to an exemplary embodiment. [Figure 10] 1 shows a prototype of a high-density cellular respirator with integrated cell-holding niches (compartments), spacing pillars, gas perfusion spaces, and gas manifolds, according to an exemplary embodiment. [Figure 11A]1A-1C show a high density cell respirator before and after purging of air bubbles and voids by perfusing liquid through a medium compartment above the membrane of the high density cell respirator, according to an exemplary embodiment. [Figure 11B] 1A-1C show a high density cell respirator before and after purging of air bubbles and voids by perfusing liquid through a medium compartment above the membrane of the high density cell respirator, according to an exemplary embodiment. [Figure 11C] 1A-1C show a high density cell respirator before and after purging of air bubbles and voids by perfusing liquid through a medium compartment above the membrane of the high density cell respirator, according to an exemplary embodiment. [Figure 12] A membrane prototype for a medium-scale, high-density cell respirator is shown. [Figure 12A] FIG. 12B is an enlarged view of a portion of FIG. 12A according to an exemplary embodiment. [Figure 13A] 13A shows a 24-well plate with silicone inserts having patterned grooves adhered to each of the wells, according to an exemplary embodiment, and FIG. 13A is a close-up view of a groove in one of the silicone inserts of FIG. 13A. [Figure 13B] 13A shows a 24-well plate with silicone inserts having patterned grooves adhered to each of the wells, according to an exemplary embodiment, and FIG. 13A is a close-up view of a groove in one of the silicone inserts of FIG. 13A. [Figure 14] 1 shows a high-density cell respiration Petri dish according to an exemplary embodiment, where a gas-permeable membrane forms the bottom of the Petri dish, gas diffuses from below the membrane, and cells and nutrients are supplied from a deep reservoir above the membrane. [Figure 15A] An example of a Petri dish corresponding to that of Figure 14 is shown, in which HEK293 cells have been cultured on microcarriers. [Figure 15B] An example of a Petri dish corresponding to that of Figure 14 is shown, in which HEK293 cells have been cultured on microcarriers. [Figure 15C]An example of a Petri dish corresponding to that of Figure 14 is shown, in which HEK293 cells have been cultured on microcarriers. [Figure 16] 1 shows a silicone sheet bioreactor in which microcarriers flow into a membrane until stopped by a filter post at the end of the membrane, according to an exemplary embodiment. [Figure 17] FIG. 1B is a cross-sectional top view of a silicone sheet bioreactor showing cells on microcarriers within the area defined by posts, according to an exemplary embodiment. [Figure 18] FIG. 18 is an enlarged view of the silicone sheet bioreactor of FIGS. 16 and 17. [Figure 19] 1 shows the results of an experiment in which CHO-S cells were grown in suspension and Wagner (GFP+) cells were grown on microcarriers in a 24-well high density cell respiration plate with fins according to an exemplary embodiment. [Figure 20] 1 is a graph of experimental results of orthopoxvirus production in A549 in 24-well high density cell respiratory cell plates, the graph comparing per cell production in 24-well high density cell respiratory plates with tissue culture plates according to an exemplary embodiment. [Figure 21] 1 shows experimental results demonstrating that HEK293 cells can be transfected at high density using the membrane structures described in the disclosed technology, according to exemplary embodiments. [Figure 22] 1 shows experimental results of titration of orthopoxvirus (CF33) from high density A549 cells grown on high density cell respirator membranes, according to an exemplary embodiment. [Figure 23] FIG. 1 is a schematic diagram of an HDCR architecture configured to receive culture medium, exchange oxygen for carbon dioxide, and output waste at a static volume exchange rate (kLa) greater than about 60 / hr, e.g., greater than about 63+ / -12 (SEM) / hr. [Figure 24] 1 is a plot of cell density (cells / mL) (on the y-axis) versus days in culture (on the x-axis), according to an exemplary embodiment. [Figure 25] 1 is a plot of AAV (vg / cell) (on the y-axis) against packaging time (in hours) (on the x-axis) for samples having 4 million cells / mL and 16 million cells / mL, according to an exemplary embodiment. [Figure 26] 1 is a plot of AAV (vg / mL) (on the y-axis) against packaging time (in hours) (on the x-axis) for samples having 4 million cells / mL and 16 million cells / mL, according to an exemplary embodiment. [Figure 27] 1 is a plot of AAV (vg / cell) (on the y-axis) against packaging time (in hours) (on the x-axis) for samples having 4 million cells / mL, 16 million cells / mL, and 32 million cells / mL, according to an exemplary embodiment. [Figure 28] 1 is a plot of AAV (vg / mL) (on the y-axis) against packaging time (in hours) (on the x-axis) for samples having 4 million cells / mL, 16 million cells / mL, and 32 million cells / mL, according to an exemplary embodiment. [Figure 29] 1 is a chart of AAV (vg / cell) on the y-axis for samples with 30 million and 100 million cells / mL, comparing static and mixed modes, according to an exemplary embodiment. [Figure 30] 1 is an HDCR-produced AAV2 vector scaled to about 37 kD to about 150 kD according to an exemplary embodiment. [Figure 31] 1 is a plot of productivity (AAV vg / L) (on the y-axis) against cell density at transfection (cells / mL) (on the x-axis) as reported in the literature from 2010-2020 and compared to HDCR producer cells of the present invention according to exemplary embodiments. [Figure 32] 1 is a plot of titer (PFU / cell) (on the y-axis) against time post-infection (on the x-axis), comparing tissue culture (flask) production (TCP) with HDCR production, according to an exemplary embodiment. [Figure 33]1 is a side-by-side comparison of an AAV2-mCherry HDCR vector scaled to about 25 kD to about 250 kD versus a standard, according to an exemplary embodiment. [Figure 34] 1 is an HDCR-produced AAV2 vector scaled to about 37 kD to about 150 kD according to an exemplary embodiment. [Figure 35] 31 includes data from FIG. 31. FIG. 32 is another plot of productivity (AAV vg / L) (on the y-axis) versus cell density at transfection (cells / mL) (on the x-axis) compared to HDCR producer cells of the present invention reported in the literature from 2010-2020, further including results with relatively low productivity according to exemplary embodiments. [Figure 36] 1 is an image of an unstained protein standard (left) alongside HDCR-derived AAV2 (right), according to an exemplary embodiment. [Figure 37] 1 is an image of an unstained protein standard (left) aligned with an AAV2-mCherry HDCR vector (right) according to an exemplary embodiment. [Figure 38] 1 is a plan view image of a small-scale HDCR platform having a volume on the order of about 5 mL and a cell density on the order of about 10 12 vg, according to an exemplary embodiment. [Figure 39] 1 is a perspective image of a mid-scale HDCR platform having a volume of about 500 mL and a cell density of about 10 14 vg, according to an exemplary embodiment. [Figure 40] 1 is a perspective image of a large-scale HDCR platform with a volume of approximately 50 L and a cell density of approximately 10 16 -17 vg according to an exemplary embodiment. [Figure 41] 1 is a schematic side view of an HDCR platform having spaced apart fins configured to supply oxygen and receive carbon dioxide, according to an exemplary embodiment; [Figure 42] 1 is a schematic side view of an inclined HDCR platform in accordance with an exemplary embodiment; [Figure 43]1A-1C are schematic side views of an inclined HDCR platform configured to facilitate bubble removal (top) and condensation (bottom), according to an exemplary embodiment. [Figure 44] FIG. 1 is a bottom perspective view of an HDCR cartridge according to an exemplary embodiment. [Figure 45] FIG. 45 is a top perspective view of the HDCR cartridge of FIG. 44 according to an exemplary embodiment. [Figure 46] 46 is an enlarged portion of the top perspective view of FIG. 45 of an HDCR cartridge according to an exemplary embodiment. [Figure 47] FIG. 47 is a top view of the HDCR cartridge of FIGS. 44-46 according to an exemplary embodiment. [Figure 48] 1 is an image (4x magnification) of GFP(pAAV) 70 hours after transfection, according to an exemplary embodiment. [Figure 49] 10 is a phase image (4x magnification) 70 hours after transfection, according to an exemplary embodiment. [Figure 50] 10 is an image (4x) of RFP (pHelper+RC) 70 hours after transfection, according to an exemplary embodiment. [Figure 51] 48, 49, and 50 in accordance with an exemplary embodiment. [Figure 52] 1 is a cross-sectional cutaway view through four uncompressed stacks of HDCR membranes, illustrating the formation of a seal and alignment of the membranes, according to an exemplary embodiment. [Figure 53] FIG. 10 is a cross-sectional cut through a compressed (40% compression ratio) four stack of HDCR membranes according to an exemplary embodiment. [Figure 54] CAD rendering of an HDCR membrane with an enlarged cross-section (bottom left) showing the gas compartment openings and various compression ratios of possible designs (i.e., 20%, 30%, 40% compression ratios) according to an exemplary embodiment. [Figure 55] FIG. 1 is a front perspective view of a four-stack demo scale HDCR according to an exemplary embodiment. [Figure 56]FIG. 56 is a rear perspective view of the four-stack demo scale HDCR of FIG. 55 in accordance with an exemplary embodiment. [Figure 57] FIG. 57 is a front perspective view of a complete system for the four-stack demo scale HDCR of FIGS. 55 and 56 according to an exemplary embodiment. [Figure 58] 1 is a plot of glucose consumption (mol / hr) (on the y-axis) versus days (on the x-axis) comparing theoretical and experimental results according to an exemplary embodiment. [Figure 59] FIG. 1 is a front perspective view of a system for measuring flow rate and glucose-derived metabolism in a waste stream according to an exemplary embodiment. [Figure 60] FIG. 1 is a front perspective view of a system for measuring glucose metabolism by harvesting cells for DNA counting, according to an exemplary embodiment. [Figure 61] FIG. 1 is a front perspective view of a half-scale HDCR system including, from right to left, a media pump, a bubble trap, a gas bubbler, a 600 cm HDCR cartridge, a cell distributor, a waste container, a gas pump, and associated interconnecting tubing, according to an exemplary embodiment. [Figure 62] 1 is a plot of glucose consumption (mol / hr) (on the y-axis) versus days of operation (on the x-axis) comparing theoretical and experimental results according to an exemplary embodiment. [Figure 63] FIG. 1B is a front perspective view of a band of CF33-GFP in a first sucrose gradient according to an exemplary embodiment. [Figure 64] 1 is a plot of exosomes / day / membrane (on the y-axis) against date (on the x-axis), i.e., exosome production in a 600 cm2 HDCR with A549 according to an exemplary embodiment. [Figure 65] FIG. 1 is a front perspective view of a system including five stacks of 600 cm2 HDCR cartridges according to an exemplary embodiment. [Figure 66] 1 is a schematic cross-sectional front view of an HDCR membrane with an air bubble above the membrane and condensation below the membrane, according to an exemplary embodiment. [Figure 67] 1 is a schematic cross-sectional front view of a sloped HDCR membrane highlighting the removal of air bubbles above the membrane and the removal of condensation below the membrane (front, above, and back, below) in accordance with an exemplary embodiment. FIG. [Figure 68] 1 is a schematic cross-sectional side view of a non-graded high aspect ratio HDCR membrane filled with culture medium, according to an exemplary embodiment. [Figure 69] 1 is a representation of the cell growth volume of a non-graded high aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. [Figure 70] 1 is a schematic cross-sectional side view of a non-graded, low aspect ratio HDCR membrane filled with culture medium according to an exemplary embodiment. [Figure 71] 1 is a representation of the cell growth volume of a non-graded, low aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. [Figure 72] 1 is a schematic cross-sectional side view of a gradient high aspect ratio HDCR membrane filled with culture medium, according to an exemplary embodiment. [Figure 73] 1 is a representation of the cell growth volume of a gradient high aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. [Figure 74] 1 is a schematic cross-sectional side view of a gradient low aspect ratio HDCR membrane filled with culture medium, according to an exemplary embodiment. [Figure 75] 1 is a representation of the cell growth volume of a gradient low aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. [Figure 76] Establishing geometric variables for cell retention in rectangular channels versus tilt angle according to exemplary embodiments. [Figure 77] 1 is a plot of cell fraction held on the y-axis versus slope (degrees) on the x-axis, comparing different channel widths (legend), according to an exemplary embodiment. [Figure 78] 1 is a plot of total volume retention efficiency (on the y-axis) against slope (degrees) (on the x-axis) comparing different channel widths (legend), according to an exemplary embodiment. [Figure 79] FIG. 47 is an enlarged plan view of an HDCR membrane according to an exemplary embodiment. [Figure 80] FIG. 1 is a schematic diagram of pAAV-minCMV-mCherry as reported in the prior art. [Figure 81] FIG. 1 is a schematic diagram of pAAV-RC as reported in the prior art. [Figure 82] FIG. 1 is a schematic diagram of pHelper as reported in the prior art. [Figure 83] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 10% cell confluence and 400 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 84] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 33% cell confluence and 400 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 85] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 100% cell confluence and 400 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 86] 11 includes 11 plots of HDCR simulations of bioreactor cellularity versus architecture for 10% cell confluence and 700 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 87] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 33% cell confluence and 700 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 88]12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 100% cell confluence and 700 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 89] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 10% cell confluence and 1000 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 90] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 33% cell confluence and 1000 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment. [Figure 91] 12 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 100% cell confluence and 1000 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0174] Wherever practical, like reference numerals refer to like structures, features, or elements.

[0175] In the following detailed description, reference is made to the accompanying drawings, which form a part of this disclosure. In the drawings, like reference numerals typically identify like elements, unless the context dictates otherwise. The exemplary embodiments described in the detailed description, the drawings, and the claims are not intended to be limiting. The detailed description is intended as an illustration of exemplary embodiments and is not intended to represent the only embodiments that may be practiced. As used herein, the term "exemplary" means "serving as an example, instance, or illustration" and should not necessarily be construed as preferred or advantageous over other embodiments. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the features of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are expressly contemplated by and form a part of this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0176] The present systems, devices and methods increase cell density while maintaining metabolically efficient producer or packaging cells. By increasing cell density while maintaining metabolically efficient producer or packaging cells, space-time productivity is improved in terms of productivity per cell.

[0177] As used herein, the term "high density" in the context of high density cell culture (and the like) can have specific meanings, including: In most processes developed for AAV production, the cell density is about 10 6 A cell density of 10 cells / mL was achieved, which translates to approximately 0.1% by volume, or 1 cell per nL of medium. 7 and 10 8 The cell densities are 1% and 10% by volume, respectively. 9The cell density of 10 corresponds to the tissue level density. Then, we increased the cell density in the bioreactor to 10 6 We focused on those that limit the range to cells / mL.

[0178] As a first approximation, the need for gas exchange means that the cell density in the bioreactor must be 10 6 In terms of the metabolic demands of cells, the required volumetric mass transfer coefficient for oxygen can be calculated and compared to, for example, glucose. 6 For 10 cells / mL, oxygen in the medium should be exchanged approximately once per hour. 7 For 10 cells / mL, oxygen in the medium should be exchanged approximately 8 times per hour, and for 10 8 For cells / mL, oxygen in the medium needs to be exchanged approximately once per minute. In contrast, when considering the mass transfer volumetric coefficient for glucose, the exchange rate is 100 times lower than oxygen exchange, and the same is true for waste excretion and other nutrients. Therefore, existing developed bioreactors (STR and fixed-bed) operate under a mixing / perfusion regime due to the need to oxygenate cells, but with a 100-fold excess for soluble nutrients. Ultimately, shear limitations limit the oxygen exchange potential. Decoupling the exchange of gases and soluble nutrients greatly reduces the need for perfusion / mixing, enabling higher density nodes.

[0179] The high density cellular respiration (HDCR) systems, devices and methods of the present invention, as well as rational variations thereof, separate gas and nutrient exchange, reduce the need for perfusion / mixing, and achieve higher density notes than in existing developed technologies.

[0180] Uses and Needs Embodiments of the high density cell respirator (HDCR) described herein can be used to generate bioproducts for a wide variety of applications, including vectors such as adeno-associated viruses (AAVs) for gene transfer and editing for inborn errors of metabolism and neurological diseases (e.g., hemophilia, sickle cell anemia, thalassemia, Parkinson's disease, Alzheimer's disease), oncolytic viruses for cancer killing and immunotherapy, stem cell expansion and gene editing for liver failure, artificial skin, and replacement of damaged tissue, including HIV infection, among others, antibodies and therapeutic proteins, and metabolites and other cell-based products.

[0181] As used herein, the term "bioproduct" and variations can refer to a virus, virus-like particle, bacteriophage, protein, recombinant protein, antibody, metabolite, cell, eukaryotic cell, bacterial cell, algae, organelle, vacuole, mitochondria, lipid, exosome, DNA, RNA, plasmid, antibiotic, etc.

[0182] As used herein, the term "virus" and variations include adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah Forest virus, Bunyamwera virus, Bunyavirus La Crosse, snowshoe hare bunyavirus, cellular copiethecin herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A, cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dorivirus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus C / G hepatitis virus, Hantaan virus, Hendra virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, hepatitis delta virus, horsepox virus, human adenovirus, human astrovirus, human coronavirus, human cytomegalovirus, human enterovirus 68, 70, human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human Papillomavirus type 1, human papillomavirus type 2, human papillomavirus types 16 and 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Lassa virus, Rhodesdale virus, louping ill virus, lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, molluscum contagiosum virus, monkeypox virus, mumps virus,Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Sheeppox virus, Olopouche virus, Pichinde virus, Poliovirus, Punta Toro phlebovirus, Puumala virus, Rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sandfly fever Sicilian virus, Sapporo virus, SARS coronavirus 2, Semliki Forest virus, Seoul virus, Simian virus It may refer to natural, modified, chimeric, and recombinant viruses and virus-like particles based on Woamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, tick-borne Powassan virus, Torque teno virus, Toscana virus, Ukuniemi virus, vaccinia virus, varicella-zoster virus, smallpox virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, yellow fever virus, Zika virus, etc.

[0183] The use of AAV vectors for gene therapy and editing is being actively investigated and is approaching clinical treatment, including the treatment of inborn errors of metabolism and genetic manipulation of stem cells. Phase I / II trials are also seeing approximately 10 17More than 100 vector genomes (vg) may be required. AAV vector production capacity and cost remain the biggest obstacles to widespread use of AAV gene therapy. Similarly, the production of sufficient vectors and the associated costs pose a major obstacle to making cancer-fighting immuno-oncolytic viruses accessible to all humankind. Recently, immuno-oncolytic viruses have been approved in the US and EU for the treatment of melanoma, and many more viruses are approaching approval. The disclosed technology enables a cell culture platform that can disrupt existing vector production methods in gene vectors and immuno-oncolytic viruses, two fields that have produced clinical products in the 21st century. It significantly increases efficiency and reduces both the production time (2-10 fold) and cost (10 fold) of therapeutic vectors.

[0184] Viruses and viral vectors have become extremely important not only as experimental drugs but also as pharmaceuticals for gene therapy and cancer treatment, as well as vaccines for infectious diseases. AAV vectors serve as effective gene transfer and gene editing tools that hold promise for the treatment of many inherited and acquired diseases. AAV vectors are being intensively investigated as treatments for, for example, inborn errors of metabolism, defective structural and secreted protein production, and potentially for the treatment of diverse diseases such as hemophilia, thalassemia, sickle cell anemia, mucopolysaccharidoses, cystic fibrosis, and muscular dystrophy. One AAV-based therapeutic (Glybera, uniQure, NV, Amsterdam) has already been approved by the European Union for the treatment of lipoprotein lipase deficiency. It was also approved in 2017 for the treatment of hereditary blindness (Spark Therapeutics). The dosage of Glybera's recombinant AAV viral particles is 10 16The cost per patient is $1.2 million, due to the small number of viruses. Similarly, Spark's blindness treatment Luxturna costs approximately $850,000 per patient. Novartis' AAV Zolgensma for SMA costs $2.1 million per dose, due in part to the cost of virus production. Recent successful clinical trials have exponentially increased the demand for high-quality, functionally active vectors. A common problem throughout the field is producing enough viral vectors for clinical trials. Even Phase I / II trials require approximately 10 17 ~10 18 Each virus particle requires 1000,000, typically costing millions of dollars and taking more than a year to achieve. During this lengthy production process, a single contaminant can decimate an entire program or company. Recent successes in clinical trials have dramatically increased the demand for high-quality, functional, active vectors, a trend expected to continue for decades to come. Therefore, designing faster and cheaper production methods will enable more products to be tested in clinical trials, making clinically proven gene editing tools more affordable for society.

[0185] Immuno-oncolytic virotherapy treats cancer with viruses that can not only directly infect and kill cancer cells but also reduce recurrence by inducing durable immunological memory. An immuno-oncolytic herpesvirus (T-VEC, Amgen, Thousand Oaks, California) has been approved for use in melanoma in the United States and Europe. An adenovirus (Oncorine, SunwayBio, Shanghai, China) has been approved for use in China for head and neck cancer. Many other viruses are in clinical trials, both as single agents and in combination with other immune-stimulating molecules, with promising results. These and most other viruses under investigation cost thousands of dollars per dose. Producing many promising viral constructs for clinical trials has been challenging. Keeping the cost of viral drugs low enough so that these drugs are accessible to cancer patients in poor countries has also been challenging. Therefore, methods to increase production efficiency and reduce costs could significantly contribute to saving lives. The development of an orthopoxvirus production process also serves as proof of concept for rapid vaccine development in response to pandemics or bioterrorism.

[0186] AAV is a replication-deficient, nonpathogenic parvovirus with a single-stranded DNA genome that infects both dividing and non-dividing cells. Because AAV can safely and efficiently transduce the liver, heart, muscle, and other organs, the development of gene therapy vectors for the treatment of genetic diseases has been intensively pursued over the past 20 years. The therapeutic benefits of AAV gene therapy are predicted to be sustained. Clinical gene therapy trials of AAV have yielded highly promising results, particularly in spinal muscular atrophy (SMA), hemophilia, and multiple forms of blindness. More recently, AAV vectors have also been used as delivery agents for components of genome editing platforms and have been shown to be effective and efficient, particularly when used in vivo. Therefore, AAV vectors are likely to play an important role in clinical genome editing. They may also be useful vectors for the genetic manipulation of stem cells for therapeutic purposes.

[0187] Despite the great promise of AAV vectors for genetic medicine, the single biggest limitation to their widespread use is the inability to produce sufficient quantities of AAV vectors in a timely and cost-effective manner. Currently, clinical GMP-grade AAV is produced in a few commercial and academic facilities worldwide.

[0188] However, the relative inefficiency of production and the use of labor-intensive processes have resulted in widespread inability to meet current demand. As a result, there is currently a wait time of approximately two years for clinical-grade AAVs. This situation has been exacerbated by major pharmaceutical companies acquiring AAV-producing CMOs to accelerate their own timelines. The ability to rapidly produce high-titer AAV vectors at low cost is expected to be a game changer in the fields of gene therapy and genome editing. Without increased production capacity and the ability to produce AAV vectors commercially available, many discoveries may never be commercialized.

[0189] Even in Phase II clinical trials, approximately 10 17 More than 100,000 vector genomes are needed. Currently, these drugs cost anywhere from hundreds of thousands to over a million dollars, so vector production and cost are major obstacles to making these vectors affordable for humanity. Such staggering figures are not sustainable for global therapeutics. Production would need to be sped up approximately 2-10 times, and efficiency and yield would need to be increased approximately 10-fold. If these goals are achieved, more candidates would be able to participate in clinical trials, and products would become more affordable.

[0190] The HDCR embodiments described herein can also be used for virus and vaccine production. Increasing viral productivity is useful in the growing field of oncolytic virotherapy. Oncolytic viruses are viruses engineered to specifically infect, replicate within, and kill cancer cells. These viruses can carry genetic payloads that stimulate the host's immune system to produce proteins at the cancer site that lead to a robust, systemic, and sustained immunological response to cancer. Two such viruses (one based on adenovirus and one based on herpes simplex virus) have been approved for human use as cancer treatments. Many other viruses are currently under investigation, including those based on vaccinia, myxoma, vesicular stomatitis, and measles, among others. Clinical trials combining oncolytic viruses with immune checkpoint inhibitors have also shown great promise as potential human cancer therapies. Cost is also a consideration in the production of oncolytic viruses. The cost of a six-month course of oncolytic virus therapy using TVEC currently is approximately $65,000. Reducing production costs will lead to increased global availability. Reducing virus production time will also lower barriers to clinical trials of novel agents.

[0191] Improvements in virus production technology also impact vaccine development. Viruses routinely produced for vaccines include vaccinia, VSV, measles, and HSV. Current influenza vaccines are made using an egg-based production process. Currently, egg-based vaccine production suffers from problems such as virus strain mutations, which contribute to low vaccine efficacy, low vaccine immunogenicity, and egg allergies, which limit the number of people eligible for vaccination. The HDCR described herein optimizes virus production to exceed the efficiency of current egg-based production methods, potentially enabling higher yields, faster production, and access to vaccines for all humanity.

[0192] Due to cost and volume pressures, cell-based production of viral vectors has evolved from Petri dishes to flasks, roller bottles, cell factories, stirred tank reactors, and finally perfusion-based reactors (e.g., hollow fiber), a technology that has persisted for decades. These systems rely on the transport of nutrients, growth factors, waste products, and oxygen / carbon dioxide through the culture medium. Maximum cell density in bioreactor systems has historically been limited by gas exchange due to the low solubility of gases in the medium and the high oxygen uptake of cells. Efforts to increase oxygen delivery by mixing or gas sparging are ultimately limited by the negative effects of shear forces on cell viability. Approximately 10 6 At 100 cells / mL (a common density in many systems), only approximately 0.1–1% (v / v) of the reactor space is occupied by the cells. Recent attempts at high-density cell culture for vaccine production using perfusion bioreactors have overcome past density limitations of continuous and batch-fed cultures, but the associated costs have not decreased significantly, due in part to increased use of specific media and reduced virus yields in these particular systems.

[0193] HDCR The disclosed technology advances bioproduction by decoupling gas and soluble nutrient delivery in a unique design that increases oxygen delivery, protects cells from shear forces, and efficiently utilizes media to achieve significantly improved space-time production capacity. Embodiments include a high-density cell respirator (HDCR) comprised of a gas-perfusable, highly permeable, stackable cell-retaining membrane, which allows direct oxygenation of the cell niche via membrane permeation and diffusion. Direct oxygenation is important because molecular oxygen (O) has low solubility in cell media and may be considered the most limiting metabolite for cell growth. By decoupling gas convective delivery from other nutrient delivery, oxygen delivery to cells can be significantly increased without increasing harmful shear forces. The HDCR cartridge is immersed in an independently perfusable media stream, which allows for the delivery of soluble nutrients or reagents, waste removal, and bioproduct harvest. These designs are suitable for cell densities within the compartments of >approximately 10 8 They can accommodate tissue-level cell counts of cells / mL, or Vcell / Vbioreactor ratios of approximately 10%-25%, representing a 100-fold improvement over conventional bioreactors. Augmenting cell-based production processes allows for more efficient use of expensive and limited Good Manufacturing Practice ("GMP") space.

[0194] Various implementations described herein include the integration of surface features into the membrane design. For example, the surface features may be fins, wells, or posts. These surface features serve two important functions:

[0195] First, the surface features create shear-protected compartments in which adherent, suspension, or microcarrier-attached cells can grow. In some embodiments, adjacent fins form grooves that correspond to the compartments. In other embodiments, wells correspond to the compartments. In still other embodiments, posts form compartments for cells that adhere to the posts. These compartments are essential for virus production because, in conventional systems, cells lose their adherence (cytopathic effect (CPE)) during virus production, resulting in washout. Because existing methods, including acoustic filters, tangential flow filtration ("TFF"), and continuous centrifugation, are expensive and complex, calls for new means of cell retention in high-density cell culture systems have repeatedly appeared in the literature.

[0196] Second, the surface features are highly gas permeable, facilitating gas exchange around / within the cell niche. This increases the oxygen mass transfer volumetric coefficient (i.e., kLa), allowing for higher cell densities to be achieved. Furthermore, the surface features constrain the size of cell aggregates, ensuring that all cells receive the necessary amount of oxygen (i.e., they are not diffusion limited). Overcoming gas / nutrient gradients has been a major challenge in the literature, but this disclosure overcomes this challenge.

[0197] term As used herein, a "compartment" is a portion of a membrane structure that has the ability to spatially confine one or more cells (or similarly sized substances, including viruses, organelles, liposomes, carriers (such as beads), etc.) to a portion of the membrane surface relative to the total surface of the membrane. Compartments can include a variety of structures and shapes, including, but not limited to, fins, posts, wells, channels, cages, and combinations of these features. Compartments can have any shape, structure, geometry, or architecture, including those described herein. For example, compartments may include circular, oval, elliptical, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, irregular or other shapes, openings, sides, bottoms, etc.

[0198] "Gas-permeable" or "semi-permeable" materials, such as membranes, include those that allow gas to pass through but prevent the passage of liquid water, or as otherwise applicable and known in the art. Permeability can be measured at standard room temperature (i.e., 25°C) and atmospheric pressure, or other applicable temperature and pressure. For example, the standard temperature for mammalian cell culture is 37°C. For thermophiles, the temperature can be as high as 122°C. Pressure can be adjusted higher or lower than standard atmospheric pressure, such as by hydrostatic pressure from submersion. The gas can be molecular oxygen, carbon dioxide, carbon monoxide, nitrogen, normal air, carbon monoxide, nitrous oxide, hydrogen sulfide, ethylene oxide, ozone, chlorine dioxide, nitrogen dioxide, or other applicable gas.

[0199] A "fin" includes a long, thin, raised structure protruding above a surface. The fin may have a substantially constant polygonal and / or curved cross-sectional shape along the length of the fin.

[0200] An "array of fins" includes a series of fins projecting above a surface with a substantially constant spacing along the length of the fins between adjacent fins in the series. The array of fins may be aligned and substantially parallel to one another.

[0201] A "groove" is an elongated recess formed by adjacent fins. The cross-sectional shape of the groove can be defined by the shape of the walls of the adjacent fins that define the groove and the portions of the surface from which the adjacent fins protrude. The adjacent fin walls and portions of the surface can form three sides of the groove's cross-section. The groove can have an opening facing the surface. The opening can be narrower or wider than the width of the groove between the walls of the adjacent fins that form the groove. If the surface is horizontal and the fins protrude upward, the opening is at the top of the groove.

[0202] An "array of grooves" includes a series of grooves formed by adjacent fins of an array of fins. The grooves may have a substantially constant spacing along the length of the groove between adjacent grooves in the series of grooves. The array of grooves may be aligned and substantially parallel to one another.

[0203] "Pillars" or "micropillars" include columns or other structures that extend perpendicularly from a surface or as otherwise known in the art. "Micropillars" include small pillars and are not limited to pillars on the micrometer (micron) or microinch scale.

[0204] An "array of spacers" includes micropillars or other spacers in a geometrically regular or irregular pattern, having a spacer height and center-to-center spacing configured to keep the sheets at a fixed distance from one another, or otherwise known in the art.

[0205] A "crush-resistant bag" includes a bag that has spacers inside it to prevent the interior surfaces of opposing sides from contacting each other during normal operation.

[0206] An "expansion-resistant bag" includes a bag having internal welds, attached spacers, or other connections that prevent the bag from assuming a balloon-like shape when pressurized, or other connections as otherwise known in the art. Expansion-resistant bags may be substantially flat. They may have convex and concave depressions and curvatures.

[0207] "Hermetically sealed" simply means airtight, or at least impermeable to liquids, but possibly permeable to gases, or otherwise sealed in other ways known in the art.

[0208] In an embodiment, a cell culture device is provided, the cell culture device including: a membrane that may include a plurality of surface features on a first side of the membrane, the surface features including one or more compartments capable of trapping one or more cells, the membrane including a material that is at least partially permeable to gas; a second side of the membrane that defines a boundary of a gas region, the membrane configured to allow gas to pass through the second side of the membrane to the first side of the membrane; and a medium region having a boundary on the first side of the membrane and configured to pass medium across the first side of the membrane, the one or more compartments configured to at least partially reduce medium flow shear forces within the compartments.

[0209] In embodiments, the device may further include one or more medium inlets and one or more medium outlets fluidly connected to the medium region, the one or more medium inlets configured to facilitate introduction of medium into the medium region on the first side of the membrane, and the one or more medium outlets configured to facilitate removal of medium from the medium region.

[0210] In embodiments, at least one of the one or more medium inlets is fluidly connected to at least one of the medium outlets to facilitate reintroduction of medium into the medium region on the first side of the membrane.

[0211] In an embodiment, the medium introduced into the medium region is in a liquid state.

[0212] In embodiments, the medium removed from the medium region comprises at least one of a plurality of cells, spent medium, cellular waste products, secretions, and biological material from the plurality of cells.

[0213] In embodiments, the device is configured to minimize medium flow shear forces in one or more compartments upon introduction or removal of medium from the medium region.

[0214] In an embodiment, the apparatus further includes one or more gas inlets and one or more gas outlets in fluid contact with the gas region, the one or more gas inlets configured to introduce gas into the gas region and the one or more gas outlets configured for removal of gas from the gas region.

[0215] In an embodiment, the device further includes one or more channels formed on a second side of the membrane, at least a first channel of the one or more channels configured to deliver gas through the membrane to at least one of the compartments by permeation and diffusion through the membrane.

[0216] In embodiments, the device further includes an enclosure disposed above the first side of the membrane.

[0217] In an embodiment, the enclosure defines a second boundary of the medium region.

[0218] In embodiments, the device is configured to be stacked with at least a second cell culture device.

[0219] In embodiments, one or more of the medium inlets or gas inlets or medium outlets or gas outlets are configured such that when stacked, the one or more of the medium inlets or gas inlets or medium outlets or gas outlets are in fluid communication with the inlet or outlet of an immediately adjacent device.

[0220] In an embodiment, one or more of the medium inlets or gas inlets are configured such that when stacked, medium or gas introduced into the device is combined and enters a combined inlet line for delivery to multiple membranes.

[0221] In embodiments, one or more of the medium outlets or gas outlets are configured such that when stacked, medium or gas removed from the device is combined and enters a combined outlet line for delivery from the multiple membranes.

[0222] In embodiments, liquid medium and gas are perfused independently over each membrane of the stacked culture device by separate gas and medium delivery tubes connected to a liquid-tight container for containing the stackable membranes.

[0223] In an embodiment, the device further comprises a liquid-tight container for holding the device.

[0224] In embodiments, the membrane comprises pores having a size that allows the permeation and diffusion of oxygen through the membrane.

[0225] In embodiments, perfusion of liquid medium over the first surface of the membrane introduces cells into one or more compartments.

[0226] In embodiments, culture medium perfused over the first surface of the membrane delivers nutrients or reagents to one or more compartments.

[0227] In embodiments, medium perfusion over the first surface of the membrane allows for waste removal from one or more compartments.

[0228] In embodiments, medium perfusion over the first surface of the membrane allows for harvesting of a bioproduct from one or more compartments.

[0229] In embodiments, one or more compartments contain about 10 per milliliter 8 Support tissue-level cell densities above 1000kJ / Vcell / Vbioreactor, or above approximately 10%-25% Vcell / Vbioreactor, where Vcell is the volume of cells and Vbioreactor is the volume of reactor space cell growth.

[0230] In embodiments, at least one of the plurality of surface features is formed from micromachined silicone and facilitates gas exchange via permeation of the membrane surrounding one or more compartments.

[0231] In embodiments, the silicone surface features are configured to facilitate gas exchange that occurs via permeation of the membrane into one or more compartments.

[0232] In an embodiment, the negative pressure deforms the bottom surface of at least one of the one or more compartments downward, drawing liquid medium into the at least one compartment through an opening at the top of the at least one cell niche, and / or the positive pressure deforms the bottom surface of at least one of the one or more cell niches upward, pushing liquid medium out of the at least one compartment through an opening at the top of the at least one compartment.

[0233] In embodiments, mechanical stretching of the membrane widens at least one of the one or more compartments to facilitate the release of one or more cells from at least one cell niche through an opening in the top of the at least one compartment.

[0234] In embodiments, the device further comprises a plurality of microdiffusers configured to rectify the pulsating pressure into a net flow normal to the first surface of the membrane.

[0235] In embodiments, the device further comprises at least one porous wick configured to transport the fluid medium to one or more compartments.

[0236] In embodiments, the membrane comprises a micropatterned architecture having multiple compartments designed to provide a significantly high gas exchange area-to-volume ratio to maximize the oxygen permeation rate into the compartments formed in the membrane.

[0237] In an embodiment, the membrane is configured to expand or contract in response to changes in pressure or flow associated with the gas.

[0238] In embodiments, the membrane is configured to expand or contract in response to changes in pressure or flow associated with the liquid medium.

[0239] In embodiments, the membrane is configured to expand or contract in response to changes in pressure or flow associated with at least one of the gas and liquid medium to promote uniform distribution of reagents or cells in one or more compartments.

[0240] In embodiments, the membrane is configured to expand or contract in response to changes in pressure or flow associated with at least one of the gas and liquid medium to facilitate efficient cultivation or harvesting of cells located within one or more compartments within the membrane.

[0241] In embodiments, the fluid path for the medium is gravitationally supported by one or more flow regulators that regulate the flow of liquid medium across the first side of the membrane.

[0242] In embodiments, the cell culture medium further comprises a plurality of microcarriers to which the cells can adhere.

[0243] In an embodiment, the plurality of surface features include a plurality of fin structures extending substantially parallel to one another, the substantially parallel fin structures defining grooves between adjacent fin structures, at least one groove on the first side of the membrane providing at least one of the one or more compartments for cell positioning, and a longitudinal direction of the at least one groove corresponding to a longitudinal direction of the one or more compartments.

[0244] In embodiments, the one or more medium inlets are configured to introduce medium into the medium region to create a medium flow that is not parallel to the length of the channel.

[0245] In embodiments, the one or more medium inlets are configured to introduce medium into the medium region to create a medium flow substantially perpendicular to the grooves.

[0246] In embodiments, the one or more medium inlets are configured to introduce medium into the medium region to create a medium flow substantially aligned with the grooves.

[0247] In embodiments, the opening at the top of one or more of the one or more cell niches is narrower than the width of the opening below.

[0248] In embodiments, a plurality of fins protrude from the base of the membrane to retain and protect one or more of the cells within the one or more cell niches from media flow shear forces generated by media delivery.

[0249] In an embodiment, the membrane has a multi-layer monolithic structure such that one or more channels for delivering gas are formed in a first layer of the membrane and compartments are formed between multiple fins in a second layer of the membrane above the first layer.

[0250] In an embodiment, a dedicated space is provided above the compartments formed between the fins to support a fluid path for liquid medium flowing above the fins and substantially perpendicular to the fins.

[0251] In an embodiment, a dedicated space is provided above the compartments formed between the fins to support a fluid path for liquid medium flowing above the fins and substantially aligned with the fins.

[0252] In embodiments, the fluid path has relatively low resistance to liquid medium flowing over the plurality of fins, thereby eliminating or reducing the need for the use of pumps to regulate liquid medium flow.

[0253] In embodiments, the surface features include a plurality of well structures, at least one of the plurality of well structures providing at least one of the one or more compartments for cell placement.

[0254] In embodiments, an opening in the top of at least one of the well structures includes a diametric width that is narrower than the diametric width of the well structure below the opening.

[0255] In embodiments, one or more of the well structures includes an opening that is circular, oval, square, rectangular, hexagonal, or octagonal.

[0256] In embodiments, the well structure protects one or more cells within one or more cell niches of the well structure from shear forces caused by media delivery.

[0257] In an embodiment, the membrane has a multi-layer monolithic structure such that one or more channels for delivering gas are formed in a first layer of the membrane and compartments are formed within well structures in a second layer of the membrane above the first layer.

[0258] In an embodiment, a dedicated space is provided above the plurality of well structures to support a fluid path for liquid medium flowing above the plurality of well structures.

[0259] In embodiments, the fluid pathway has relatively low resistance to liquid medium flowing over the multiple well structure, thereby eliminating or reducing the need for the use of pumps to regulate liquid medium flow.

[0260] In an embodiment, a dedicated space is provided above the plurality of well structures to support a fluid path for liquid medium flowing above the plurality of well structures.

[0261] In an embodiment, at least one well structure has a circular opening.

[0262] In an embodiment, at least one well structure has a polygonal opening.

[0263] In an embodiment, at least one well structure has a curved opening.

[0264] In embodiments, the surface structure comprises a plurality of post structures, at least one post structure providing at least one of a plurality of compartments for cell placement.

[0265] In embodiments, the cells are attached to at least one post structure.

[0266] In embodiments, the membrane has a multi-layer monolithic structure such that one or more channels for delivering gas are formed in a first layer of the membrane and a cell niche is formed proximal to the post structure in a second layer of the membrane above the first layer.

[0267] In an embodiment, a dedicated space is provided above the plurality of post structures to support a fluid path for liquid medium flowing above the plurality of post structures.

[0268] In embodiments, the fluid pathway has relatively low resistance to liquid medium flowing over the plurality of post structures, thereby eliminating or reducing the need for the use of pumps to regulate liquid medium flow.

[0269] In an embodiment, a dedicated space is provided above the plurality of post structures to support a fluid path for liquid medium flowing above the plurality of post structures.

[0270] In an embodiment, at least one post structure has a circular cross-section.

[0271] In an embodiment, at least one well post has a polygonal cross-section.

[0272] In embodiments, at least one post structure has a curved, angled, crescent, or U-shaped cross-section.

[0273] In embodiments, the device further includes a Petri dish configured to receive the membrane.

[0274] In an embodiment, the membrane forms the bottom surface of a Petri dish.

[0275] In embodiments, the membrane further includes a tension ring configured to keep the membrane in tension and provide additional stiffness.

[0276] In embodiments, the membrane further comprises spacing pillars protruding from a second surface of the membrane opposite the first surface, the spacing pillars configured to allow gas exchange through the membrane to the cell niche.

[0277] In an embodiment, a Petri dish is provided that includes a bottom surface and sidewalls forming a dish, the bottom surface comprising at least a partially gas permeable material, the bottom surface comprising a first side and a second side, the first side comprising a plurality of structures extending from a base of the first side to form one or more cell niche regions below an upper surface of the plurality of structures, and the sidewalls connected to the bottom surface to form a continuous and substantially vertical wall around the periphery of the bottom surface.

[0278] In an embodiment, the bottom surface and the sidewalls comprise the same material.

[0279] In an embodiment, the bottom surface and sidewalls comprise at least a portion of a gas permeable material.

[0280] In an embodiment, the sidewall comprises at least one material different from the bottom surface.

[0281] In embodiments, the device further includes one or more retaining elements that provide support to the sidewalls.

[0282] In embodiments, the bottom surface of the Petri dish exposed to the external environment comprises an at least partially gas-permeable material such that gas from the external environment can pass at least partially through the gas-permeable membrane to the first side and / or gas from the first side can pass from the first side at least partially through the gas-permeable membrane to the external environment.

[0283] In embodiments, the bottom of the Petri dish is placed on a mesh, cloth, or other open-pore material to allow gases from the external environment to exchange with the membrane.

[0284] In embodiments, the bottom surface of the Petri dish, which is exposed to the external environment, includes one or more variations in its shape that allow gas to pass at least partially underneath the bottom surface when the dish is placed on a flat surface.

[0285] In embodiments, the shapes include pillars, channels, grooves, bumps, protrusions, or legs.

[0286] In an embodiment, the features include one or more spacing pillars.

[0287] In an embodiment, the Petri dish further comprises a top surface, the top surface comprising a sealing membrane.

[0288] In an embodiment, the top surface comprises a silicone-based membrane.

[0289] In embodiments, the Petri dish includes a sealable port for transferring media to or from the Petri dish.

[0290] In embodiments, a membrane is provided that includes a plurality of surface features on a first side of the membrane, the surface features including one or more compartments capable of entrapping one or more cells, and the membrane includes a material that is at least partially permeable to gas.

[0291] In embodiments, a multi-well cell growth device is provided that includes a plurality of wells, the wells including a bottom having a first side that contacts the interior of the well and a second side that contacts the exterior of the multi-well cell growth device, the first side including a topography that provides a plurality of cell growth compartments, the bottom comprising a material that is at least partially permeable to gas, and the bottom configured such that gas from outside the multi-well growth device can pass from the exterior to the interior of the well to contact one or more of the plurality of cell growth niches, and / or gas from within one or more cell growth niches can pass from the first side to the second side.

[0292] In embodiments, the topography of the first side includes one or more of fins, subwells, and pillars, the fins, subwells, and pillars at least partially defining a plurality of cell growth compartments.

[0293] In an embodiment, the sidewalls of the plurality of wells at least partially comprise a gas permeable material.

[0294] In embodiments, the second side includes one or more variations in its shape that allow gas to pass at least partially beneath the bottom surface when resting on a flat surface.

[0295] In embodiments, the shapes include pillars, channels, grooves, bumps, protrusions, or legs.

[0296] In an embodiment, the features include one or more spacing pillars.

[0297] In an embodiment, a cell culture device is provided, the cell culture device including: a membrane that may include a plurality of fin structures extending substantially parallel to one another, the substantially parallel fin structures defining grooves between adjacent fin structures, at least one groove on a first side of the membrane providing a compartment for at least cell placement, the membrane including a material that is at least partially permeable to gas; a second side of the membrane defining a gas region, the second side of the membrane being separated from the first side of the membrane by the membrane, allowing gas to pass through the membrane; and a medium region on the first side of the membrane configured to receive medium containing one or more cells that can be deposited within the compartment.

[0298] In embodiments, a cellular respirator device is provided, the cellular respirator device including one or more membranes and at least one membrane of the one or more membranes having a plurality of fins including at least a first fin and a second fin protruding from a first surface of the membrane to form a first compartment of a plurality of compartments configured to hold a plurality of cells, the membrane being formed from a gas-permeable and air-permeable material to facilitate delivery of gas to the plurality of compartments through one or more channels formed below the first surface of the membrane below the plurality of fins, the at least first channel being configured to deliver gas directly to the first compartment.

[0299] In an embodiment, there is provided a method for culturing biological cells, the method comprising providing a device according to any one of claims 1 to 83, introducing biological cells into the device, and providing a cell culture medium on a first side of the device such that the cell culture medium contacts the biological cells.

[0300] In an embodiment, the method further comprises providing a gas to a second side of the membrane.

[0301] In embodiments, the method further comprises flowing culture medium over one or more cell niches.

[0302] In an embodiment, flowing the medium comprises introducing the medium through an inlet and removing the medium through an outlet.

[0303] In an embodiment, the method further comprises flowing a gas onto a second side of the membrane.

[0304] In an embodiment, flowing the gas includes introducing the gas through an inlet and removing the gas through an outlet.

[0305] In embodiments, the method further comprises causing medium, biological cells, or another substance to flow into one or more cell niches by generating negative pressure in one or more compartments.

[0306] In embodiments, the method further comprises maintaining the cells in the one or more compartments by creating a negative pressure in the one or more compartments.

[0307] In embodiments, the method further comprises causing medium, biological cells, or another substance to flow out of one or more cell niches by generating a positive pressure in one or more compartments.

[0308] In embodiments, the method further comprises directing the cells out of the one or more compartments by creating a positive pressure within the one or more compartments.

[0309] In embodiments, the method further comprises mixing one or more of the biological cells in the one or more compartments by creating positive and / or negative pressure in the one or more compartments.

[0310] In embodiments, positive and / or negative pressure is generated by flowing gas into a gas region, flowing medium into a medium region, flowing both gas and medium into their respective regions, preventing the flow of gas into the gas region, and / or preventing the flow of medium into the medium region, thereby creating a pressure differential between the two regions.

[0311] The snowflake is a snowflake and a snowflake Layers, keys, 3T3-L1, 4T1, 9L, A20, A1 72. A253, A431, A549, A2780, A2780ADR, A2780cis, AB9, AHL-1, ALC, B 16. B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BOSC23, BT-20, BxPC-3, C2 C12, C3H-10T1 / 2, C6, C6 / 36, Caco-2, Cal-27, Calu-3, CGR8, CHO, CML T1, CMT12, COR-L23, COR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23, COS-7, COV-434, CT26, D17, DAOY, DH82, DU145, DuC aP, E14Tg2a, vEL4, EM-2, EM-3, vEMT6 / AR1, EMT6 / AR10.0, FM3, GL261, H1299, HaCaT, HCA2, HEK293, HEK293T, HeLa, Hep G2, Hepa1c1c7, High Five, HL-60, HT-29, HT-1080, J558L, Jurkat, JY, K562, KBM-7, KCL-22, KG1, Ku812, KYO-1, L243, L1210, LNCa P, MA-104, Ma-Mel, MA2.1, MC-38, MCF-7, MCF-10A, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, MDCK II, MG63, MIA PaCa-2, Mono-Mac-6, MOR / 0.2R, MRC-5, MTD-1A, MyEnd, NALM-1, NCI-H69, NCI-H69 / CPR, NCI-H69 / LX4, NCI-H69 / LX10, NCI-H69 / LX20, Neuro-2a, Neuro2a, NIH-3T3, NK-92, NTERA-2, NW-145, OK, OPCN / OPCT cell line, P3X63Ag8, PANC-1, PC-3, PC12, Peer, PNT1A, PNT2, Pt Selected from the group consisting of K2, Raji, RBL-1, RenCa, RIN-5F, RMA-S, S2, SaOS-2, Sf9, Sf21, SH-SY5Y, SiHa, SK-BR-3, SK-N-SH, SK-OV-3, T-47D, T2, T84, T98G, THP-1, U2OS, U87, U373, U937, VCaP, Vero, VG-1, WM39, WT-49, YAC-1, and YAR cells.

[0312] In an embodiment, the gas is selected from the group consisting of oxygen, carbon dioxide, nitrogen, carbon monoxide, nitrous oxide, hydrogen sulfide, ethylene oxide, ozone, chlorine dioxide, and nitrogen dioxide.

[0313] In embodiments, the culture medium is selected from the group consisting of DMEM, FCS, 293SFM II, AEM, CDM4HEK293, SFM4HEK293, Ex-Cell293, SFM4Transfx-293, Freestyle293, ESF SFM, CDM4CHO, CHO Medium, MEM, MEM Alpha, RPMI, F-10, F-12, IMDM, Medium 199, Leibovitz L-15, McCoy's 5A, MCDB Medium, William's Medium, CMRL Medium, OptiMEM, OptiPro, AIM V, OptiPEAK T Lymphocyte, ExCellerate Human T Cell Expansion Medium, StemXVivo Serum-Free Human T Cell Base Medium, ExpiSf CD Medium, Sf-900 II / III SFM, and TC-100 Insect Medium.

[0314] In embodiments, the other material is selected from the group consisting of waste materials, materials secreted by cells, materials inside cells, cell debris, and gases.

[0315] In embodiments, the method further includes introducing a cryogen into the gas region to freeze cells and cellular components in the multiple compartments, wherein the cryogen has a temperature below 0°C and the cryogen is in a gaseous or liquid state.

[0316] In embodiments, the membranes and devices disclosed herein can be used in methods for isolating cells. For example, the method for isolating cells can include pulsating with medium to generate turbulent flow and flow to a collection outlet. Alternatively, the method for isolating cells can include generating pressure from a gas region to push a portion of the membrane upward and / or inward from the side of one or more compartments, thereby urging cells out of one or more compartments. Furthermore, the method for isolating cells can include a combination of pulsating with medium to generate turbulent flow and generating pressure from a gas region to push a portion of the membrane upward and / or inward from the side of one or more compartments, thereby urging cells out of one or more compartments. Furthermore, the method for isolating cells can include mechanically stretching the membrane in combination with flowing medium across the membrane. Alternatively, the method for isolating cells can include mechanically stretching the membrane, causing the membrane to collapse and release cells from one or more compartments.

[0317] In embodiments, the membranes and devices disclosed herein can be used in methods for isolating non-cellular biological materials, such as virus particles, proteins, and cellular metabolites, from cultures. In embodiments, the methods can be used to isolate proteins. In embodiments, the methods can be used to isolate viruses (virus particles). In embodiments, the methods can be used to isolate cellular metabolites.

[0318] In embodiments, a method for isolating biological material may include flowing a medium through one or more compartments to flush the biological material from one or more compartments while cells remain within the one or more compartments, and flowing the medium to a collection outlet. For example, a method for isolating biological material may include applying pressure from a gas region of a membrane. Alternatively, a method for isolating biological material may include flowing a medium through one or more compartments and applying pressure from a gas region. In embodiments, a method for isolating biological material includes separating the biological material from cells. For example, a method may include continuously separating the biological material during culture. Alternatively, a method may include separating the biological material after culture.

[0319] HDCR membrane FIG. 1 provides a functional overview of an HDCR 100 that separates nutrient and oxygen delivery for high viral vector yields. The cellular respirator 100 includes a membrane 110, gas channels 125 that penetrate the membrane 110, and surface features in the form of fin structures 120 on a first side of the membrane 110. The first side of the membrane 110 corresponds to the upper surface of the membrane in FIG. 1. The fin structures 120 are shown in cross section in FIG. 1 and in perspective view in FIG. 7 (see fin 720 in FIG. 7). The fin structures 120 extend substantially parallel to one another along the length of the fin structures 120. The substantially parallel fin structures 120 define grooves 130a-d between adjacent fin structures. The grooves 130 between adjacent fin structures provide compartments that can be used for cell placement and production.

[0320] Gas channels 125 penetrate membrane 110 as shown in FIG. 1 . Spacing pillars 190 also provide an interface for gases outside membrane 110 from below. Various exemplary embodiments may include none, one, or more than one gas channel 125 and / or spacing pillars 190. In some exemplary embodiments, spacing pillars 190 may be located on top of membrane 110 so that a gap remains for medium perfusion when stacked. Spacing pillars 190 located on top of membrane 110 are the implementation used in cartridge-style HDCRs (e.g., FIGS. 8D, 8E, and / or 8F). Bottom spacing pillars may be used in 24-well and Petri-style HDCR devices.

[0321] The membrane 110 is formed from a material that is permeable to gases so that oxygen and other gases can be exchanged with the compartments formed by the grooves 130a-d. This allows gas exchange from below and on the sides of the compartments formed by the grooves 130a-d.

[0322] The membrane 110 may be permeable to gases but less permeable or non-permeable to liquid or suspension culture media. The culture medium region 140 allows the flow of culture medium into the grooves 130a-d. The culture medium flows in a direction substantially perpendicular to the fin structures 120 and the grooves 130a-d. In embodiments, for cell harvesting, the culture medium may flow in a direction substantially aligned with the fin structures 120. The medium flow substantially perpendicular to the fin structures 120 facilitates cell retention within the grooves 130a-d. The medium flow substantially aligned with the fin structures 120 facilitates cell harvesting from the grooves 130a-d.

[0323] In a first phase, cell-containing medium may flow across medium region 140 into the seed compartment formed by channels 130a-d. Cells 150a may then flow into the compartment, as shown in channel 130a. To illustrate the production timeline, five cell production stages are numbered: initial cell flow 160, cell retention 165 within the compartment, gas exchange 170 through gas-permeable membrane 110, cell production 175, and cell harvest 180. For clarity, cell retention 165 is shown in channel 130a, gas exchange is shown in channel 130b, production with medium exchange is shown in channel 130c, and harvest is shown in channel 130d. However, each of these stages occurs chronologically across all of channels 130a-d. For example, cells are harvested from all compartments substantially simultaneously after production.

[0324] During initial cell flow 160, medium containing cells 150a transported into channels 130a-d flows through medium region 140. As represented in channel 130a, cells transported to channels 130a-d are largely retained within the compartments 165. Medium flowing across medium region 140 to the compartments within channels 130a-d may also supply nutrients or reagents to cells 150a-d within the compartments.

[0325] The medium flows in a direction substantially perpendicular to the length of the fin structures 120 and grooves 130a-d. For example, in FIG. 1 , the medium flows from left to right through the medium region on the first surface of the membrane 110, which has the fins 120 and grooves 130a-d. This medium flow, which is substantially perpendicular to the compartments formed within the grooves 130a-d, transfers less momentum from the medium region to the compartments within the grooves 130a-d than if the medium flow were aligned with the grooves a-f or if no compartments with depth existed. Thus, momentum transfer from the medium region to the compartments is lower than if the medium flow were aligned with the length of the grooves or if no compartments formed by the surface structures on the membrane 110 existed. Reducing or minimizing momentum transfer and flow to the compartments reduces or minimizes shear forces on the cells 150a within the compartments formed by the grooves, thereby reducing cytopathic effects on the cells 150a within the compartments and preventing cell washout during a stage of cell or bioproduct production.

[0326] Gas exchange 170 occurs between the compartments through gas-permeable membrane 110 with fins 120 while medium flows from above through medium region 140. For example, oxygen from gas channels 125 or pillars 190 may enter the cell niches in grooves 130b to oxygenate cells 150b, and carbon dioxide waste gas from the cells may exit the cell niches in grooves 130b through membrane 110 with fins 120. This gas exchange allows cells 150b to remain oxygenated while preventing carbon dioxide buildup.

[0327] The cells grow to high density and produce or are induced to produce bioproduct (e.g., protein, virus, metabolite) production 175. In addition to gas exchange, cell, virus, and bioproduct production 175 also require the delivery of nutrients for cell growth and the removal of waste products. Nutrients for cell growth can be delivered in the medium via medium region 140 to cells 150c within the cell niche formed by grooves 130c. Additionally, waste products from respiration of cells 150c can be transported away in the medium via medium region 140.

[0328] By separating oxygen supply from the delivery of soluble nutrients in the medium, the HDCR100 can maintain much higher cell densities than current bioreactors. Integrated cell retention grooves also protect cells from shear forces that typically affect cell viability in conventional bioreactors, thereby increasing production capacity. This can result in higher yields, faster production, and lower costs.

[0329] For example, the cell volume (Vcell) may occupy about 10% to 25% of the reactor volume available for cell growth (Vbioreactor). The reactor volume available for cell growth may correspond to the volume of channels 130a-d. For some cells in some embodiments, this may be about 10% to 25%. 8 This can be converted to a density of cells / mL.

[0330] Once produced, the bioproduct, such as cells or virus-containing cells 150d, can be harvested using trypsin, EDTA, or turbulence.

[0331] Membrane 100 may be formed from a material that allows high gas permeability (a function of solubility and diffusion rate), such as silicone, including polydimethylsiloxane (PDMS).

[0332] Gas permeability rates are generally inversely proportional to the membrane thickness. At very thin dimensions, the permeability of a material can also increase significantly, as observed with thin parylene (<10 μm). This fact can be used to make polymers that are traditionally considered gas barriers suitable for gas permeability. Porous membrane materials can also be used in which the breakthrough point of the small pores is high enough to allow pressure-driven flow within the hollow membrane without gas bubbles forming at the surface. A combination of approaches can also be used to achieve the desired high gas permeability properties of the membrane.

[0333] The membrane 100 and surface features can be surface treated to facilitate cell attachment and growth. A widely applicable approach is to coat the membrane with a thin layer of parylene and plasma etch the membrane using oxygen and / or ammonia plasma treatment to make the membrane hydrophilic and / or improve cell attachment.

[0334] Further methods include coating with proteins (eg, agarose, collagen, fibronectin, fibrin) or other coatings (eg, lactic acid, laminin, poly-D-lysine, or poly-L-lysine).

[0335] HDCRs containing membranes consistent with Figure 1 have the following features and advantages. First, gas exchange and nutrient delivery are decoupled. In particular, oxygen and carbon dioxide exchange occurs via permeation of the membrane and surface features (such as fins) in the compartment, while medium is independently perfused throughout the compartment. This allows for approximately 10 8 It allows for the production of cell densities on the order of cells / mL, or Vcell / Vbioreactor ratios of approximately 10%-25%, which allows for more efficient media use than traditional methods of cell production and eliminates the need for base addition as required in traditional methods of cell production.

[0336] Second, the compartments and / or patterned recesses in the HDCR membrane provide integrated cell retention. In this case, the membrane 100 retains cells and protects them from shear forces and washout caused by perfusion of medium over the compartments. This structure of the membrane 100 reduces or eliminates cell loss due to cytopathic effects. It also eliminates the need for auxiliary acoustic, TFF, or other auxiliary filtration systems. The compartments and / or patterned recesses are suitable for growing cells in suspension, adherent cells, or microcarrier-compatible cells.

[0337] Third, the membrane 100 provides uniform conditions with the micropatterned architecture of HDCR membranes, with virtually identical compartments or cell growth compartments engineered to stay below both gas and medium diffusion limits, even at high cell densities. The extremely high gas exchange area-to-volume ratio maximizes oxygen permeability, often the limiting factor for cellular respiration. These membranes enable uniform conditions throughout the device for cell production. They also provide precise control of oxygen partial pressure for faster cell growth and higher viral titers. The uniform conditions allow for open-loop control of oxygen, e.g., oxygen volumetric mass transfer coefficients of kLa > 40 / hr.

[0338] The shape of the cell niche can be tailored to the metabolic demands of the cells and the production process. For example, for cultures with low metabolic rates, a niche with deep and wide grooves can be used to achieve a high volumetric ratio Vcell / Vbioreactor for cell growth. Conversely, cultures with high metabolic rates (e.g., bacteria) can be accommodated by using shallower and narrower grooves at the expense of Vcell / Vbioreactor. The optimal trade-off between niche volume, gas exchange rate, cell retention, and nutrient / waste exchange is performed using an integrated multiphysics finite element model.

[0339] Fourth, the membrane allows for space-efficient packaging, with bed lengths exceeding 40 cm, for example, compared to 4 cm in existing packed-bed designs. This allows for the creation of more optimal form factors to increase volumetric production capacity in GMP spaces. Furthermore, the membrane 100 can be stacked, packaged, and fitted into standard cell incubators. Simulations and preliminary data indicate that membranes can be stacked in approximately 10 8 Support the goal of achieving extremely high densities of cells / mL or a Vcell / Vbioreactor ratio of approximately 10%-25%. 4 ~10 5 The yield of vector genome (vg / cell) was 10 13 This is converted to a production of approximately 10 16 ~ 17 Production of a vg batch of cGMP vectors can be achieved with approximately 1-10 liters of medium, instead of the approximately 500-1000 liters required with current methods. What previously required a room's worth of production space can now be produced in a single incubator. With such high efficiencies, the embodiments described herein can reduce production costs, speed production, and disrupt the field of virus, vector, and vaccine production, increasing volume by approximately 10-fold and increasing production rates by approximately 2-10-fold.

[0340] Fifth, membrane 100 has a mass-producible and scalable design, compatible with, for example, liquid silicone rubber (LSR) injection molding. Membranes can be easily scaled and stacked. Therefore, membranes 100 can be produced at low cost in established production ecosystems. They can be implemented in batch sizes scalable up to clinical scale.

[0341] Sixth, membrane 100 provides low fluid resistance, with dedicated medium space above the compartment allowing for a low-resistance fluid path. This eliminates the need for expensive pumps or power pumps. Cartridges with membrane 100 can have gravity-fed medium with flow regulators.

[0342] Seventh, membrane 100 is configured to expand and contract in response to fluctuations or pulsations in gas or fluid pressure to induce mixing within the compartment. Such mixing allows for more uniform distribution of reagents or viruses within the compartment. Such mixing capability may also facilitate cell harvesting.

[0343] Modeling high density cell regulators Figure 2 shows a model of the HDCR device of Figure 1. Development of a multiphysics finite element model (FEM) (Comsol Inc, MA, USA) allows for understanding of the interrelationships between cell growth, nutrient and gas transport, cell retention, perfusion rate, and pressure drop in the HDCR device 200 of Figure 2. The HDCR 200 includes a membrane 210 with fins 20 and compartments within grooves 230a-d.

[0344] At 240, liquid medium flows through the medium region above membrane 210. At 265, cells in the liquid medium settle and are retained in the cell niche within groove 230a. The thin solid line above the niche (U-shaped, descending meniscus toward the bottom of the cell niche) indicates the fluid boundary (i.e., washout boundary) where the upward component of the flow velocity is less than the cell settling velocity. Thus, cells below the boundary are retained. In this experiment, a medium inlet velocity of approximately 100 μm / s was assumed. Devices and systems can be configured for medium inlet velocities on the order of approximately 1 μm / s to approximately 1 m / s. Relatively slow operating speeds can correspond to a fluid steady state within the compartment (i.e., retention mode). Relatively high operating speeds can correspond to a fluid turbulent state within the compartment (i.e., flushing mode). The 100 μm / s medium inlet velocity was sufficient to promote laminar or near-laminar nutrient flow, glucose input, cell growth and retention, waste output, and sufficient mixing in the primary medium pathway without excessive cell loss compared to the perfusion medium compartment. Note that during the growth process, cell-cell and cell-substrate adhesion increases, which may result in a higher "true" washout boundary. The length of the arrows in the perfusion medium compartment is proportional to the flow rate, so smaller arrows correspond to lower flow rates.

[0345] By providing dedicated medium paths, there is a great deal of flexibility to operate at different medium velocities. Flow rates can range from as low as 1 μm / s to several cm / s, or as high as 1 m / s. Furthermore, medium perfusion can be pulsatile or intermittent (i.e., a bolus of fresh medium is fed into the system, then stopped, consumed, and replaced with a bolus of fresh medium, etc.). The medium flow rate can be set so that the medium loses approximately 50% of its glucose content as it leaves the reactor. The flow rate can be feedback-controlled. If the cell density is relatively low, the need for perfusion can be proportionally lower. For example, a bioreactor with a length (y-axis) of approximately 40 cm can achieve a perfusion rate of approximately 10 8 A velocity of approximately 100 μm / sec was determined to be sufficient to produce cells / mL. Also, a velocity of approximately 1 μm / sec was found to produce cells / mL during the seeding step. 6It was determined that this was sufficient to produce 100 cells / mL (i.e., a reasonable seeding density).

[0346] At 225, gas perfuses from subsurface gas channels in membrane 210 through membrane 210 with fins 220 to and from cell niches (compartments) in grooves 230b. At 270, this allows for the delivery of oxygen to and removal of carbon dioxide from the cell niches in grooves 230b. At 245, soluble nutrients, including glucose, are delivered by diffusion from the medium stream to the cell niches in grooves 230c. At 280, soluble waste products are removed by diffusion from the cell niches in grooves 230a to the medium stream. For clarity of presentation, these steps 265, 270, 275, and 280 are shown as occurring within individual compartments in grooves 230a, 230b, 230c, and 230d, respectively; however, in embodiments, similar to a finite element model, these steps occur across multiple compartments in the grooves throughout membrane 210.

[0347] The finite element model models each of the cell delivery and retention 265, gas exchange 270, nutrient delivery 275, and waste removal 280 operations for the dense gas respirator 200. The model can adjust dimensions, flow rates, concentrations, orientations, and pressures to aid in the design of membranes for different applications.

[0348] For example, embodiments may be implemented with a medium flow rate of about 0.1 mm / sec, resulting in a flow rate of about 2×10 8 cells / cm 3 The system includes a design that supports cell densities of 1000-1500 nm. This medium flow rate is slow enough for single cell retention and can deliver membranes greater than approximately 40 cm long. This embodiment requires a pressure head of only approximately 5 Pa and ensures that all cells experience an oxygen partial pressure greater than the Michaelis-Menten constant for oxygen (KM,O2 = approximately 1 μM) of a typical cell.

[0349] The membrane for this embodiment can be fabricated using a CNC-machined mold fabrication process, which allows for precise membrane construction via silicone casting or LSR injection molding. This allows for the direct integration of pillars, connectors, niches, and manifolds into the membrane with high yield and low cost. Chemical vapor deposition and plasma processes can be used to render the membrane surface hydrophilic / wettable, allowing cell attachment.

[0350] Membranes for this embodiment can be connected and stacked to create HDCR cartridges such as the cartridge described below with respect to Figures 8A-8F.

[0351] Dynamic characteristics of the membrane Figure 3 shows the deformation of the membrane of Figure 1 in the presence of pressure fluctuations, resulting in the flow of medium into and out of the compartments of the HDCR device of Figure 1. Generally, the membrane can deform in response to changes in pressure. For example, it can deform upward or downward toward or away from the pressure.

[0352] In 300a of Figure 3, membrane 310a is at neutral pressure. Therefore, membrane 310a with fins 320a and grooves 330a does not deform. Cells, nutrients, or waste products in the medium flowing over membrane 310a can diffuse between grooves 330a and the medium flowing above the grooves, but cells or other liquids are not drawn into or expelled from grooves 330a due to pressure fluctuations.

[0353] In 300b of Figure 3, membrane 310b is under negative pressure. Thus, the underside of membrane 310b in the region of grooves 330b between fins 320b is deformed downward by the negative pressure acting on membrane 310b. Cells, nutrients, or other materials are drawn into grooves 330b due to the deformation of membrane 310b and the negative pressure (downward in Figure 3).

[0354] In 300c of Figure 3, membrane 310c is under positive pressure. Thus, the underside of membrane 310c in the region of grooves 330c between fins 320c is deformed upward by the positive pressure acting on membrane 310c. Cells, waste, or other material are drawn out of grooves 330b due to the deformation of membrane 310c and the positive pressure (upward in Figure 3).

[0355] The pressure may be varied, cycled, or pulsed between neutral, negative, and positive pressure states in channels 300a, 300b, and 300c. This variation, cycling, or pulsation may allow for periodic or pulsating movement, as illustrated by the stages in channels 300a, 300b, and 300c. Such variation, cycling, or pulsation may be used to mix the contents of the compartments within channels 330a-c.

[0356] FIG. 4 illustrates the use of pulsating flow to enhance mixing and nutrient transport according to various exemplary embodiments of the disclosed technology. Periodic pulsation of fluid pressure through / across a membrane can induce growth and medium mixing or eruptions in the cell compartment, enhancing nutrient and oxygen transport. Planar (sheet) membranes can incorporate microdiffusers to rectify the pulsating pressure into a net flow perpendicular to the main flow path. Phases of periodic pulsation are shown in phases 400a, 400b, and 400c. In phase 400a, membrane 410a is in a neutral state and fluid pressure is increasing, resulting in upward flow 420a. The increased pressure in phase 400b results in membrane 410b expanding in size, forcing fluid to flow upward with greater intensity 420b, resulting in more mixing. The pressure is then reduced at 400c, causing the fluid to flow downward 420, reducing the pressure on the upper edge of the membrane and causing it to shrink in size back to the size of the membrane at 410a. These steps 400a, 400b, and 400c can be repeated, resulting in mixing in the cell niche.

[0357] Membrane Architecture and Surface Features Figure 5 shows an open membrane architecture.

[0358] Membrane 510a has fins 520a and compartments in grooves 530a between fins 520a. Culture medium flows through culture medium region 540a. Cells from the culture medium settle into grooves 530a. A fluid manifold supplies culture medium to culture medium region 540a. The culture medium can be collected, recycled, or discarded.

[0359] Membrane 510b has fins 520b and compartments in grooves 530b between fins 520b. Medium flows through medium region 540b. The membrane is angled so that medium flow is driven by gravity as the medium flows downhill. A fluid manifold supplies medium to medium region 540b. The medium can be collected, recycled, or discarded.

[0360] Membrane 510c has fins 520c and compartments in grooves 530c between fins 520c. Medium flows through medium region 540b. The membrane is angled so that the medium flow is driven by gravity as the medium flows downhill. The medium flow acts to draw in air. A fluid manifold supplies medium to medium region 540b. The medium can be collected, recycled, or discarded.

[0361] Membrane 510d has fins 520d and compartments in grooves 530d between fins 520d. Medium flows through porous or fibrous membrane 550 in medium region 540d. Porous or fibrous membrane 550 acts as a wick to draw fluid into grooves 530d and deliver fluid to and from grooves 530d. The membrane is angled so that medium flow is gravity-driven as the medium flows downhill. The medium flow acts to draw in air. A fluid manifold supplies medium to medium region 540b. The medium can be collected, recycled, or discarded.

[0362] 6 shows a membrane 610a having fins 620a with a keystone shape, which forms grooves 630a in which the openings 635a at the top of the grooves 630a are narrower than at least some points closer to the surface of the membrane 610a. This keystone fin shape differs from the fin shapes shown in FIGS. 1, 2, 3, and 5.

[0363] Other fin shapes are also possible. The fins may be tapered, curved to form a teardrop-shaped groove, prism-shaped, or any other shape that creates a compartment that protects cells in the niche from shear. One feature of the keystone shape that applies to some other shapes, such as the teardrop shape, is that the opening at the top of the groove can be narrower than the cross-sectional width at a lower height closer to the surface of the membrane. Such narrow openings can further protect cells from shear and improve the ability to retain proliferating cells.

[0364] Mechanical stretching of membrane 610b stretches the length of membrane 610b in a direction along the cross section of fins 620b and grooves 630b, widening openings 635b at the tops of grooves 630b and facilitating removal of the contents of grooves 630b, as shown in FIG.

[0365] In addition to mechanical stretching, the membrane 610c may be inverted so that the fins 620c and corresponding grooves 630c are inverted (facing downward instead of upward). The grooves 630c have downward-facing openings 635c so that the contents of the grooves 630c fall by gravity. The membrane 610c can be stretched and / or inverted.

[0366] FIG. 7 shows various membranes corresponding to different embodiments.

[0367] Membrane 700a includes a flat surface 710 with protruding fins 720. The fins 720 are substantially parallel. Grooves 730 are formed between adjacent fins 720. The fins 720 have a rectangular or prismatic cross-section, as do the grooves 730 formed between the fins. As discussed with respect to FIG. 6 , other fin cross-sectional shapes are possible, such as keystone, teardrop, prismatic, tapered, polygonal, or curved. Some fins 720 may be shaped with openings that are narrower than the width of their cross-sections at lower heights (closer to surface 710). The membrane 710 and fins 720 are gas permeable, allowing gas to perfuse through the membrane and / or fins into the compartments within the grooves 730. Culture medium can flow across the tops of the fins 720 and grooves 730 in a direction that is not parallel to the length of the grooves 730. Alternatively, culture medium may flow in a direction substantially perpendicular to the length of the fins 720 and grooves 730 to reduce shear forces on cells within the compartments within the fins.

[0368] Membrane 700b contains a two-dimensional array of wells 740. Wells 740 function similarly to grooves 720 in that they form protective compartments that protect cells from shear as media flows over the surface. Membrane 700b is gas permeable, so that gases can perfuse into and out of the wells through the membrane and well walls.

[0369] Membrane 700c does not include surface features. In embodiments without surface features, membrane 700c is gas permeable.

[0370] Membrane 700d includes an array of posts 750. The posts 750 form compartments adjacent to the posts. Posts with crescent, chevron, or U-shapes can provide better protection in one direction of flow than posts with other shapes. These compartments are more open than the niches in wells 740 or grooves 730, but still provide some protection for adherent cells that may attach to the posts 750. The posts 750 are gas permeable, allowing gas to perfuse to and from cells attached to or near the posts 750.

[0371] Fins 720, wells 740, and posts 750 are examples of surface features that protrude from or are recessed into the membrane, all of which may be gas permeable.

[0372] The HDCR may use one or a combination of membranes with fins and grooves 700a, membranes with wells 700b, membranes without surface features 700c, and / or membranes with posts 700d.

[0373] HDCR cartridge Figure 8A shows the structure and formation of an HDCR device. Figure 8A shows a quarter view of a medium layer 810 bonded to a gas layer 820. Medium layer 810 and gas layer 820. Gas layer 820 is bonded below medium layer 810 so that gas exchange and oxygen supply from below are physically separated from liquid medium perfusion from above. Medium layer 810 and gas layer 820 may be formed from polydimethylsiloxane (PDMS).

[0374] FIG. 8B shows spacing pillars defining spaces for medium perfusion (dark) above the compartments formed by the surface features of the membrane of FIG. 8A.

[0375] FIG. 8C shows multiple membranes 860a, 860b, 860c, and 860d stacked on top of each other with gas manifolds 850 at the corners of the stacked membranes 860a-d, each of which corresponds to the membranes of FIGS. 8A and 8B.

[0376] Figure 8D shows an enclosure 870 housing the stacked membranes 860a-d of Figure 8C, with a gas access port 890 and a medium access port 880. Each of the gas access ports 890 can function as a gas inlet or a gas outlet. Similarly, each of the medium access ports 880 can function as a medium inlet or a medium outlet. While Figure 8D shows two medium access ports 880 and four gas access ports 890, other embodiments may have a different number of gas and / or medium access ports.

[0377] FIG. 8E shows an incubator 895 in which the HDCR cartridge of FIG. 8D can be enclosed for cell incubation.

[0378] Figure 8F shows an example of an HDCR cartridge containing the components described above with respect to Figures 8A–8D. The HDCR optimizes the cell growth niche, enabling very high-density production cell line growth. The HDCR is composed of a stackable, gas-permeable, and gas-permeable polydimethylsiloxane (PDMS) membrane encased in an independently perfusable medium compartment. Cells settle into precisely patterned grooves in the membrane, which retain the cells, protect them from fluid shear forces, and provide sufficient oxygen supply via membrane permeation. Integrated spacing posts maintain a thin medium film over the cell niche for delivery of soluble nutrients, reagents, and / or viruses, as well as for waste removal and cell / bioproduct harvest. The membranes can be inexpensively manufactured in large sheets by industry-standard liquid silicone rubber (LSR) injection molding. A coating process renders them hydrophilic, promoting cell attachment. Such a device forms the basis of a fully enclosed, autoclavable vector production cartridge. The integrated cell retention technology makes the disclosed design compatible with both adherent and suspension cells.

[0379] Gas-permeable membranes that allow gas perfusion provide a large surface area while delivering sufficient oxygen and gas exchange for high-density cell growth. These membranes can be folded or stacked to achieve a high surface-to-volume ratio. Cells can be grown directly on the membrane surface or on a substrate sandwiched between the membranes. The gaps between stacked membranes can be perfused with solutions to deliver or remove components into or from the membrane stack. The perfusion gap and rate can be selected to maintain a suitable shear rate within the device. A network of tubes or channels can be used throughout the system to deliver media separately from the gas source. The tubes or channels can contain pores that allow particles of various sizes, ranging from molecules to cells, to pass into or out of the tubes. This is a means by which cells can be seeded into the device and / or viruses can be delivered to infect cells. Pores can be formed in the membrane itself to provide a means for perfusing stacked membranes perpendicular to their surfaces. In this configuration, the flow induces minimal shear forces on the cells because the flow direction is not parallel to the cells. Nutrients then reach the cells by diffusing away from these pores.

[0380] The role of oxygen in virus production is well established and has been reviewed. The so-called "cell density effect" refers to the tendency for cell-specific virus yields to decrease in response to limited oxygen or nutrient availability. Interestingly, transient transfection of HEK293 suspension cells with PEI was observed at low densities (approximately 10 6 cells / mL) than 7It has been demonstrated to be simpler and twice as efficient at a production rate of 1000 cells / mL. Therefore, overcoming metabolic limitations is key to achieving improved yields. For example, a volume-expanded fed-batch process, which uses fresh medium during the viral infection step to provide large amounts of oxygen and nutrients, has been reported to increase production capacity per cell by up to 40-fold for Parapoxvirus ovis in adherent bovine kidney cells. Similar improvements have been reported for nearly all clinically relevant viruses.

[0381] Furthermore, increased DNA and protein synthesis significantly increases the oxygen uptake rate during viral replication. For example, in baculovirus-infected Sf9 cells, the oxygen uptake rate increased 1.3-fold.

[0382] Bioreactors must adapt to this increased oxygen demand following infection. Existing perfusion systems (such as iCellis) rely on cell retention to adhere to stationary supports, and increasing flow rates to maintain oxygen supply is impractical because cells become less adherent due to cytopathic effects and detach with increased shear. Our high-density bioreactor decouples gas and nutrient supply and integrates cell retention, creating an optimal environment for virus production. Based on preliminary results and modeling, our HDCR has significantly improved space efficiency (>10-fold) at a fraction of the cost (<4-fold) compared to the industry-leading iCellis bioreactor system.

[0383] Figure 9 shows an example of an open membrane high-volume cellular respirator device 900a with ten stacked membranes. Region 910 of 900a is shown in 900b, which shows a fluid manifold 930 that supplies media to membrane 960 via media channels 940. Grooves 950 in the membrane serve as compartments for cell retention and growth. 900c shows a side view of high-volume cellular respirator device 900a with a stack of ten membranes 960.

[0384] FIG. 10 shows a prototype HDCR with an integrated cell retention niche, spacing pillars, gas perfusion space, and gas manifold. 1000a shows a top view of the HDCR. 1000b is an enlarged view of region 1010 of 1000a. 1000b shows a detailed view of the cell niche grooves and underlying gas compartment ties. 1000c is an enlarged view of region 1020 of 1000a. 1000c shows the stackable gas manifold. 1000d is an enlarged view of the side view corresponding to 1030 of 1000a. This side view 1000d shows the cell and medium channels and spacing pillars.

[0385] FIG. 11 illustrates the management of bubbles, voids, condensation, and evaporation for an HDCR 1100a. 1100b is a photograph of a membrane with bubbles 1110. The membrane was placed in an imaging- and autoclavable-compatible enclosure. Purging liquid through the medium compartment effectively eliminated the bubbles 1110 and voids. Pre-humidifying gas prevents evaporation. Pre-heating gas prevents condensation in the gas compartment. After purging, the membrane no longer has bubbles or voids, as shown in photograph 1100c.

[0386] Figure 12 is a photograph 1200 of a medium-sized HDCR membrane prototype approximately 34 cm long and 10 cm wide, which was fabricated using injection molding to mitigate risk in the production of the stackable HDCR membranes described herein. Figure 12A is a close-up 1200a of a portion of the membrane prototype of Figure 12.

[0387] 24-well HDCR plate Figure 13 shows a 24-well plate 1310 with a silicone insert 1320 with patterned grooves bonded to each well. The 24-well plate can be used for million-cell scale cell culture. The 24-well plate can be used as a high-throughput screening tool for optimizing and experimenting with cell seeding density, growth curves, media feed rates, oxygen tension, transfection methods, production / packaging, and other parameters before scaling up to billion-cell scale Petri dishes or trillion-cell scale cartridges with stacked membranes. The wells within the well plate are not uniform (meaning, for example, that groove and / or fin dimensions vary across the wells to identify effective configurations and useful shapes) and are at a much larger scale compared to the wells 740 in Figure 7. Figure 13B shows a close-up of a groove 1330 in one of the silicone inserts in Figure 13.

[0388] Petri dish HDCR FIG. 14 shows an HDCR Petri dish 1400 in which a gas-permeable membrane 1410 with fins 1420 forms the bottom of the Petri dish 1400. Gas diffuses from below the membrane 1410, and cells and nutrients are supplied from a deep reservoir 1450 above the membrane. Spacing pillars 1440 at the bottom of the membrane 1410 allow gas to flow underneath the Petri dish 1400. A tension ring 1460 keeps the membrane taut, adding rigidity to the Petri dish 1400. The fins 1420 define compartments within the grooves 1430. The membrane 1410 and fins 1420 are gas-permeable. For example, oxygen may enter the membrane 1410 through the spacing pillars 1440 or the sides of the membrane 1410 and permeate through the membrane 1410 and fins 1420 to the compartments within the grooves 1430. Carbon dioxide is removed from the compartments within the grooves 1430 by permeating through the fins 1420 and membrane 1410 and can exit the membrane 1410 near the spacing pillars 1440 or the sides of the membrane 1410. The reservoirs 1450 above the membrane 1410 and the compartments within the grooves 1430 are deep, reducing the need to replenish or replace the nutrient-rich medium. In many applications, it may not be necessary to replenish or replace the medium in the reservoirs 1450.

[0389] Figure 15 shows an example of a Petri dish 1500a, corresponding to the Petri dish in Figure 14, in which HEK293 cells have been cultured on microcarriers. Microfabricated grooves and fins provide an oxygen-optimized niche for cell growth. In the example of Figure 15, HEK293 cells on microcarriers 1500b concentrate in the grooves of Petri dish 1500a, resulting in the cultivation and harvest of approximately 400 million HEK293 cells 1500c from a single HDCR Petri dish. This is a much larger quantity than is possible from a conventional Petri dish. Each HDCR Petri dish produces the same number of cells as 25 Petri dishes culturing cells using conventional methods. This frees up incubator space and technician time by approximately a 25:1 ratio. The plates are autoclavable and reusable.

[0390] Silicone Sheet Bioreactor Figure 16 shows a silicone sheet-type bioreactor 1600 with an HDCR membrane 1600. Media enters membrane 1600 through inlet 1630 and exits membrane 1640. Figure 1600b shows a close-up view of cell-growing microcarriers 1660 and membrane ties 1670 perpendicular to the membrane. Membrane ties 1670 secure membrane 1610 in place. Filter posts 1680 perpendicular to the membrane provide a boundary or fence that defines the area accessible to microcarriers 1660. Filter posts 1680 allow perfusion of media without microcarrier loss because media flows more freely in areas of the membrane free of microcarriers, where fluid resistance is lower. Filter posts 1680 can be arranged to prevent microcarriers from migrating past filter posts 1680.

[0391] Figure 17 is a cross-sectional top view of a silicone sheet bioreactor 1700, such as the silicone sheet bioreactor of Figure 18. The top view of membrane 1710 includes an array 1720 of filter posts similar to filter posts 1680 of Figure 16. Filter posts 1720 define regions 1740 accessible to microcarriers 1770, as well as regions 1730 inaccessible to microcarriers 1770. This allows microcarriers 1780 with attached cells to accumulate in region 1740. Media flow enters at arrow 1750 and flows through membrane 1710 from left to right in Figure 17 toward arrow 1760. Media flows more freely in region 1730 without microcarriers than in region 1740 with microcarriers, reducing microcarrier loss. Additionally, microcarriers are blocked by filter posts 1720 adjacent to arrow 1760, thereby halting washing into the region indicated by arrow 1760.

[0392] Figure 18 shows a close-up of the silicone sheet bioreactor of Figure 17. 1800a is a photograph of the silicone membrane filled with microcarriers and medium. 1800b is a close-up of the inlet to the silicone membrane inlet. 1800c is a close-up of the silicone membrane outlet with microcarriers. 1800d is a close-up of the filter channel outlined by the filter posts holding the microcarriers.

[0393] Experimental results The HDCR described herein has been used to demonstrate confluent cell growth on a high surface area membrane bioreactor with separated gas and nutrient supplies. CV-1 cells were grown at approximately 1.2 × 10 cells perfused on microcarriers within the HDCR membrane over 5 days of growth. 8 The cells were grown to a density of approximately 0.86 ± 0.17 SEM × 10 cells / mL in 24-well HDCR plates. 8 cells / mL), Wagner39652-1 (approximately 1.1 ± 0.1 SEM × 10 8 cells / mL), and suspended CHO-S (approximately 0.82 ± 0.02 SEM 8 HEK293 cells were grown to approximately 500 million cells / mL in approximately 150 mm HDCR Petri dishes on Cultisphere G microcarriers, and HEK293-S cells were similarly grown to approximately 250 million cells (approximately 30% of niche capacity).

[0394] Figure 19 shows cell growth in a 24-well HDCR plate over a 7-day period. 1900a on the left shows CHO-S cells at the start of the experiment, with compartment 1910 seeded with CHO-S cells. After 7 days of growth, compartment 1930 appears dark in 1900c as it has filled with cells as shown in 1900c. 1900b on the right shows adherent GFP+ Wagner 39652-1 cells on Cultisphere G microcarriers under fluorescent light at the start of the experiment (day 0). As expected, the field in 1900b is dark. After 7 days, Wagner 39652 cells on Cultisphere G microcarriers are visible under fluorescent light along compartment 1950 in 1900d.

[0395] Figure 20 shows that approximately 2 x 10 cells were cultured in A549 cells in 24-well HDCR plates on Cultisphere microcarriers. 7 Figure 20 shows graphs of experimental results for orthopoxvirus production up to a niche density of approximately 10 cells / mL. Cells were infected with HOV-2 at a multiplicity of infection (MOI) of approximately 0.01 (approximately 1 viral particle per 10 cells) and harvested at approximately 24, 48, and 72 hours. The measured titers (pfu / cell) were comparable to virus production in A549 cells grown on tissue culture plates (Figure 20), alleviating concerns that virus infection may be compromised at high cell densities. This data demonstrates successful scaling of cell growth from approximately 5 million cells / well in a 24-well HDCR plate to approximately 500 million cells in a 150 mm HDCR Petri dish, demonstrating successful orthopoxvirus production in the HDCR device described herein. Furthermore, A549 cells were infected at approximately 4 x 10 niche densities in a 24-well HDCR plate. 7 The cells were grown at a cell density of 100 cells / mL and achieved a yield of approximately 64±9 pfu / cell of CF33 per cell.

[0396] Figure 21 shows experimental results demonstrating that HEK293 cells can be transfected at high densities using the membrane structures described in the disclosed technology. Using jetPRIME reagent, approximately 2 x 10 cells were transfected into cells grown directly on HDCR membranes, as well as on solid (Cytodex-1 and Cytodex-3) and microcarriers (Cultisphere G). 7 The mCherry plasmid was transfected at cells / mL. Other embodiments may include different macrocarriers, such as non-porous or macroporous macrocarriers. Figure 21 shows phase 2110, fluorescence 2120, and overlay 2130 views of transfection of high-density HEK293 cells with RFP plasmid grown on Cultisphere G microcarriers using jetPRIME in a 24-well HDCR plate.

[0397] FIG. 22 shows the results of an experiment to titrate orthopoxvirus (CF33) derived from high density A549 cells grown on HDCR membranes.

[0398] background Historically, deep tank fermentation techniques began in the 1940s with the successful scale-up of penicillin production. Stirred tank reactors (STRs) have been used to generate cells important for gene therapy. However, the demand for AAVs for systemic therapy has outpaced the production capacity offered by STRs.

[0399] To date (e.g., over the past 30 years), much effort has been devoted to improving cell-specific production capacity, which has nominally resulted in improved space-time production capacity. Cell-specific AAV production capacity in the published literature is estimated to have an upper limit of 10 6 Existing development techniques such as flasks, STRs, and fixed-bed reactors have demonstrated a cell density of 10 6Some developed systems, such as fixed-bed reactors, have been reported to have relatively high local cell densities in the reactor bed (10 8 Although there are some (cells / mL) available, the relatively large volume of the reactor, which is virtually cell-free, limits the effective cell density of the system to within an order of magnitude, certainly similar to other reactors.

[0400] Scaling saves resources: space, labor, and cash. Scaling reduces costs and achieves production capacity goals. Production efficiency can be measured in terms of the space-time capacity continuum, i.e., how much of a given product can be produced in a given volume in a given period of time. Space-time capacity is the product of both the cell-specific capacity of the cells and the cell density of the bioreactor.

[0401] As described above, the present systems, devices, and methods increase cell density while maintaining metabolically efficient producer or packaging cells. By increasing cell density while maintaining metabolically efficient producer or packaging cells, space-time productivity is improved in terms of productivity per cell. [Example]

[0402] Figure 23 is a schematic diagram of an HDCR architecture configured to receive culture medium, exchange oxygen for carbon dioxide, and output waste with a static volume exchange rate (kLa) greater than about 60 / hr, e.g., greater than about 63 + / - 12 (SEM) / hr. Specifically, as represented by the schematic diagram in Figure 23, for example, the HDCR architecture achieves decoupling by utilizing membrane oxygenation and CO2 exchange, whereby gas permeates the membrane to reach cells, while relying on gentle perfusion of culture medium through the cell growth niche to provide exchange of soluble nutrients and waste. By stacking multiple HDCR membranes on top of each other, space is utilized efficiently, and the system can be scaled up.

[0403] Using the HDCR architecture, static volumetric oxygen exchange rates (kLa) of greater than approximately 60 / hr are achieved compared to flasks, STRs, and fixed beds, which operate on the order of 1-8 / hr for mammalian cell culture. Exchange rates of approximately 60 / hr are achieved in 10 8 Provide sufficient gas exchange to maintain above 100 cells / mL.

[0404] As shown in Figure 16, a microscopic image of an exemplary HDCR membrane, in which medium perfuses from left to right and gas exchange occurs inside and outside the screen. The HDCR membrane supports a microcarrier-supported culture, which may be packed into the HDCR membrane and held by lines, for example, on a filter post. A representative example is a 145 cm 2 Approximately 500 million HEK293 cells were grown on a single HDCR membrane (i.e., the size of a 15 cm Petri dish), which would normally require an order of magnitude of a 25 x 15 cm Petri dish for culture. In other words, the HDCR membrane can achieve approximately a 25-fold increase in cell density per unit area.

[0405] The HDCR membrane is effective across cell lines. Using the present system, device, and method, HEK293 cells were cultured for AAV applications, HEK293 cells were cultured on microcarriers, CHO suspension cells were cultured for protein production applications, NS0 cells were cultured for antibody production applications, and A549 and CV-1 cells were cultured for oncolytic virus production. All of the above cells were tested and appeared to grow robustly. Doubling times were comparable to traditional flask culture. The HDCR architecture and platform can be considered universal for AAV, oncolytic viruses, antibodies, vaccines, and the like. Specifically, Figure 24 is a plot of cell density (cells / mL) (on the y-axis) versus days in culture (on the x-axis) according to an exemplary embodiment. In Figure 24, HEK293 cells cultured for AAV applications are represented by squares, HEK293 cells on microcarriers are represented by filled triangles, CHO suspension cells are represented by filled circles, and NS0 cells are represented by filled diamonds. Using the HDCR architecture of the present invention, cell densities can be increased to approximately 10 in approximately 2-8 days. 6 About 10 orders 8 The number of cases increased to the order of .

[0406] Growing cells to high density involves the AAV production process using the HDCR system. For example, a three-plasmid and PEI-based transfection approach was used to transfect cells with the AAV2-mCherry vector (3 PEI: 1 DNA, 3 µg DNA / 10 6A proof-of-concept experiment using a 4000-cell, 2 pHelper:1.5 Rep / Cap:1 AAV2-CMV-mCherry cell line was used. DNAse-resistant particles in the resulting crude cell lysates were observed by qPCR. Yields per cell were comparable at conventional and higher cell densities. Thus, in terms of volumetric productivity, a multiplicative improvement with increasing cell density is observed using the HDCR architecture. Specifically, Figure 25 is a plot of AAV (vg / cell) (on the y-axis) versus packaging time (in hours) (on the x-axis) for samples with 4 million cells / mL and 16 million cells / mL, according to an exemplary embodiment. Figure 26 is a plot of AAV (vg / mL) (on the y-axis) versus packaging time (in hours) (on the x-axis) for samples with 4 million cells / mL and 16 million cells / mL, according to an exemplary embodiment.

[0407] Transfection can be a limiting factor, e.g., at a density of 3 x 10 7 A decrease in productivity per cell was observed as the cell density increased to 100 million cells / mL. As a result, at these high densities, volumetric productivity achieved diminishing returns with increasing cell density. There are many factors to consider, but transfection may be the limiting factor. Specifically, Figure 27 is a plot of AAV (vg / cell) (on the y-axis) versus packaging time (in hours) (on the x-axis) for samples having 4 million cells / mL, 16 million cells / mL, and 32 million cells / mL, according to an exemplary embodiment. Figure 28 is a plot of AAV (vg / mL) (on the y-axis) versus packaging time (in hours) (on the x-axis) for samples having 4 million cells / mL, 16 million cells / mL, and 32 million cells / mL, according to an exemplary embodiment.

[0408] Adding a dispersion / mixing step to the transfection process improved the yield per cell, especially at high cell densities. Because of the relatively high density, the plasmid complex may not interact with all cells. Therefore, the low productivity per cell may be the result of averaging between normally producing and non-producing cell populations. Specifically, Figure 29 shows a chart of AAV (vg / cell) on the y-axis for samples with 30 million and 100 million cells / mL, comparing static and mixed modes, according to an exemplary embodiment. The dispersion / mixing step significantly improved AAV (vg / cell). Figure 30 shows an HDCR-produced AAV2 vector scaled from approximately 37 kD to approximately 150 kD according to an exemplary embodiment.

[0409] Figure 31 is a plot of productivity (AAV vg / L) (on the y-axis) versus cell density at transfection (cells / mL) (on the x-axis) reported in the literature from 2010-2020 and compared to HDCR producer cells of the present invention according to exemplary embodiments. Productivities in the literature from 2010-2020 are approximately 10 13 ~about 10 15 The cell density for transfection was reported to be approximately 10 5 ~about 10 6 (cells / mL). On the other hand, the productivity of the HDCR architecture was approximately 10 15 ~about 10 16 AAV (vg / L), and the cell density at transfection was approximately 10 7 ~about 10 8 (cells / mL). In summary, HDCR increases AAV volumetric productivity by 10 15 Boost to the vg / L regime.

[0410] HDCR supports the production of CF33-hNIS orthopoxvirus. Oncolytic virus production in HDCR has been demonstrated. For example, CF33-hNIS, a replication-competent chimeric orthopoxvirus with efficacy against colon cancer, was generated. A549 cells were cultured at 2 × 10 in HDCR.7 The cells were grown to 10 cells / mL and infected with CF33 at an MOI of 0.01. Figure 32 is a plot of titer (PFU / cell) (on the y-axis) versus time post-infection (on the x-axis) comparing tissue culture (flask) production (TCP) with HDCR production, according to an exemplary embodiment. Figure 32 shows equivalent CF-33 production per cell in HDCR compared to conventional production in tissue culture (flask) production (TCP). As noted above, increasing cell density leads to a multiplicative improvement in volumetric production capacity. (Note that HOV-2 orthopoxvirus is a chimeric poxvirus that encodes the human sodium iodide symporter (hNIS) at a redundant tk locus (also known as CF33-hNIS). HOV-2 titers (pfu / cell) from production in A549 cells were also compared using a high cell density (2 x 10) versus standard monolayer production. 7 Cells were grown in 24-well HDCR plates at 1000 cells / mL. Cells were harvested at 24, 48, and 72 hours post-infection.

[0411] Thus, decoupling of gas and medium supply in HDCR architecture is useful for supporting high-density cell culture and has applications in AAV and oncolytic virus production.

[0412] Further Examples FIG. 33 is a side-by-side comparison of an AAV2-mCherry HDCR vector scaled to about 25 kD to about 250 kD versus a standard, according to an exemplary embodiment.

[0413] FIG. 34 is an HDCR-produced AAV2 vector scaled to about 37 kD to about 150 kD according to an exemplary embodiment.

[0414] Figure 35 is another plot (including the data in Figure 31) of productivity (AAV vg / L) (on the y-axis) versus cell density at transfection (cells / mL) (on the x-axis) compared to HDCR producer cells of the present invention reported in the literature from 2010-2020, and further including results at relatively low productivity according to exemplary embodiments.

[0415] FIG. 36 is an image of unstained protein standards (left) alongside HDCR-derived AAV2 (right), according to an exemplary embodiment.

[0416] FIG. 37 is an image of unstained protein standards (left) aligned with an AAV2-mCherry HDCR vector (right) according to an exemplary embodiment.

[0417] Figure 80 is a schematic diagram of pAAV-minCMV-mCherry reported by Zhang et al. in "Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription" in Nat Biotechnol on January 19, 2011.

[0418] Figure 80 is a schematic diagram of pAAV-RC as reported in the prior art. In relation to Figure 80, Table 1 below summarizes the features and their corresponding nucleotide positions. [Table 1]

[0419] Figure 81 is a schematic diagram of pHelper as reported in the prior art. In relation to Figure 81, Table 2 below summarizes the features and their corresponding nucleotide positions. [Table 2]

[0420] Overview of the embodiment First, note that Figures 38, 40-45, 54-56, 61, 65-75, and 79 include example coordinate axes for convenience. The directions are not intended to be limiting. For example, as seen in Figure 40, device width may be provided along the x-axis, where "inside the page" is the -x-direction and "off the page" is the +x-direction. Device length may be provided along the y-axis, where left of the page is the -y-direction and right of the page is the +y-direction. Device height may be provided along the z-axis, where top of the page is the +z-direction and bottom of the page is the -z-direction. Figures 38, 40-45, 54-56, 61, 65-75, and 79 use similar notation, rotated and labeled appropriately for the figures.

[0421] FIG. 38 illustrates a volume of approximately 5 mL and a volume of approximately 10 mL according to an exemplary embodiment. 12 1 is a plan view image of a small-scale HDCR platform with a cell density of approximately vg. Small-scale HDCR may be suitable for research and development applications.

[0422] FIG. 39 shows a volume of approximately 500 mL and a volume of approximately 10 mL according to an exemplary embodiment. 14 1 is a perspective image of a mid-scale HDCR platform with a cell density of approximately vg. Mid-scale HDCR may be suitable for animal research applications.

[0423] FIG. 40 shows a volume of approximately 50 L and a volume of approximately 10 L according to an exemplary embodiment. 16~17 Figure 1 shows a perspective image of a large-scale HDCR platform with a cell density of approximately 15 inches. In this exemplary embodiment, the plate extends approximately 15 inches from inlet to outlet. Large-scale HDCR may be suitable for clinical use.

[0424] Some embodiments of the present systems, devices, and methods include a gas-perfusable microfabricated membrane for high-density cell culture. Oxygen can be the most limiting nutrient, and has been observed to be approximately 155 times more limiting than the next most limiting nutrient (i.e., GLN). Some embodiments of the present systems, devices, and methods overcome the oxygen delivery limitations of conventional culture systems by providing oxygen directly from the gas-perfusable membrane on which the cells grow. With feasibility in mind, the membrane may be constructed solely from relatively inexpensive medical-grade materials to mitigate concerns about contaminant leaching.

[0425] Illustrative Embodiments FIG. 41 is a schematic side view of an HDCR platform having spaced apart fins configured to supply oxygen and receive carbon dioxide, according to an exemplary embodiment.

[0426] FIG. 42 is a schematic side view of a tilted HDCR platform according to an exemplary embodiment. The top of FIG. 42 is a high-density embodiment of the tilted HDCR platform with seven fins between the ends of the device. The bottom of FIG. 42 is a low-density embodiment of the tilted HDCR platform with no fins between the ends of the device. Both the high-density and low-density embodiments are tilted at an angle of approximately 10 degrees. See FIGS. 70-78 for an analysis of the effect of various tilt angles.

[0427] FIG. 43 is a schematic side view of a sloped HDCR platform configured to facilitate bubble removal (top) and condensation (bottom), according to an exemplary embodiment.

[0428] 44-47, 52-54 and 79 show exemplary embodiments of HDCR devices according to exemplary embodiments.

[0429] FIG. 44 is a bottom perspective view of an HDCR cartridge according to an exemplary embodiment.

[0430] FIG. 45 is a top perspective view of the HDCR cartridge of FIG. 44 according to an exemplary embodiment. The HDCR device may have multiple stacked plates configured to be stacked on top of each other. In FIG. 45, a stack of three plates 4502, 4504, and 4506 is provided. Each plate 4502, 4504, and 4506 may have one or more channels. In FIG. 45, three channels 4512, 4514, and 4516 are provided. The three channels 4512, 4514, and 4516 may be bounded by a boundary ridge 4522. The boundary ridge 4522 may include a continuous wall that bounds the entire perimeter of the three channels 4512, 4514, and 4516. The boundary ridge 4522 may have a relatively thick portion at each corner of the plate. Each corner of the plate may have an opening 4524 to allow a connector (not shown) to pass therethrough. The openings 4524 may be bounded by circular ridges 4526. Each opening 4524 in each plate may communicate with an opening in an adjacent plate.

[0431] 46 is an enlarged portion of the top perspective view of FIG. 45 of an HDCR cartridge according to an exemplary embodiment. The first channel 4512 may be separated from the second channel 4514 by a first ridge 4605. The second channel 4514 may be separated from the third channel 4516 by a second ridge 4610. Each of the first ridge 4605 and the second ridge 4610 may extend continuously across the entirety or substantially the entirety of each plate (e.g., 4502). The first channel 4512 may be separated from the second channel 4514 by the first ridge 4605.

[0432] Figure 47 is a top view of the HDCR cartridge of Figures 44-46 according to an exemplary embodiment. Each plate (e.g., 4502, 4504, 4506) can have multiple openings through its base channel surface (e.g., 4601) for communication with openings located in adjacent plates or adjacent structures other than plates. The openings in one plate can communicate with the openings in another plate. The top of one plate can be configured to nest and / or structurally cooperate with the bottom of another plate to form an integrated sandwich structure as shown in detail in Figures 52-54.

[0433] Figure 79 is an enlarged plan view of an HDCR membrane according to an exemplary embodiment (see Figure 47). Specifically, a three-layer 50 cm 2 HDCR membranes are installed. Note the asymmetry of the left and right media inlet and outlet channels. In this configuration, the membranes are rotated 180° relative to the membranes above and below them. This creates a serpentine flow path, which can improve mixing of the components added to the bioreactor to achieve better uniformity.

[0434] For example, the first plate 4502 may include a first opening 4705, a second opening 4710, and a third opening 4715 within an area surrounded by a boundary region 4522 adjacent to a first end of the first ridge 4605 and a first end of the second ridge 4610, and further, the first plate 4502 may include a fourth opening 4720, a fifth opening 4725, a sixth opening 4730, and a seventh opening 4735 within an area surrounded by a boundary region 4522 adjacent to a second end of the first ridge 4605 and a second end of the second ridge 4610. The first opening 4705 in the first plate 4502 can communicate with one end of the fourth opening 4720 and one end of the fifth opening 4725 in the second plate 4504 to form a first common fluid passageway through the first plate 4502 and the second plate 4504. The second opening 4710 in the first plate 4502 can communicate with the other end of the fifth opening 4725 and one end of the sixth opening 4730 in the second plate 4504 to form a second common fluid passageway through the first plate 4502 and the second plate 4504. The third opening 4715 in the first plate 4502 can communicate with the other ends of the sixth opening 4730 and the seventh opening 4735 in the second plate 4504 to form a third common fluid passageway through the first plate 4502 and the second plate 4504. The fourth opening 4720 in the first plate 4502 can communicate with one end of the first opening 4705 in the second plate 4504 to form a fourth common fluid passageway through the first plate 4502 and the second plate 4504. The fifth opening 4725 in the first plate 4502 can communicate with the other end of the first opening 4705 and one end of the second opening 4710 in the second plate 4504 to form a fifth common fluid passageway through the first plate 4502 and the second plate 4504. The sixth opening 4730 in the first plate 4502 can communicate with the other end of the second opening 4710 in the second plate 4504 and one end of the third opening 4715 to form a sixth common fluid passageway through the first plate 4502 and the second plate 4504.The seventh opening 4735 in the first plate 4502 can communicate with another end of the third opening 4715 in the second plate 4504 to form a seventh common fluid passageway through the first plate 4502 and the second plate 4504. The first opening 4705, the second opening 4710, the third opening 4715, the fifth opening 4725, and the sixth opening 4730 can have a generally similar shape. That is, they can each be a relatively long slot with rounded ends, as shown in FIG. 47. The fourth opening 4720 and the seventh opening 4735 can have a generally similar shape. That is, they can each be a relatively short slot with rounded ends, as shown in FIG. 47. As best seen in FIG. 47, the overlap of one set of openings with another set of openings can effectively form a generally circular through opening when looking straight down through the plates.

[0435] Thus, at each end of the first plate 4502, the first channel 4512 of the first plate 4502 may be configured to communicate with the first channel 4512 and the second channel 4512 of the second plate 4504, the second channel 4514 of the first plate 4502 may be configured to communicate with the first channel 4512 and the third channel 4516 of the second plate 4504, and the third channel of the first plate 4502 may be configured to communicate with the second channel 4514 and the third channel 4514 of the second plate 4504.

[0436] Each plate (e.g., 4502) can be configured with a plurality of structures in the active region 4699, which are configured to promote oxygen and carbon dioxide exchange and other features of the HDCR system described herein. The plurality of structures in the active region 4699 may be a series of alternating protruding ridges and recessed depressions formed on and in the base channel surface 4601 of each plate. Side cross-sectional views of various exemplary ridges and depressions are shown in Figures 1, 2, 3, 5, 6, 14, 41, 42, 43, 66, 67, 68, and 72 (inclusive). Other structures may be provided, such as grooves / fins shown in the upper left corner of Figure 7, wells shown in the lower left corner of Figure 7, and / or posts shown in the lower right corner of Figure 7. Region 4699 may have a smooth structure, as shown in the upper right corner of Figure 7. The structures in region 4699 may be any suitable shape, including pillars, channels, grooves, bumps, protrusions, and / or legs in any suitable combination. Each structure may be substantially similar to the other structures within region 4699, or different shapes may be provided within region 4699.

[0437] Plasmid transfection in 2-HDCR In an exemplary embodiment, a demo-scale HDCR bioreactor according to an exemplary embodiment was assembled with a glass bottom for microscope compatibility. Approximately 1.7 mL of blue-dyed Cytodex 3 microcarriers was pumped through the bioreactor membrane using two approximately 50 mL syringes. The bioreactor and components were autoclaved on a liquid cycle for approximately 15 minutes.

[0438] On day 1, the final connection to the three-way stopcock was made under a biohood. The fluid pathways in the HDCR bioreactor were flushed with degassed PBS + 1% P / S / A at 37°C to remove air bubbles, including the loop. The HDCR bioreactor was then flushed with DMEM + 10% FBS + 1% P / S / A (20 mL) on day 2. The stopcock was rotated to purge any air bubbles in the dead volume. 3.6 million P21 HEK293 AAV cells were loaded into the bioreactor (3 PM) through the gas trap line and circulated via a static pump for 3 × 1 min to evenly seed the cells within the membrane. The gas flow rate was set to approximately 0.1 mL / min, and the medium flow was set to approximately 0.02 mL / min. Air bubbles were removed from the trap daily by purging with medium.

[0439] On day 5, 4 mg DNA / million cells at 4x10 7 A transfection cocktail was prepared assuming a density of 100 cells / mL (232 μL pDP2rs, 52 μL pAAV-ssG FP, 1216 μL DMEM combined with 567 μL PEI and 933 μL DMEM, vortexed for approximately 5 seconds, and allowed to stand at room temperature for approximately 10 minutes). On day 5, the transfection cocktail was injected into the loop and circulated for 3 × 1 minute. The perfusion was stopped, and the bioreactor was returned to the incubator. At 30, 60, and 90 minutes after transfection, the medium was mixed on the loop for approximately 1 minute. Approximately 4 hours after transfection, the medium flow was resumed at 0.05 mL / min.

[0440] On day 8, the membranes were removed and imaged in a Petri dish by filling the gas chamber with PBS to improve light transmission. Cells and supernatant were collected for purification from half of the membrane that showed the best transfection (the other half had low transfection). The cells / microcarriers were pelleted at 500 g for 5 minutes, and the supernatant was removed. The pellets were frozen at 80°C.

[0441] For the above clinical trial, Figure 48 is an image (4x magnification) of GFP (pAAV) 70 hours after transfection according to an exemplary embodiment. Figure 49 is an image (4x magnification) of the phase 70 hours after transfection according to an exemplary embodiment. Figure 50 is an image (4x magnification) of RFP (pHelper+RC) 70 hours after transfection according to an exemplary embodiment. Figure 51 is an overlay of Figures 48, 49, and 50 according to an exemplary embodiment.

[0442] Integral O-ring seal HDCR compartment FIGS. 52-54 include various views of O-ring seals for HDCR compartments between HDCR plates. In FIG. 52, one or more peripheral integral O-rings 5201 facilitate alignment and stacking of HDCR membranes, such as plates 4502, 4504, and 4506. When compressed, as shown in FIG. 53, the O-rings 5201 seal to form independent gas and fluid manifolds. Specifically, FIG. 52 is a cross-sectional cut through an uncompressed four-stack HDCR membrane, according to an exemplary embodiment, illustrating the membrane seal and alignment. FIG. 53 is a cross-sectional cut through a compressed (40% compression ratio) four-stack HDCR membrane, according to an exemplary embodiment. FIG. 54 is a CAD rendering of an HDCR membrane with an enlarged cross-section (bottom left) showing the gas compartment openings and various compression ratios of possible designs (i.e., 20%, 30%, and 40% compression ratios) according to an exemplary embodiment. The gas compartment openings may be reinforced (eg, with rigid tubing) to maintain their patency under compressive forces from O-rings above and below the gas compartment opening.

[0443] 4-Stack Demo Scale HDCR Run Figures 55-57 show four stacks of 50cm assembled into a functional bioreactor. 2 HDCR membrane shown. 5% CO 2Air containing supplements is perfused through the membrane through the gas compartment, while medium is perfused through the cell-medium compartment and waste is disposed of through the medium outlet. A mixing loop is formed by tubing connected between the medium inlet and outlet using an adjustable valve. Connecting the tubing to a peristaltic pump allows for circulation of medium within the cell-medium compartment, useful for uniform introduction of cells, microcarriers, viruses, transfection agents, or other additives. Rapid perfusion of fluid through the circulation loop, optionally combined with tilting or inversion of the bioreactor, allows cells or bioproducts to be harvested from the membrane. Bubble traps can be added to the bioreactor to mitigate the introduction of air bubbles into the cell-medium compartment and facilitate their removal. Perfusion of medium and gas can be controlled by peristaltic, syringe, or other pumps (e.g., active or passive, such as gravity-fed). After loading the membrane with Cytodex-3 microcarriers stained with trypan blue for visualization, A549 cells were seeded and grown over 10 days from approximately 700,000 to 100,000,000 cells. Specifically, Figure 55 is a front perspective view of a four-stack demo-scale HDCR according to an exemplary embodiment, Figure 56 is a rear perspective view of the four-stack demo-scale HDCR of Figure 55 according to an exemplary embodiment, and Figure 57 is a front perspective view of the complete system for the four-stack demo-scale HDCR of Figures 55 and 56 according to an exemplary embodiment.

[0444] Glucose-mediated metabolic monitoring Glucose consumption by the cells in the bioreactor is monitored by sampling the input and output medium streams using a glucose meter. Glucose consumption is determined from the difference between the glucose concentration and the known perfusion rate. Glucose levels can be maintained within a specific range by adjusting the medium and gas supply rates to the bioreactor. Specifically, FIG. 58 is a plot of glucose consumption (mol / hr) (on the y-axis) versus days (on the x-axis) comparing theoretical and experimental results according to an exemplary embodiment, and FIG. 59 is a front perspective view of a system for measuring flow rates and glucose-derived metabolism in the effluent stream according to an exemplary embodiment.

[0445] FIG. 60 is a front perspective view of a system for measuring glucose metabolism by harvesting cells for counting in DNA units, according to an exemplary embodiment, with harvested cells and microcarriers appearing at the bottom of a vial after 10 days of growth of A549 cells in an HDCR bioreactor.

[0446] Half scale (600cm 2 ) Running HDCR 600cm 2An HDCR bioreactor system based on a membrane design is provided. The HDCR cartridge includes top and bottom compression plates, fluid couplings and valves for the medium and gas compartments, as well as circulation tubing, an HDCR membrane, and bolts and spacers. In this example, a syringe pump controls the perfusion of medium into the bioreactor. A bubble trap removes air bubbles from the perfusion stream and prevents them from entering the system. A peristaltic pump controls the perfusion of gas into the bioreactor. A gas bubbler can also be incorporated to humidify the gas and reduce water pervaporation from the medium compartment. Spent medium is collected in a waste bottle, while depleted gas is vented to the atmosphere. By appropriately positioning additional peristaltic pumps and fluid valves, circulation of medium through the bioreactor can be achieved to facilitate seeding, infection / transfection, and harvesting. For example, Figure 61 shows, from right to left, a medium pump, a bubble trap, a gas bubbler, and a 600 cm 2 FIG. 1 is a front perspective view of a half-scale HDCR system, including an HDCR cartridge, a cell distributor, a waste container, a gas pump, and associated interconnecting tubing.

[0447] Production of CF33-GFP Production of the orthopoxvirus CF33-GFP was performed in an HDCR bioreactor by growing A549 cells to approximately 400 million cells / membrane under controlled media and gas perfusion. Cell growth was monitored using theoretical and experimental measurements of glucose consumption during the production run. On day 11, CF33-GFP virus was introduced into the bioreactor at a multiplicity of infection of 0.03 plaque-forming units per cell. Three days post-infection, cells and viral material were harvested from the bioreactor for titration and purification. Figure 62 is a plot of glucose consumption (mol / hour) (on the y-axis) versus days of operation (on the x-axis), comparing theoretical and experimental results according to an exemplary embodiment. Figure 63 shows the CF33-GFP band after the first sucrose gradient centrifugation. Table 3 below shows the 600 cm concentration of CF33-GFP after purification. 2The yield of CF-33 GFP in the HDCR membrane and in the unpurified flow-through medium is shown. [Table 3]

[0448] Exosome extraction 600cm 2 Spent media from the HDCR bioreactor was collected over days 8, 9, 10, and 11 of A549 cell growth. Exosomes were purified from the spent media and subsequently quantified and characterized using nanoparticle tracking analysis. The results demonstrate that significant amounts of exosomes can be collected from the media exiting the HDCR bioreactor. Figure 64 shows a plot of exosomes / day / membrane (on the y-axis) versus date (on the x-axis), i.e., for a 600 cm membrane with A549 cells according to an exemplary embodiment. 2 Exosome production in HDCR.

[0449] 5-Stack HDCR Five stacks of 600cm operated in a standard cell incubator 2 An HDCR bioreactor setup including a membrane is provided. Microcarriers stained with trypan blue are seen to be uniformly distributed within the membrane. Specifically, Figure 65 shows a five-stack 600 cm membrane according to an exemplary embodiment. 2 FIG. 1 is a front perspective view of a system including an HDCR cartridge.

[0450] Tilting of HDCR bioreactor Positioning the HDCR bioreactor membrane at an angle facilitates the removal of air bubbles from the cell-medium compartment and the removal of condensation from the gas compartment. In the horizontal position, there is minimal force from the perfusion medium to displace and sweep the air bubbles out of the medium compartment, whereas in the tilted position, the hydrostatic pressure gradient propels the air bubbles toward a higher elevation, where they eventually reach the fluid manifold and are transported to waste collection. Although not required, it is advantageous to perfuse the medium into a low-elevation fluid manifold and collect it from a high-elevation fluid manifold. This is because, in this configuration, both the hydrostatic force on the air bubbles and the hydrostatic force are in the same direction. Similarly, in the gas compartment, condensation and water droplets are more easily removed when the membrane is in an inclined position due to their tendency to move down a gravitational potential gradient. Although not required, it is advantageous for the perfusion of gas in the gas compartment to enter and exit the high-elevation fluid manifold. This is because, in this configuration, both the hydrostatic force on the droplets and the gravitational force are in the same direction. Figure 66 is a schematic cross-sectional front view of an HDCR membrane with air bubbles above the membrane and condensation below the membrane, according to an exemplary embodiment. In contrast, Figure 67 is a schematic cross-sectional front view of a sloped HDCR membrane highlighting the removal of the air bubbles above the membrane and the removal of the condensation below the membrane, according to an exemplary embodiment (the pre-removal state is shown at the top of Figure 67, and the post-removal state is shown at the bottom of Figure 67).

[0451] Figures 68-75 illustrate membrane architectures that facilitate cell retention. The architectures are configured for membrane-based bioreactor operation in a non-horizontal orientation due to the tendency of cells to clump or pool together at masses too large for efficient mass transport of nutrients and gases. HDCR membranes incorporate such architectures (e.g., wells, grooves, cups, pillars, overhangs, etc.) on the side of the membrane facing the cell culture medium to overcome this problem. A comparison of membranes with high- and low-aspect ratio architectures in a horizontal orientation (Figures 68 and 70) shows how the introduction of cell retention structures (e.g., fins) reduces the volume available for cell growth (Figures 69 and 71). However, in a tilted orientation (Figures 72 and 74), the growth volume lost due to the presence of the retention structures is more than compensated for by increased cell retention (Figures 73 and 75). Specifically, Figure 68 is a schematic cross-sectional side view of a non-tilted, high-aspect ratio HDCR membrane filled with culture medium, according to an exemplary embodiment. Figure 69 is a representation of the cell growth volume of a non-graded, high-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. Figure 70 is a schematic cross-sectional side view of a non-graded, low-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. Figure 71 is a representation of the cell growth volume of a non-graded, low-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. Figure 72 is a schematic cross-sectional side view of a gradient, high-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. Figure 73 is a representation of the cell growth volume of a gradient, high-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. Figure 74 is a schematic cross-sectional side view of a gradient, low-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment. Figure 75 is a representation of the cell growth volume of a gradient, low-aspect ratio HDCR membrane filled with medium, according to an exemplary embodiment.

[0452] Figure 76 establishes geometric variables for cell retention in a rectangular channel versus tilt angle, according to an exemplary embodiment. In the equation below, W is the width of the fin, A is the area of ​​the fin, h is the height of the fin, W is the width of the cell, A is the area of ​​the cell, and θ is the tilt angle of the cell. Relative cell retention efficiency:

number

number

number

[0453] Figure 77 is a plot of the relative cell retention efficiency in a 0.8 mm deep rectangular channel of various widths (mm) as a function of membrane tilt angle, which increases exponentially with widths ranging from about 0.5 mm to about 128 mm.

[0454] Figure 78 is a plot of the overall cell retention efficiency in 0.8 mm deep rectangular channels of various widths (mm) as a function of membrane tilt angle, which increases exponentially with widths ranging from about 0.5 mm to about 128 mm.

[0455] FIG. 83 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 10% cell confluence and 400 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0456] FIG. 84 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 33% cell confluence and 400 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0457] FIG. 85 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 100% cell confluence and 400 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0458] FIG. 86 includes 11 plots of HDCR simulations of bioreactor cellularity versus architecture for 10% cell confluence and 700 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0459] FIG. 87 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 33% cell confluence and 700 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0460] FIG. 88 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 100% cell confluence and 700 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0461] FIG. 89 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 10% cell confluence and 1000 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0462] FIG. 90 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 33% cell confluence and 1000 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0463] FIG. 91 includes 12 plots of HDCR simulations of bioreactor cellularity versus architecture for 100% cell confluence and 1000 μm fin height with varying cell niche width and fin width, according to an exemplary embodiment.

[0464] For each of the simulations referenced above, the cell niche width was varied from 200 μm to 500 μm in 100 μm increments, and the fin width was varied from 300 μm to 500 μm in 100 μm increments.

[0465] The results of the simulations are summarized in Tables 4, 5 and 6 below. [Table 4] [Table 5] [Table 6]

[0466] In conclusion, in terms of viable reactor cellularity (VRC) per mL, the highest VRC per mL, i.e., 3.65 × 10 8 The lowest VRC / mL, i.e., 1.49x10, was observed with a fin height of 400 μm, a fin width of 500 μm, a niche width of 200 μm, a cell confluence of 10%, and a reactor length of 9.15 m. 7VRC / mL was observed. Generally speaking, higher cell density and metabolism may contribute to the availability of relatively shorter bioreactor lengths due to the associated consumption of soluble nutrients in the higher cell density and metabolism. Example

[0467] 600cm 2 Four stacks of HDCR membranes were assembled into a bioreactor cartridge and sterilized. The bioreactor was wetted and filled with 80 mL of microcarriers. The bioreactor and mixing lines were flushed with approximately 400 mL of medium (i.e., F-12K + 10% FBS + 1% P / S). Approximately 166 million A549 cells at passage 22 were seeded into the bioreactor in approximately 20 mL and gently circulated to settle into the cell niche of the membrane. A bubble trap line was fluidly connected to a syringe pump to remove accumulated gas (set at approximately 1 mL / hr). The gas flow rate was set at approximately 0.5 mL / min, and the medium flow rate was set at approximately 0.5 mL / hr. The medium exiting the waste stream was measured for glucose daily, and the flow rate was adjusted to target a concentration of approximately 100 mg / dL. Gas perfusion was scaled proportionally to the medium perfusion. Prior to virus infection, the medium was replaced with approximately 500 mL of reduced serum (F-12K + 2.5% FBS + 1% P / S). CF33 hNIS-anti-PDL1 virus was added at an MOI of 0.1 to approximately 25 mL of 2.5% FBS + F-12K medium and circulated on a loop at approximately 50 mL / min for approximately 15 minutes. The bioreactor was returned to the incubator, and medium perfusion was resumed at approximately 0.5 mL / min for 48 hours post-infection (F-12K + 10% FBS + 1% P / S). After virus addition, the effluent stream was collected and subsequently stored at approximately 4°C to analyze virus escape from the host cells. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0468] The foregoing description details specific embodiments of the systems, devices, and methods disclosed herein. However, no matter how detailed the foregoing appears in the text, it will be understood that the devices and methods can be practiced in many ways. Also, as noted above, the use of specific terms in describing a particular feature or characteristic of technology should not be construed as meaning that the terms have been redefined herein so as to be limited to including any particular characteristics of the feature or characteristic of the relevant technology. Accordingly, the scope of the present disclosure should be construed according to the appended claims and any equivalents thereof.

[0469] Those skilled in the art will understand that various modifications and changes can be made without departing from the scope of the described technology. Such modifications and changes are intended to be included within the scope of the embodiments as defined by the appended claims. Those skilled in the art will also understand that parts included in one embodiment can be interchanged with other embodiments, and that one or more parts from an illustrated embodiment can be included with other illustrated embodiments in any combination. For example, any of the various components described herein and / or shown in the drawings can be combined with, interchanged with, or excluded from other embodiments.

[0470] With respect to the use of any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0471] It will be understood by those skilled in the art that, in general, the terms used herein, and particularly in the appended claims, are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the terms "comprising" and "having" should be interpreted as "comprising at least" and "having at least," respectively, the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those skilled in the art that if a specific numerical recitation is intended in the appended claims, such intention will be expressly recited in the claims; in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitation. However, the use of such phrases should not be construed as suggesting that introducing a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to embodiments containing only one such recitation, even if that same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an." Generally, "a" and / or "an" should be construed to mean "at least one" or "one or more," and the same is true for the use of definite articles used to introduce claim recitations.

[0472] Although at least one exemplary embodiment is described as using multiple units to perform an exemplary process, it will be understood that the exemplary process may be performed by one or more modules.

[0473] The use of terms such as "first," "second," "third," etc. herein is provided to distinguish various structures, dimensions, or operations, and does not describe any order; structures, dimensions, or operations may be performed in an order different from that stated, unless a particular order is clearly dictated by the context.

[0474] Throughout this specification and the claims, approximate expressions can be used to modify any quantitative expression that can be permissibly varied without causing a change in the basic function to which it relates. Thus, values ​​modified by one or more terms such as "about" and "substantially" should not be limited to the exact value specified. In at least some cases, approximate language can correspond to the precision of the instrument used to measure the value. Here, and throughout this specification and the claims, range limitations can be combined and / or interchanged, and such ranges include all subranges identified and contained therein, unless the context or language indicates otherwise.

[0475] Unless specifically stated or clear from the context, the term "about" as used herein is understood to mean within normal tolerances in the art, for example, within two standard deviations of the mean. "About" may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0476] Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, or C together, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, wherever it appears in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B." The subject matter described herein may be embodied in systems, devices, methods, and / or articles, depending on the desired configuration. The embodiments set forth in the foregoing description do not represent all embodiments consistent with the subject matter described herein. Instead, they are merely some examples consistent with aspects related to the described subject matter. While some variations have been described in detail above, other modifications or additions are possible. In particular, additional features and / or variations may be provided in addition to those described herein. For example, the above-described embodiments may be directed to various combinations and subcombinations of the disclosed features and / or combinations and subcombinations of multiple additional features described above.Additionally, the logic flow depicted in the accompanying figures and / or described herein does not necessarily require the particular order shown or sequential order to achieve desirable results. Other embodiments may be within the scope of the following claims.

[0477] The technology disclosed herein has many applications, and while particular embodiments of the technology have been described in detail, it will be apparent to those skilled in the art that the disclosed embodiments can be modified in light of the design considerations discussed herein. Accordingly, the foregoing description should be considered illustrative rather than limiting, and the true scope of the invention is defined in the following claims.

Claims

1. a membrane comprising a plurality of surface features on a first side of the membrane, the surface features comprising one or more compartments capable of entrapment of one or more cells; a membrane, the membrane comprising a material that is at least partially permeable to gas; a second side of the membrane defining one boundary of a gas region, the membrane configured to allow gas to pass through the second side of the membrane to the first side of the membrane; a medium region having a boundary on the first side of the membrane and configured to pass medium across the first side of the membrane, wherein the one or more compartments are configured to at least partially reduce medium flow shear forces within the compartments.

2. 10. The device of claim 1, further comprising one or more medium inlets and one or more medium outlets fluidly connected to the medium region, wherein the one or more medium inlets are configured to facilitate introduction of medium into the medium region on the first side of the membrane and the one or more medium outlets are configured to facilitate removal of medium from the medium region.

3. 3. The device of claim 1, wherein at least one of the one or more medium inlets is fluidly connected to at least one of the medium outlets to facilitate reintroduction of medium into the medium region on the first side of the membrane.

4. 4. The device of claim 1, wherein the device is configured to minimize medium flow shear forces in the one or more compartments upon introduction or removal of medium from the medium region.

5. 5. The apparatus of claim 1, further comprising one or more gas inlets and one or more gas outlets in fluid contact with the gas region, the one or more gas inlets configured to introduce gas into the gas region and the one or more gas outlets configured for removal of gas from the gas region.

6. 6. The device of claim 1, further comprising one or more channels formed in the second side of the membrane, wherein at least a first of the one or more channels is configured to deliver gas through the membrane to the at least one of the compartments by permeation and diffusion through the membrane.

7. The device of any one of claims 1 to 6, further comprising an enclosure disposed above the first side of the membrane.

8. The device of claim 7 , wherein the enclosure defines a second boundary of the medium region.

9. The device of any one of claims 1 to 8, wherein the device is configured to be stacked with at least a second cell culture device.

10. 10. The device of claim 9, wherein one or more of the medium inlet or gas inlet or medium outlet or gas outlet are configured such that when stacked, the one or more of the medium inlet or gas inlet or medium outlet or gas outlet are in fluid communication with an inlet or outlet of an immediately adjacent device.

11. 11. The device of claim 9 or 10, wherein one or more of the medium inlets or gas inlets are configured such that, when stacked, medium or gas introduced into the device is combined and enters a combined inlet line for delivery to multiple of the membranes.

12. 12. The device of any one of claims 9 to 11, wherein one or more of the medium outlets or gas outlets are configured such that, when stacked, medium or gas removed from the device is combined into a combined outlet line for delivery from a plurality of the membranes.

13. The device of any one of claims 1 to 12, further comprising a liquid-tight container for holding the device.

14. 14. The device of any one of claims 1 to 13, wherein the membrane comprises pores having a size that allows permeation and diffusion of oxygen through the membrane.

15. 15. The device of any one of claims 1-14, wherein the one or more compartments support tissue-level cell densities such that a volume of cells occupies at least about 10% of the volume of the one or more compartments capable of enclosing one or more cells.

16. 16. The device according to claim 1, wherein the negative pressure deforms a bottom surface of at least one of the one or more compartments downward, drawing liquid medium into the at least one compartment through an opening in an upper part of the at least one cell niche, and / or the positive pressure deforms a bottom surface of the at least one cell niche upward, pushing the liquid medium out of the at least one compartment through the opening in the upper part of the at least one compartment.

17. 17. The device of any one of claims 1 to 16, wherein mechanical stretching of the membrane expands at least one of the one or more compartments to facilitate release of one or more cells from the at least one cell niche through the opening in the top of the at least one compartment.

18. The apparatus of any one of claims 1 to 17, further comprising a plurality of microdiffusers configured to rectify the pulsating pressure into a net flow normal to the first surface of the membrane.

19. The device of any one of claims 1 to 18, further comprising at least one porous wick configured to transport said fluid medium to said one or more compartments.

20. 20. The device of any one of claims 1 to 19, wherein the membrane comprises a micropatterned architecture having a plurality of compartments designed to provide a significantly high gas exchange area to volume ratio to maximize oxygen permeation rates into the compartments formed in the membrane.

21. 21. The device of any preceding claim, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with the gas.

22. 22. The device of any one of claims 1 to 21, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with the liquid medium.

23. 23. The device of any one of claims 1 to 22, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with at least one of the gas and the liquid medium to promote uniform distribution of reagents or cells in the one or more compartments.

24. 24. The device of any one of claims 1-23, wherein the membrane is configured to expand or contract in response to changes in pressure or flow associated with at least one of the gas and the liquid medium to facilitate efficient cultivation or harvesting of cells located within the one or more compartments within the membrane.

25. 25. The apparatus of any one of claims 1 to 24, wherein the fluid path for the culture medium is gravitationally supported by one or more flow regulators that regulate liquid culture medium flow across the first side of the membrane.

26. 26. The device of any one of claims 1 to 25, further comprising a plurality of microcarriers to which cells can adhere.

27. 27. The device of any one of claims 1 to 26, wherein the plurality of surface features comprises a plurality of fin structures extending substantially parallel to one another, the substantially parallel fin structures defining grooves between adjacent fin structures, at least one groove on a first side of the membrane providing at least one of the one or more compartments for cell placement, and a longitudinal direction of the at least one groove corresponding to a longitudinal direction of the one or more compartments.

28. 28. The apparatus of claim 27, wherein the one or more medium inlets are configured to introduce medium into the medium region to create a medium flow that is not parallel to the longitudinal direction of the channel.

29. 29. The apparatus of claim 27 or 28, wherein the one or more medium inlets are configured to introduce medium into the medium region to create a medium flow substantially perpendicular to the groove.

30. 29. The apparatus of claim 27 or 28, wherein the one or more medium inlets are configured to introduce medium into the medium region to create a medium flow substantially aligned with the grooves.

31. The device of any one of claims 27 to 30, wherein the upper opening of one or more of the one or more cell niches is narrower than the width of the lower opening.

32. 32. The device of any one of claims 27-31, wherein the plurality of fins protrude from a base of the membrane to retain and protect one or more of the cells within the one or more cell niches from media flow shear forces generated by media delivery.

33. 33. The apparatus of any one of claims 27 to 32, wherein the membrane has a multi-layer monolithic structure such that the one or more channels for delivering the gas are formed in a first layer of the membrane and the compartments are formed between the plurality of fins in a second layer of the membrane above the first layer.

34. 34. The device of any one of claims 27 to 33, wherein a dedicated space is provided above the compartments formed between the fins to support a fluid path for the liquid medium flowing above the fins and substantially perpendicular to the fins.

35. 35. The device of any one of claims 27 to 34, wherein a dedicated space is provided above the plurality of compartments formed between the plurality of fins to support a fluid path for the liquid medium to flow above the plurality of fins and substantially aligned with the plurality of fins.

36. 36. The apparatus of any one of claims 27-35, wherein the fluid pathway has relatively low resistance to the liquid medium flowing over the plurality of fins, thereby eliminating or reducing the need for the use of a pump to regulate the liquid medium flow.

37. 37. The device of any one of claims 1 to 36, wherein the surface features comprise a plurality of well structures, at least one of the plurality of well structures providing at least one of the one or more compartments for cell placement.

38. 38. The apparatus of claim 37, wherein an opening in an upper portion of at least one of the well structures includes a diametric width that is narrower than a diametric width of the well structure below the opening.

39. 39. The apparatus of claim 37 or 38, wherein one or more of the well structures comprises an opening that is circular, oval, square, rectangular, hexagonal, or octagonal.

40. 40. The device of any one of claims 37 to 39, wherein the well structure protects the one or more cells within the one or more cell niches of the well structure from shear forces caused by the medium delivery.

41. 41. The apparatus of any one of claims 37 to 40, wherein the membrane has a multi-layer monolithic structure such that the one or more channels for delivering the gas are formed in a first layer of the membrane and the compartments are formed in well structures in a second layer of the membrane above the first layer.

42. 42. The apparatus of any one of claims 37 to 41, wherein a dedicated space is provided above the plurality of well structures to support a fluid path for the liquid medium to flow above the plurality of well structures.

43. 43. The apparatus of any one of claims 37-42, wherein the fluid pathway has relatively low resistance to the liquid medium flowing over the plurality of well structures, thereby eliminating or reducing the need for the use of a pump to regulate the liquid medium flow.

44. 44. The apparatus of any one of claims 37 to 43, wherein a dedicated space is provided above the plurality of well structures to support a fluid path for the liquid medium to flow above the plurality of well structures.

45. 43. The device of any one of claims 37 to 42, wherein at least one well structure has a circular opening.

46. 44. The device of any one of claims 37 to 43, wherein at least one well structure has a polygonal opening.

47. 45. The device of any one of claims 37 to 44, wherein at least one well structure has a curved opening.

48. 38. The device of any one of claims 1 to 37, wherein the surface structure comprises a plurality of post structures, at least one post structure providing at least one of the plurality of compartments for cell placement.

49. 49. The device of claim 48, wherein cells are attached to the at least one post structure.

50. 50. The device of claim 48 or 49, wherein the membrane has a multi-layer monolithic structure such that the one or more channels for delivering the gas are formed in a first layer of the membrane and the cell niche is formed proximal to a post structure in a second layer of the membrane above the first layer.

51. 51. The apparatus of any one of claims 48 to 50, wherein a dedicated space is provided above the plurality of post structures to support a fluid path for the liquid medium to flow above the plurality of post structures.

52. 52. The apparatus of any one of claims 48-51, wherein the fluid pathway has relatively low resistance to the liquid medium flowing over the plurality of post structures, thereby eliminating or reducing the need for the use of a pump to regulate the liquid medium flow.

53. 53. The apparatus of any one of claims 48 to 52, wherein a dedicated space is provided above the plurality of post structures to support a fluid path for the liquid medium to flow above the plurality of post structures.

54. 54. The apparatus of any one of claims 48 to 53, wherein at least one post structure has a circular cross-section.

55. 55. The device of any one of claims 48 to 54, wherein at least one well post has a polygonal cross-section.

56. 54. The apparatus of any one of claims 48 to 53, wherein at least one post structure has a curved, angled, crescent-shaped, or U-shaped cross-section.

57. 57. The apparatus of any one of claims 1 to 56, further comprising a Petri dish configured to receive the membrane.

58. 58. The device of claim 57, wherein the membrane forms the bottom of the Petri dish.

59. 59. The device of claim 57 or 58, further comprising a tension ring configured to keep the membrane in tension and provide additional rigidity.

60. 60. The device of any one of claims 57 to 59, further comprising spacing pillars protruding from a second surface of the membrane opposite the first surface, the spacing pillars being configured to allow gas exchange through the membrane to the cell niche.

61. 1. A Petri dish comprising a bottom surface and a sidewall forming a dish, the bottom surface comprises at least a portion of a gas permeable material, the bottom surface comprising a first side and a second side, the first side comprising a plurality of structures extending from a base of the first side to form one or more cell niche regions below an upper surface of the plurality of structures; The sidewalls are connected to the bottom surface and form a continuous and substantially vertical wall around the periphery of the bottom surface.

62. 62. The petri dish of claim 61, wherein the bottom surface and the sidewalls comprise the same material.

63. 63. The petri dish of claim 62, wherein the bottom surface and the sidewalls comprise the at least partially gas permeable material.

64. 64. The petri dish of any one of claims 61 to 63, wherein the sidewall comprises at least one material different from the bottom surface.

65. 65. The Petri dish of any one of claims 61 to 64, further comprising one or more retaining elements that provide support to the side walls.

66. 66. A Petri dish according to any one of claims 61 to 65, wherein the bottom surface of the Petri dish exposed to the external environment comprises the at least partially gas-permeable material such that gas from the external environment can pass at least partially through a gas-permeable membrane to the first side and / or gas from the first side can pass from the first side through the at least partially gas-permeable membrane to the external environment.

67. 67. A Petri dish according to any one of claims 61 to 66, wherein the bottom surface of the Petri dish is placed on a mesh, cloth, or other open-pore material to allow gases from the external environment to exchange with the membrane.

68. 68. The Petri dish of claim 67, wherein the bottom surface of the Petri dish exposed to the external environment includes one or more deformations in its shape that allow gas to pass at least partially underneath the bottom surface when the dish is placed on a flat surface.

69. 69. The petri dish of claim 68, wherein the shapes comprise pillars, channels, grooves, bumps, protrusions, or legs.

70. 69. The petri dish of claim 68, wherein the shape includes one or more spacing pillars.

71. 62. The petri dish of claim 61, further comprising a top surface, said top surface comprising a sealing membrane.

72. 72. The petri dish of claim 71, wherein the top surface comprises a silicone-based membrane.

73. 72. The Petri dish of claim 71, further comprising a sealable port for transferring media to or from said Petri dish.

74. 1. A multi-well cell growth device comprising a plurality of wells, the wells comprising a bottom surface including a first side in contact with the interior of the well and a second side in contact with the exterior of the multi-well cell growth device, the first side comprising a topography providing a plurality of cell growth compartments, the bottom surface comprising a material that is at least partially permeable to gas, the bottom surface being configured to allow gas from the exterior of the multi-well growth device to pass from the exterior to the interior of the wells to contact one or more of the plurality of cell growth compartments, and / or to allow gas from within the one or more cell growth compartments to pass from the first side to the second side.

75. 75. The multi-well cell growth device of claim 74, wherein the topography of the first side comprises one or more of fins, subwells, and pillars, the fins, subwells, and pillars at least partially defining the plurality of cell growth compartments.

76. 76. The multi-well cell growth device of claim 74 or 75, wherein the sidewalls of the plurality of wells comprise at least a partially gas-permeable material.

77. 77. The multi-well cell growth device of any one of claims 74-76, wherein the second side includes one or more variations in its shape that allow gas to pass at least partially beneath the bottom surface when resting on a flat surface.

78. 78. The multi-well cell growth device of claim 77, wherein the shapes comprise pillars, channels, grooves, bumps, protrusions, or legs.

79. 79. The multi-well cell growth device of claim 77 or 78, wherein the shape comprises one or more spacing pillars.

80. a membrane comprising a plurality of fin structures extending substantially parallel to one another, the substantially parallel fin structures defining grooves between adjacent fin structures, at least one groove on a first side of the membrane providing at least a compartment for cell placement; a membrane, the membrane comprising a material that is at least partially permeable to gas; a second side of the membrane defining a gas region, the second side being separated from the first side by the membrane and through which gas can pass; a medium region on the first side of the membrane configured to receive medium containing one or more cells that can be deposited within the compartment.

81. one or more membranes; at least one membrane of the one or more membranes having a plurality of fins including at least a first fin and a second fin protruding from a first surface of the membrane to form a first compartment of a plurality of compartments configured to hold a plurality of cells; a membrane formed from a gas-permeable and air-permeable material to facilitate delivery of gas to the plurality of compartments through one or more channels formed beneath the first surface of the membrane below the plurality of fins, and at least a first channel of the one or more channels configured to deliver the gas directly to the first compartment;

82. 100. A method of culturing biological cells, the method comprising: providing a device according to any one of claims 1 to 81; introducing biological cells into the device; and providing cell culture medium on the first side of the device such that the cell culture medium contacts the biological cells.

83. 83. The method of claim 82, further comprising providing a gas to the second side of the membrane.

84. 84. The method of claim 82 or 83, further comprising flowing culture medium over one or more cell compartments.

85. 85. The method of any one of claims 82 to 84, wherein flowing the medium comprises introducing the medium through an inlet and removing the medium through an outlet.

86. 86. The method of any one of claims 82 to 85, further comprising flowing a gas to the second side of the membrane.

87. 87. The method of any one of claims 82 to 86, wherein flowing the gas comprises introducing the gas through an inlet and removing the gas through an outlet.

88. 88. The method of any one of claims 82-87, further comprising flowing culture medium, biological cells, or another substance into the one or more cell compartments by creating a negative pressure within the one or more compartments.

89. 89. The method of any one of claims 82 to 88, further comprising maintaining cells within said one or more compartments by creating a negative pressure within said one or more compartments.

90. 90. The method of any one of claims 82-89, further comprising creating a positive pressure within the one or more compartments, thereby causing medium, biological cells, or another substance to flow out of the one or more cell compartments.

91. 91. The method of any one of claims 82 to 90, further comprising directing cells out of said one or more compartments by creating a positive pressure within said one or more compartments.

92. 92. The method of any one of claims 82-91, further comprising mixing one or more of the biological cells in the one or more compartments by creating positive and / or negative pressure in the one or more compartments.

93. 93. The method of any one of claims 82-92, wherein the positive and / or negative pressure is generated by flowing gas through the gas region, flowing medium through the medium region, flowing both gas and medium through their respective regions, preventing flow of gas into the gas region and / or preventing flow of medium into the medium region, thereby generating a pressure differential between the two regions.

94. The living cells are human, non-human mammals, insects, bacteria, fungi, yeast, 3T3-L1, 4T1, 9L, A20, A172, A253, A431, A549, A2780, A2780ADR, A2780cis, AB9, AHL-1, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BOSC23, BT-20, BxPC-3, C2C12, C3H-10T1 / 2, C6, C6 / 36, Caco-2, Cal-27, Calu-3, CGR8, CHO, CML T1, CMT12, COR-L23, COR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23-, COS-7, COV-434, CT26, D17, DAOY, DH82, DU145, DuCaP, E14Tg2a, vEL4, EM-2, EM-3, vEMT6 / AR1, EMT6 / AR10.0, FM3, GL261, H1299, HaCaT, HCA2, HEK293, HEK293T, HeLa, Hep G2, Hepa1c1c7, High Five, HL-60, HT-29, HT-1080, J558L, Jurkat, JY, K562, KBM-7, KCL-22, KG1, Ku812, KYO-1, L243, L1210, LNCaP, MA-104, Ma-Mel, MA2.1, MC-38, MCF-7, MCF-10A, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, MDCK II, MG63, MIA PaCa-2, Mono-Mac-6, MOR / 0.2R, MRC-5, MTD-1A, MyEnd, NALM-1, NCI-H69, NCI-H69 / CPR, NCI-H69 / LX4, NCI-H69 / LX10, NCI-H69 / LX20, Neuro-2a, Neuro2a, NIH-3T3, NK-92, NTERA-2, NW-145, OK, OPCN / OPCT cell line, P3X63Ag8, PAN-1, PC-3, PC12, Peer, PNT1A, PNT2, Pt94. The method of any one of claims 82 to 93, wherein the cell is selected from the group consisting of K2, Raji, RBL-1, RenCa, RIN-5F, RMA-S, S2, SaOS-2, Sf9, Sf21, SH-SY5Y, SiHa, SK-BR-3, SK-N-SH, SK-OV-3, T-47D, T2, T84, T98G, THP-1, U2OS, U87, U373, U937, VCaP, Vero, VG-1, WM39, WT-49, YAC-1, and YAR cells.

95. 95. The method of any one of claims 82 to 94, wherein the gas is selected from the group consisting of oxygen, carbon dioxide, nitrogen, carbon monoxide, nitrous oxide, hydrogen sulfide, ethylene oxide, ozone, chlorine dioxide, and nitrogen dioxide.

96. The medium may be DMEM, FCS, 293SFM II, AEM, CDM4HEK293, SFM4HEK293, Ex-Cell293, SFM4Transfx-293, Freestyle293, ESF SFM, CDM4CHO, CHO medium, MEM, MEM Alpha, RPMI, F-10, F-12, IMDM, Medium 199, Leibovitz L-15, McCoy's 5A, MCDB medium, William's medium, CMRL medium, OptiMEM, OptiPro, AIM V, or OptiPEAK.

96. The method of any one of claims 82-95, wherein the T lymphocytes are cultured in a medium selected from the group consisting of ExCellerate Human T Cell Growth Medium, StemXVivo Serum-Free Human T Cell Base Medium, ExpiSf CD Medium, Sf-900 II / III SFM, and TC-100 Insect Medium.

97. 91. The method of claim 88 or 90, wherein the other material is selected from the group consisting of waste materials, materials secreted by the cell, materials inside the cell, cell debris, and gases.

98. 98. The method of any one of claims 82-97, further comprising introducing a cryogen into the gas region to freeze cells and cellular components in the plurality of compartments, wherein the cryogen has a temperature below 0°C, and wherein the cryogen is in a gaseous or liquid state.

99. 61. The device of any one of claims 1 to 60, wherein at least one of the surface features has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

100. 100. The apparatus of claim 99, wherein the at least one of the surface features has a height of about 700 μm to about 1000 μm.

101. 101. The apparatus of claim 100, wherein the at least one of the surface features has a height of about 700 μm.

102. 61. The apparatus of any one of claims 1 to 60, wherein at least one of the surface features has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

103. 103. The apparatus of claim 102, wherein the height to width to pitch ratio is about 7-10 to about 3-5 to about 2-5.

104. 104. The apparatus of claim 103, wherein the height to width to pitch ratio is about 7 to about 3-5 to about 2-5.

105. 74. The petri dish of any one of claims 61 to 73, wherein at least one of the plurality of structures has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

106. 106. The petri dish of claim 105, wherein the at least one of the plurality of structures has a height of about 700 μm to about 1000 μm.

107. 107. The petri dish of claim 106, wherein said at least one of said plurality of structures has a height of about 700 μm.

108. 74. The petri dish of any one of claims 61-73, wherein at least one of the plurality of structures has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

109. 109. The Petri dish of claim 108, wherein the height to width to pitch ratio is about 7-10 to about 3-5 to about 2-5.

110. 110. The Petri dish of claim 109, wherein the height to width to pitch ratio is about 7 to about 3-5 to about 2-5.

111. 80. The multi-well cell growth device of any one of claims 74-79, wherein at least one of the plurality of cell growth compartments has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

112. 112. The multi-well cell growth device of claim 111, wherein said at least one of said plurality of cell growth compartments has a height of about 700 μm to about 1000 μm.

113. 113. The multi-well cell growth device of claim 112, wherein said at least one of said plurality of cell growth compartments has a height of approximately 700 μm.

114. 80. The multi-well cell growth device of any one of claims 74-79, wherein at least one of said plurality of cell growth compartments has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

115. 115. The multi-well cell growth device of claim 114, wherein the height to width to pitch ratio is about 7-10 to about 3-5 to about 2-5.

116. 116. The multi-well cell growth device of claim 115, wherein the height to width to pitch ratio is about 7 to about 3-5 to about 2-5.

117. 81. The cell culture device of claim 80, wherein at least one of the plurality of fin structures has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

118. 118. The cell culture device of claim 117, wherein said at least one of said plurality of fin structures has a height of about 700 μm to about 1000 μm.

119. 119. The cell culture device of claim 118, wherein said at least one of said plurality of fin structures has a height of approximately 700 μm.

120. 81. The cell-culture device of claim 80, wherein at least one of said plurality of fin structures has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

121. 121. The cell-cultivating device of claim 120, wherein the height to width to pitch ratio is about 7-10 to about 3-5 to about 2-5.

122. 122. The cell-cultivating device of claim 121, wherein the height to width to pitch ratio is about 7 to about 3-5 to about 2-5.

123. 82. The cellular respiratory device of claim 81, wherein at least one of the plurality of fins has a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

124. 124. The cellular respiratory device of claim 123, wherein said at least one of said plurality of fins has a height of about 700 μm to about 1000 μm.

125. 125. The cellular respiration device of claim 124, wherein the at least one of the plurality of fins has a height of about 700 μm.

126. 82. The cellular respiratory device of claim 81, wherein at least one of said plurality of fins has a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

127. 127. The cellular respiratory device of claim 126, wherein the height to width to pitch ratio is about 7-10 to about 3-5 to about 2-5.

128. 128. The cellular respiratory device of claim 127, wherein the height to width to pitch ratio is about 7 to about 3-5 to about 2-5.

129. 99. The method of any one of claims 82 to 98, wherein the device comprises at least one surface feature having a height of about 400 μm to about 1000 μm, a width of about 300 μm to about 500 μm, and a pitch of about 200 μm to about 500 μm.

130. 130. The method of claim 129, wherein the device comprises at least one surface feature having a height of about 700 μm to about 1000 μm.

131. 131. The method of claim 130, wherein the device comprises at least one surface feature having a height of about 700 μm.

132. 99. The method of any one of claims 82 to 98, wherein the device comprises at least one surface feature having a height to width to pitch ratio of about 4-10 to about 3-5 to about 2-5.

133. 133. The method of claim 132, wherein the height to width to pitch ratio is about 7-10 to about 3-5 to about 2-5.

134. 134. The method of claim 133, wherein the height to width to pitch ratio is about 7 to about 3-5 to about 2-5.

135. A gas inlet; A gas outlet; a gas path connected between the gas inlet and the gas outlet; a medium inlet; a medium outlet; a medium passage connected between the medium inlet and the medium outlet; a membrane separating the gas supply path and the culture medium supply path; the membrane comprises a cytoprotective region; the cell protection region is in fluid communication with the medium pathway; the cell protection region is fluidically distinct from the medium pathway; the cell protection region is in gas communication with the gas pathway across the membrane; A bioreactor, wherein the cell protection region is configured to reduce the effect of shear force of the culture medium flowing through the culture medium path on cells produced in the cell protection region.

136. The bioreactor is ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 136. The bioreactor of claim 135, configured to produce a productivity of AAV vg / L.

137. 137. The bioreactor of claim 135 or 136, wherein the velocity of the medium at the medium inlet is from about 1 μm / sec to about 1 m / sec.

138. The velocity of the medium at the medium inlet is about 100 μm / sec, the bioreactor has a length of about 40 cm, and ８ 138. The bioreactor of any one of claims 135 to 137, which produces cells / mL.

139. The velocity of the medium at the medium inlet is about 1 μm / sec, and the bioreactor is about 10 ６ 139. The bioreactor of any one of claims 135 to 138, which produces cells / mL.

141. The device is ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 105. The apparatus of any one of claims 1 to 60 and 99 to 104, configured to produce an AAV vg / L productivity.

142. 142. The apparatus of any one of claims 1-60, 99-104, and 141, wherein the velocity of the medium at the medium inlet is from about 1 μm / sec to about 1 m / sec.

143. The velocity of the medium at the medium inlet is about 100 μm / sec, the device has a length of about 40 cm, and ８ 143. The device of any one of claims 1 to 60, 99 to 104, 141 and 142, which produces cells / mL.

144. The velocity of the medium at the medium inlet is about 1 μm / sec, and the device is ６ 144. The device of any one of claims 1 to 60, 99 to 104 and 141 to 143, producing cells / mL.

145. The Petri dish contains about 10 ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 111. The petri dish of any one of claims 61 to 73 and 105 to 110, configured to produce a productivity of AAV vg / L.

146. 146. The petri dish of any one of claims 61-73, 105-110, and 145, wherein the petri dish has a length of about 40 cm.

147. The Petri dish contains about 10 ８ 147. The petri dish of any one of claims 61 to 73, 105 to 110, 145 and 146, which produces cells / mL.

148. The Petri dish contains about 10 ６ 147. The petri dish of any one of claims 61 to 73, 105 to 110, 145 and 146, which produces cells / mL.

149. The multi-well cell growth device comprises about 10 ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 117. The multi-well cell growth device of any one of claims 74-79 and 111-116, configured to generate a productivity of AAV vg / L.

150. 150. The multi-well cell growth device of any one of claims 74-79, 111-116, and 149, wherein the multi-well cell growth device has a length of about 40 cm.

151. The multi-well cell growth device comprises about 10 ８ 151. The multi-well cell growth device of any one of claims 74-79, 111-116, 149 and 150, producing cells / mL.

152. The multi-well cell growth device comprises about 10 ６ 151. The multi-well cell growth device of any one of claims 74-79, 111-116, 149 and 150, producing cells / mL.

153. The cell culture device is ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 123. The cell culture device of any one of claims 80 and 117 to 122, configured to produce AAV vg / L of production capacity.

154. 154. The cell-cultivating device of any one of claims 80, 117-122, and 153, wherein the velocity of the culture medium at the culture medium inlet is from about 1 μm / sec to about 1 m / sec.

155. The velocity of the medium at the medium inlet is about 100 μm / sec, the cell culture device has a length of about 40 cm, and ８ The cell culture device according to any one of claims 80, 117 to 122, 153 and 154, which produces cells / mL.

156. The velocity of the medium at the medium inlet is about 1 μm / sec, and the cell culture device is ６ The cell culture device according to any one of claims 80, 117 to 122 and 153 to 155, which produces cells / mL.

157. The cellular respiratory device ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 129. The cellular respiration device of any one of claims 81 and 123-128, configured to produce AAV vg / L.

158. 158. The cellular respiratory device of any one of claims 81, 123-128, and 157, wherein the velocity of the medium at the medium inlet is from about 1 μm / sec to about 1 m / sec.

159. The velocity of the medium at the medium inlet is about 100 μm / sec, the cellular respiration device has a length of about 40 cm, and ８ 159. The cellular respiration device of any one of claims 81, 123-128, 157 and 158, producing cells / mL.

160. The velocity of the medium at the medium inlet is about 1 μm / sec, and the cellular respiratory device is about 10 ６ 160. The cellular respiration device of any one of claims 81, 123-128 and 157-159, producing cells / mL.

161. The method comprises: ７ cells / milliliter (cells / mL) to about 10 ９ The cell density at the time of transfection was 10 cells / mL, and approximately 10 １５ Adeno-associated virus genomes / liter (AAV vg / L) to about 10 １６ 135. The method of any one of claims 82-98 and 129-134, configured to produce an AAV vg / L productivity.

162. 162. The method of any one of claims 82-98, 129-134, and 161, wherein the velocity of the medium at the medium inlet is from about 1 μm / sec to about 1 m / sec.

163. the velocity of the medium at the medium inlet is about 100 μm / sec, the method has a length of about 40 cm, and ８ The method of any one of claims 82 to 98, 129 to 134, 161 and 162, wherein the method produces cells / mL.

164. the velocity of the medium at the medium inlet is about 1 μm / sec, and the method comprises: ６ The method of any one of claims 82 to 98, 129 to 134 and 161 to 163, wherein the method produces cells / mL.

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