Fixed-bed cell culture and harvesting system and method of using the same

Abstract

A cell culture system is provided that includes a cell culture vessel having an internal cavity that accommodates a cell culture substrate within a cell culture space and at least one port for at least one of fluid inlet and outlet from the internal cavity. The system further includes a piston having a distal end positioned within the cell culture vessel above the cell culture space, the distal end of the piston being sealed by an airtight seal within the internal cavity. The system also includes a drive coupled to the piston that moves the piston to increase or decrease the distance between the distal end and the cell culture space. The drive can pressurize the internal cavity by actuation of the piston to harvest cells from the cell culture space through the at least one port.

Description

Description of Related Applications

[0001] This application claims the benefit of priority under 35 U.S.C. § 120 to U.S. Provisional Patent Application No. 62 / 941,010, filed November 27, 2019, the contents of which are relied upon and incorporated herein in their entirety.

Technical Field

[0002] The present disclosure relates generally to the field of bioprocesses, and more particularly to packed bed bioreactors and methods of using such bioreactors for performing cell culture, including cell harvesting.

Background Art

[0003] In the bioprocess industry, large-scale cell culture is carried out for the purpose of producing hormones, enzymes, antibodies, vaccines, and developing cell therapies. A significant portion of the cells used in bioprocesses are anchorage-dependent, meaning that the cells require a surface to which they can attach in order to grow and function. Conventionally, the culture of adherent cells has been performed on two-dimensional (2D) cell adhesion surfaces incorporated into one of several container formats, such as T-flasks, Petri dishes, cell factories, cell stack containers, roller bottles, HYPERStack® containers, and the like. These approaches can have significant drawbacks, such as difficulty in achieving cell densities high enough to enable therapies or large-scale production of cells.

[0004] Alternative methods have been proposed to increase the volumetric density of cultured cells. These include microcarrier culture performed in stirred tanks. In this method, cells attached to the surface of the microcarriers are subjected to a certain shear stress, and as a result, their growth and culture performance are significantly affected. Another example of a high-density cell culture system is a hollow fiber bioreactor, where cells may form large three-dimensional aggregates while growing in the fiber interstices. However, due to nutrient deficiencies, cell growth and performance are significantly inhibited. To mitigate this problem, these bioreactors have been miniaturized and are not suitable for large-scale manufacturing.

[0005] Another example of a high-density culture system for scaffold-dependent cells is a packed-bed bioreactor system. For example, a packed-bed bioreactor system including a packed bed of a support or matrix system for capturing cells has been previously disclosed in Patent Documents 1 to 3. The packed-bed matrix is usually made of porous particles as a substrate or a non-woven microfiber of a polymer. Such a bioreactor functions as a recirculation flow-through type bioreactor. One of the major problems of such a bioreactor is the non-uniform cell distribution inside the packed bed. For example, the packed bed functions as a depth filter, and cells are mainly captured in the inlet region, resulting in a gradient of cell distribution during the seeding process. Furthermore, since the fibers are randomly installed, the flow resistance and cell capture efficiency across the cross-section of the packed bed are not uniform. For example, in a region where the cell packing density is low, the culture medium flows fast, and in a region where the number of captured cells is large and the resistance is high, the culture medium flows slowly. For this reason, a channeling effect is brought about in which nutrients and oxygen are efficiently supplied in a region with a low volume cell density, and a suboptimal culture state is maintained in a region with a high cell density. Another major drawback of the packed-bed systems disclosed in the prior art is that viable cells may not be efficiently harvested at the end of the culture process. Patent Document 4 discloses a design of a bioreactor for improving the cell recovery efficiency from a packed bed during the cell harvesting process. This is based on loosening the packed-bed matrix, stirring or agitating the packed-bed particles, and colliding the porous matrix to thereby separate the cells. However, this method is time-consuming and may cause significant damage to the cells, resulting in a decrease in the overall cell viability.

[0006] Among other current solutions available on the market, cells cannot be easily harvested from a perfusion bioreactor using standard enzymatic methods. Instead, virus recovery by cell lysis with detergents is used, which adds time and complexity to the downstream purification of the virus.

Prior Art Documents

Patent Document

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] The production of viral vectors for early-stage clinical trials is possible with existing platforms, but a platform that can produce more high-quality products is needed to reach the scale of late-stage commercial production. In particular, there is a need for a platform and method for partitioning a packed bed while managing the fluid flow of cells and nutrients through the packed bed and the air permeability of the cell culture medium.

Means for Solving the Problems

[0009] Disclosed herein is a cell culture system comprising a cell culture container having an internal cavity for containing a cell culture substrate within a cell culture space and at least one port for at least one of a fluid inlet to the internal cavity and a fluid outlet from the internal cavity. The system further comprises a piston having a distal end disposed within the cell culture container above the cell culture space, the distal end of the piston being sealed by an airtight seal within the internal cavity. The system also comprises a drive device coupled to the piston for moving the piston to increase or decrease the distance between the distal end and the cell culture space. The drive device can pressurize the internal cavity by actuation of the piston to harvest cells from the cell culture space through at least one port.

[0010] A method of using a cell culture system to culture cells and harvest cells from the container is also disclosed, using the operation of a piston disposed at least partially inside the cell culture container. This piston is used to draw one or more solutions into the container, including a dissociation solution for separating cells from a washing solution and a substrate. Next, the piston is used to draw a fluid containing the dissociation solution, cells, and any cell by-products from the container into a harvester.

[0011] Additional aspects of the present disclosure are set forth in part in the detailed description, the drawings, and any of the following claims, and in part are derived from the detailed description or can be learned by the practice of the present disclosure. It should be understood that both the foregoing general description and the following detailed description are merely examples and explanations and are not limitations of the present disclosure as disclosed.

Brief Description of the Drawings

[0012] The present disclosure will be more fully understood by reference to the following detailed description when interpreted in conjunction with the accompanying drawings.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0013] Referring to the drawings showing various aspects of a packed bed bioreactor system according to non-limiting embodiments of the present disclosure and related methods of using such bioreactor systems, various embodiments of the present disclosure will be discussed. The following description is intended to provide a possible description of such bioreactor systems, and various aspects of the bioreactor systems and methods are specifically discussed in detail throughout the present disclosure with reference to non-limiting embodiments, and these embodiments are interchangeable with each other within the context of the present disclosure.

[0014] Referring to FIG. 1, a schematic diagram of a packed bed bioreactor system 100 according to one or more embodiments of the present disclosure is provided. The bioreactor system 100 enables the cultivation of adherent cells and the harvesting of cell by-products (e.g., transfected viruses), as well as the harvesting of the cells themselves. As can be seen from the figure, the bioreactor system 100 includes a container 102 containing a cell culture substrate 104. This cell culture substrate can take various forms, but is preferably a porous material provided in an integral structure (such as a foam substrate or a single woven mesh sheet) or in a packed bed form of a plurality of fragments of the substrate material 103. In one preferred embodiment, for example, the plurality of fragments of the substrate material 103 are sheets or disks of a porous polymer made from one or more polymeric fibers. For example, the porous polymeric material can be a woven mesh substrate material provided as a laminate of sheets or disks within the container 102.

[0015] The container 102 of FIG. 1 is a simplified plunger design for explaining the general structure and operation of a cell culture container that enables cell harvesting according to an embodiment of the present disclosure. Specifically, the container 102 includes a plunger 106 disposed to move at least partially within the container in directions D1 and D2 toward and away from the cell culture substrate 104. This pumping action pressurizes or depressurizes the interior of the container 102, as described below, to draw one or more solutions from sources 108 and 110 (e.g., harvest solution or DPBS (Dulbecco's phosphate buffered saline) wash), assist in releasing adherent cells from the cell culture substrate 104, and discharge those released cells from the interior of the container, all without damaging the cells or viral products. Sources 108 and 110 can contain different enzymatic and non-enzymatic dissociating agents to target cell-substrate and cell-cell interactions.

[0016] The bioreactor systems and methods of the present disclosure provide advantages related to automation, viable cell harvest quantity, and scalability. According to aspects of some embodiments, the bioreactor systems disclosed herein can pressurize the container to produce a safe flow rate that does not damage the cells or harvest products. The pressurization operation for harvesting can be automated and fully integrated into the bioreactor control system. For example, the plunger can be actuated by a pneumatic piston or some other electromechanical drive device controlled by a controller. Further, the solutions provided herein can scale up to larger bioreactors (e.g., containers of 50 L or more) used in the industry. Embodiments of the present disclosure can create high pressure and flow within the bioreactor system, for example, by using a pneumatic cylinder that pushes down and then lifts the cell culture substrate, and continuously raising and lowering that pneumatic cylinder with a pump to cause cell separation from the substrate after treatment of the substrate with a dissociating agent.

[0017] Current commercial products tend to use non-uniform substrates, which can unevenly capture cells within the substrate during attachment. This results in non-uniform cell growth and subsequent inefficient transfection of DNA plasmids into the cells. This has an adverse effect on the total virus produced by the cells. In addition, since many of the cells are captured, the virus within the cells cannot be efficiently recovered for later use in gene therapy. Currently, as a second-best option, detergents are used to lyse the cells and release the virus in situ. However, the addition of detergents adds cost and complexity to the downstream virus purification process. In embodiments of the present disclosure, the pressure supplied from the automatic piston should promote cell release from the bioreactor substrate, eliminating the need for cell lysis. As one aspect of an embodiment of the present disclosure, an appropriate cell culture substrate promotes the uniformity of flow and the release of cells for harvesting. Examples of such substrate materials are disclosed in the specifications of U.S. Patent Application Nos. 16 / 781685, 16 / 781723, and 17 / 039218, and International Publication No. 2019 / 104069, the entire contents of which are hereby incorporated by reference.

[0018] Figure 2 is a schematic diagram of a cell culture system 200 according to one or more embodiments. The system 200 includes a cell culture vessel 202 having an internal cavity at least partially filled with a cell culture substrate, and one or more fluid inlets and / or outlets 220, 222 arranged to send and remove a fluid containing air and a cell culture medium to and from the internal cavity. A drive device 205 (such as a pneumatic drive device or an air cylinder) is arranged above the cell culture vessel 202 and is connected to a piston 206 at least partially inserted into the cell culture vessel 202 via a connection part 207 (such as a clevis). The system 200 further includes a drive device control unit 216. The drive device control unit 216 can be any control unit known in the art that can control the operation of the drive device 205. As shown in Figure 2, the system 200 also includes a pressure regulator 218 connected to a gas or air supply source. The pressure regulator 218, the gas or air supply source, the valve, and the drive device control unit 216 control the entry of air and the medium into the bioreactor. The inlets and / or outlets 220, 222 are connected to one or more fluid supply sources. According to various embodiments, the inlets and / or outlets 220, 222 can both be connected to the same fluid supply source or different fluid supply sources, and each is controlled by one or more valves to control the inflow or outflow of fluid from the inlets and / or outlets 220, 222. A control device 214 for controlling the fluid flow and the operation of the drive device during cell harvesting can be incorporated into the system 200 or housed in another computer that communicates with the system according to known wired or wireless connection methods.

[0019] During harvesting, the substrate and cells are immersed in a dissociation buffer or enzyme. Thereafter, air will come from an air supply source through regulator 218. Here, the pressure of the air is controlled by regulator 218. The valve is opened so that the pressurized air can move to drive device 205. As a result, the inflowing pressurized air will push down piston 206 sealed inside the upper part of container 202. When piston 206 moves downward, pressure is applied to the liquid in container 202. As a result, the cells move downward together with the liquid through the substrate and are discharged from the bottom of container 202 to harvest collection container 208 at outlet 222.

[0020] According to an embodiment, by entering container 202 and increasing the flow rate of the dissociating agent passing through container 202, the cell harvest amount can be improved. The flow rate of the dissociating agent can be, for example, from about 1 ml / min to about 500 ml / min, or about 250 ml / min, and a higher flow rate can result in a larger harvest amount.

[0021] The following aspects of the embodiment describe a method for harvesting cells from the above-described system 200. During cell culture, piston 206 is at least partially inside container 202. The position of piston 206 during cell culture before harvesting is supported above cell culture substrate 204 and can hold the substrate in place and immobilized during culture. Alternatively, container 202 can include another cell culture chamber or other support to limit and / or support cell culture substrate 204. Referring to FIG. 3, the initial stage of cell culture is cell adhesion stage S1. During this stage, the medium is perfused into bioreactor container 202, cells are seeded into container 202 from the inlet, the control device sets container 202 and the medium to a desired temperature (e.g., 37° C.), the cell culture medium is sent through container 202 at a specified rate, and the concentrations of relevant nutrients, oxygen, and CO2 are set.

[0022] Next, the cell culture enters the cell growth stage S2. During this period, data can be collected to monitor the progress of the cell culture. This data may include information about cell metabolites, pH levels, gas measurements, cell confluence or biomass, and temperature. The cell growth stage can continue for several days during which this data is monitored, and the system can be refilled with the conditioning medium as needed. At least one or two days after the cell culture stage, cell harvesting can be started in step S3. Piston 206 may remain pressing on top of substrate 204 at the start of harvesting, and the valves between container 212 and container 202, as well as the valve between container 208 and container 202, are opened. Next, the system is pressurized or the pump connected to container 208 is activated to supply the DPBS detergent from container 208 into container 202, and the excess DPBS detergent can be collected in container 212 in step S4. Next, the valve to container 212 is closed, and the valve at inlet 222 is switched in container 208 from the supply of the DPBS detergent to the supply of the harvesting solution. Then, piston 206 moves up and down according to the control system, thereby sucking up the dissociating agent into container 202 in step S5. The pressure from piston 206 and the resulting inflow of the dissociating agent create shear stress on the cells, causing the cells to be released from substrate 204. Piston 206 remains in the raised position for a predetermined period so that all of the dissociating agent enters container 202 and the cells are exposed to the dissociating agent for a sufficient time. After this exposure time, in step S6, piston 206 is forced down to discharge the combination of the medium, dissociating agent, cells, and cell by-products into a bottle, which becomes the "harvesting solution". This harvesting solution is collected in a section of container 208. After the harvesting solution is collected, the valves at inlets 220 and 222 are actuated so that inlet 222 is closed and inlet 220 is opened, and washing with DPBS is performed in step S7. When piston 206 is pulled up, the DPBS detergent enters container 202 from container 212 and the washing is performed.Next, the valve at the inlet 222 can be opened and the valve at the inlet 220 can be closed, so that when the piston 206 descends, the DPBS cleaning agent is collected in the harvest solution collection compartment within the container 208. According to the method described above, it is possible to harvest viable cells in high yields as described herein.

[0023] During cell culture, the medium is consumed for cell metabolism. Therefore, the system 200 may include a number of sensors (e.g., probes, disposable patches, RAMAN, etc.) for reading environmental conditions (e.g., pH, CO2, DO, temperature, fluid flow, shear stress, cell density) in order to enable monitoring and control of the bioreactor 200, for example, by a PID feedback loop to the system. As an example, if the dissolved oxygen (DO) is too low, the flow of medium refilled through the cell culture substrate can be increased. In addition, one or more inlets 220, 222 may provide inlets for rapidly supplying a liquid feed (e.g., caustic, glucose, medium, bolus addition) into the cell culture space of the container 202. In addition, one or more probes (e.g., biomass probe, Raman probe) may be provided within or near the cell culture substrate 204.

[0024] The cell culture substrate is porous so that cells, medium, nutrients, and cell by-products can be perfused through the substrate and spent medium containing cell secreted substances (e.g., recombinant proteins, antibodies, virus particles, DNA, RNA, sugars, lipids, biodiesel, inorganic particles, butanol, metabolic by-products) can be passed through and harvested from the substrate. Further details of the cell culture substrate according to embodiments are given below.

[0025] The container 202 can be made of plastic, glass, ceramic, or stainless steel. According to some embodiments, all or part of the container 202 may be made of a transparent material, or may include one or more transparent windows on the outer wall of the container 202 to enable inspection of the interior of the container 202 by the naked eye or by any of a number of sensors, probes, cameras, or monitoring devices. For example, according to aspects of some embodiments, an optical camera or a Raman spectroscopy probe can be used to monitor the progress of cell culture within the cavity of the container 202.

[0026] Figure 4 shows a bioreactor 402 as described herein incorporated into a bioprocess system 400 according to one or more embodiments. The system 400 includes, for example, a media conditioning container 411 for appropriately maintaining parameters of a cell culture medium, such as pH, temperature, and oxygenation level. An automatically controlled pump 409 is used to perfuse the media into the bioreactor 402. An additional three-way port is provided at the inlet 413 of the bioreactor to facilitate cell inoculation or collection of harvested cells. The system 400 may include in-line sensors, as well as sensors within the media conditioning container 411.

[0027] As described above, the bioreactor according to the embodiments of the present disclosure can include one or more ports and sensors for monitoring and regulating the culture medium and the cell culture environment within the container. However, according to some embodiments, the detection and conditioning of the cell culture medium can be performed within a second container external to the bioreactor. For example, FIG. 4 shows a schematic view of a bioreactor vessel 402 connected to the main external components including a medium conditioning vessel, a pump that enables the flow of the medium into the bioreactor, and an external dissolved oxygen sensor to assist in the process conditions required for a successful bioprocess. The cell culture medium is conditioned within the medium conditioning vessel 411 where appropriate pH levels, temperature levels, and dissolved oxygen concentrations are maintained. Subsequently, the medium is perfused into the bioreactor by pump 409. The flow rate of pump 409 is integrated into a feedback loop that automatically adapts to maintain a minimum predetermined concentration of dissolved oxygen in the medium exiting the bioreactor. All transfection reagents, nutrients, and additional medium supplements required for a given bioprocess can be introduced into the bulk medium and spent medium can be removed through the medium conditioning vessel 411. At the end of this process, the medium can be drained from the bioreactor and used to replenish the cell harvest solution. After incubating the packed bed in the harvest solution for a predetermined time sufficient for the cells to separate from the substrate, the cells are harvested in a countercurrent flow by applying pneumatic pressure to the outlet of the bioreactor to achieve a flow rate in the range of 70 ml / cm 2 (cross-sectional area of the packed bed) / minute. The cells are harvested at the three-way port 413 of the bioreactor. The cells can also be lysed directly within the bioreactor and the lysate solution containing the AVV particles can be collected through the three-way port 413.

[0028] The medium conditioning vessel 411 can be equipped with sensors and control components found in typical bioreactors used in the bioprocessing industry for suspension batch, fed-batch, or perfusion cultures. These can include, but are not limited to, a DO oxygen sensor, a pH sensor, an oxygenation / gas sparging device, a temperature probe, and ports for nutrient addition and base addition. The gas mixture supplied to the sparging device can be controlled by a gas flow controller for N2, O2, and CO2 gases. The medium conditioning vessel 411 also includes an impeller for medium mixing. All of the medium parameters measured by the sensors enumerated above can be controlled by a medium conditioning controller 418 that communicates with the medium conditioning vessel 411 and can measure and / or adjust the state of the cell culture medium 406 to a desired level.

[0029] The medium 406 from the medium conditioning vessel 411 is delivered through an inlet to the bioreactor 402, which may also include an injection port for the cell inoculum material to be seeded and may initiate the culturing of cells. The bioreactor vessel 402 may also include one or more outlets through which the cell culture medium exits the vessel 402. In addition, cells or cell products may be produced through that outlet. One or more sensors 412 may be provided in that line to analyze the contents of the effluent from the bioreactor 402. In some embodiments, the system 400 includes a flow control device for controlling the flow into the bioreactor 402. For example, the flow control device may receive a signal from one or more sensors 412 and, based on that signal, transmit a signal to a pump (e.g., a peristaltic pump) upstream of the inlet 408 to the bioreactor 402 to adjust the flow into the bioreactor 402. Therefore, based on one or a combination of the factors measured by the sensors 412, the pump can control the flow into the bioreactor 402 to obtain desired cell culture conditions.

[0030] The medium perfusion rate is controlled by a signal processing device that collects and compares signals from sensors in the medium conditioning vessel 411 and sensors located at the outlet of the packed bed bioreactor. Due to the nature of the perfusion flow of the medium through the packed bed bioreactor 402, gradients of nutrients, pH, and oxygen occur along the packed bed. The perfusion rate of the bioreactor can be automatically controlled by a flow control device operably connected to a peristaltic pump according to the flowchart of FIG. 5.

[0031] FIG. 5 shows an example of a method 450 for controlling the flow of a perfusion bioreactor system such as the system 400 of FIG. 4. According to the method 450, certain parameters of the system 400 are predetermined in step S1 by optimizing the operation of the bioreactor. From these optimized operations, the values of pH1, pO1, [glucose]1, pH2, pO2, [glucose]2, and the maximum flow rate can be determined. The values of pH1, pO1, and [glucose]1 are measured in the cell culture chamber of the bioreactor 402 in step S2, and the values of pH2, pO2, and [glucose]2 are measured by the sensor 412 in the medium conditioning vessel 411 (or within the bioreactor according to the embodiments described herein) in step S3. Based on these values in S2 and S3, the perfusion pump control device makes a determination in S4 to maintain or adjust the perfusion rate. For example, the perfusion rate of the cell culture medium into the cell culture chamber is such that if at least one of pH2 ≧ pH 2min , pO2 ≧ pO 2min , and [glucose]2 ≧ [glucose] 2min holds, it will continue at the current flow rate (S5). If the current flow rate is below the predetermined maximum flow rate of the cell culture system, the perfusion rate is increased (S7). Further, if the current flow rate is not below the predetermined maximum flow rate of the cell culture system, the control device of the cell culture system can re-evaluate at least one of (1) pH 2min , pO 2min , and [glucose] 2min ; (2) pH1, pO1, and [glucose]1; and (3) the height of the bioreactor vessel (S6).

[0032] Embodiments of the present disclosure include a bioreactor and a cell culture substrate used herein, including a cell growth matrix and / or a substrate for a packed bed system for anchorage-dependent cells that enables easy and effective scaling up to any realistic production scale of cells or cell-derived products (e.g., proteins, antibodies, virus particles). In one embodiment, there is provided a matrix having a structurally defined surface area for adherent cells to adhere and grow, having good mechanical strength, and forming a highly uniform and diverse interconnected fluid network structure when mounted in a packed bed or other bioreactor. In a particular embodiment, a mechanically stable non-degradable woven mesh can be used to assist in the production of adherent cells. Uniform cell seeding of such a matrix and efficient harvesting of cells or other products from the bioreactor are achievable. In addition, embodiments of the present disclosure assist in cell culture to achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, avoiding the formation of 3D cell aggregates with limited nutrient diffusion and increased metabolite concentration. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor. In another embodiment of the present disclosure, a method of culturing cells is provided using a bioreactor having a matrix for the bioprocess production of therapeutic proteins, antibodies, virus particles, or viral vectors.

[0033] In one or more embodiments, the cell culture matrix supports the attachment and growth of high volume density forms of anchorage-dependent cells. The matrix is attached to and used in a bioreactor system, such as a perfused packed bed bioreactor as disclosed herein, providing a uniform cell distribution during the seeding process while preventing the formation of large and / or uncontrollable cell aggregates within the matrix or packed bed. Therefore, the matrix eliminates diffusion limitation during the operation of the bioreactor. In addition, the matrix enables easy and efficient cell harvesting from the bioreactor.

[0034] The matrix can be formed by a substrate material in the form of a thin sheet-like structure having a first surface and a second surface separated by a relatively small thickness. In other words, the thickness of the sheet-like substrate is small compared to the width and / or length of the first and second surfaces of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is sized and shaped such that cells can adhere to the surface of the substrate material as if it were a two-dimensional (2D) surface, while allowing appropriate fluid to flow around the substrate material and through the openings. In some embodiments, the substrate is a polymeric material and can be formed as a molded polymeric sheet, a polymeric sheet with openings punched through its thickness, a number of filaments fused to a mesh-like layer, or a plurality of filaments woven into a mesh layer. The physical structure of this matrix has a high surface area to volume ratio for culturing scaffold-dependent cells. According to various embodiments, the matrix can be arranged or packed within a bioreactor in a specific manner to achieve uniform cell seeding, uniform medium perfusion, and efficient cell harvesting.

[0035] This cell culture substrate can be a woven mesh layer made of a first plurality of fibers extending in a first direction and a second plurality of fibers extending in a second direction. The woven fibers of this substrate form a plurality of openings. The size and shape of the openings can vary based on the type of weave (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments, etc.). The openings can be defined by a specific width or diameter. The woven mesh may be considered a macroscale, two-dimensional sheet or layer. However, upon closer inspection of the woven mesh, it can be seen that it has a three-dimensional structure due to the rise and fall of the intersecting fibers of the mesh. Therefore, the thickness of the woven mesh will be greater than the thickness of a single fiber.

[0036] The woven mesh can be made of single or multi-fiber polymer fibers. In one or more embodiments, the single fiber may have a diameter in the range of about 50 μm to about 1000 μm. At the microscale, due to the scale of the fibers compared to cells (e.g., the fiber diameter is larger than that of cells), the surface of the single fiber is presented as a regular 2D surface for adherent cells to attach and proliferate. Such fibers are woven into a mesh having a defined pattern and a specific amount of structural stiffness. The fibers can be woven into a mesh having openings ranging from about 100 μm × 100 μm to about 1000 μm × 1000 μm. These ranges of filament diameter and aperture size are examples of some embodiments, but are not intended to limit the possible characteristic sizes of the mesh according to all embodiments.

[0037] The substrate mesh can be manufactured from single or multi-fiber polymer materials suitable for cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh substrate may have different structural patterns or weaves, including, for example, knit, warp knit, or woven (plain weave, twill weave, basket weave, five needle weave).

[0038] The chemical nature of the surface of the mesh filaments may need to be modified to provide the desired cell adhesion properties. Such modification can be performed by chemical treatment of the polymer material of the mesh or grafting of cell adhesion molecules onto the filament surface. Alternatively, the mesh can be coated with a thin layer of a biocompatible hydrogel that exhibits cell adhesion properties, including, for example, collagen or Matrigel®. Alternatively, the surface of the filament fibers of the mesh can be made to have cell adhesion properties by a treatment process with various types of plasma, treatment gases, and / or chemicals known in the art.

[0039] The woven matrix may be provided with a number of disks having central holes made to surround the central column of the bioreactor described herein. A plurality of such disks can be stacked in the outer region of the bioreactor to form a packed bed.

[0040] According to some embodiments, the cell culture matrix is a soluble foam scaffold made from an ion channel cross-linked polygalacturonic acid compound selected from at least one of pectic acid; partially esterified pectic acid, partially amidated pectic acid and salts thereof; and at least one first water-soluble polymer having surface activity.

[0041] Embodiments of the present disclosure can achieve a practical-sized viral vector platform that can produce viral genomes on the order of about 10 15 to about 10 18 or more viral genomes. For example, in some embodiments, the viral genome yield is about 10 15 to about 10 16 viral genomes per batch, or about 10 16 to about 10 19 viral genomes per batch, or about 10 16 ~10 18 viral genomes per batch, or about 10 17 to about 10 19 viral genomes per batch, or about 10 18 to about 10 19 viral genomes per batch, or about 10 18 or more viral genomes per batch.

[0042] In addition, the embodiments disclosed herein enable harvesting of cultured cells in a viable state, in addition to cell attachment and growth on the cell culture substrate. The inability to harvest viable cells is a significant shortcoming in current platforms, which poses difficulties in creating and maintaining a significant number of cells for production capacity. According to aspects of the embodiments of the present disclosure, it is possible to harvest viable cells from the cell culture substrate, which includes 80% to 100% viability, or about 85% to about 99% viability, or about 90% to about 99% viability. For example, at least 80% of the harvested cells are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. The cells can be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase™.

[0043] It will be recognized that the various disclosed embodiments may include the specific features, elements, or steps described with respect to a particular embodiment. It will also be recognized that a particular feature, element, or step described with respect to one particular embodiment may be interchanged or combined with alternative embodiments in various combinations or orders not otherwise described.

[0044] Specific examples of the invention The following describes various aspects of implementing the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The implementations are intended to illustrate some aspects of the disclosed subject matter and should not be regarded as an exhaustive or comprehensive description of all possible implementations.

[0045] Aspect 1 relates to a cell culture system, comprising: a cell culture vessel having an internal cavity configured to contain a cell culture substrate within a cell culture space, and at least one port configured for at least one of a fluid inlet to and a fluid outlet from the internal cavity; a piston disposed within the cell culture vessel above the cell culture space, having a distal end that is sealed by an airtight seal within the internal cavity; and a drive device coupled to the piston and configured to move the piston to increase or decrease the distance between the distal end and the cell culture space, the drive device being configured to pressurize the internal cavity by actuation of the piston to harvest cells from the cell culture space through the at least one port.

[0046] Aspect 2 relates to the cell culture system of Aspect 1, further comprising a cell harvest chamber fluidly connected to the at least one port and configured to capture cells harvested from the cell culture space.

[0047] Aspect 3 relates to the cell culture system of Aspect 1 or Aspect 2, further comprising a dissociation solution chamber fluidly connected to the at least one port and configured to contain a dissociation solution.

[0048] Aspect 4 relates to the cell culture system of Aspect 3, wherein the dissociation solution is configured to release cells from the cell substrate within the cell culture space.

[0049] Aspect 5 relates to the cell culture system of Aspect 3 or Aspect 4, wherein the system is configured to increase the distance between the distal end of the piston and the cell culture space to draw the dissociation solution into the internal cavity.

[0050] Aspect 6 relates to the cell culture system of Aspect 5, wherein the system is configured to decrease the distance between the distal end of the piston and the cell culture space to release the dissociation solution from the internal cavity.

[0051] Aspect 7 relates to a cell culture system according to any of Aspects 1-6, further comprising a washing chamber fluidly connected to at least one port and configured to contain a washing solution.

[0052] Aspect 8 relates to a cell culture system according to Aspect 7, wherein the washing solution is DPBS.

[0053] Aspect 9 relates to a cell culture system according to Aspect 7 or Aspect 8, wherein the system is configured to feed a washing solution into an internal cavity through at least one port.

[0054] Aspect 10 relates to a cell culture system according to any of Aspects 1-9, further comprising a cell culture substrate disposed within the cell culture space.

[0055] Aspect 11 relates to a cell culture system according to any of Aspects 1-10, further comprising a control device configured to control at least one of a drive device, a medium temperature, a medium flow rate, and a supply of at least one of a medium, oxygen, caustic alkali, and cell nutrients to an internal cavity.

[0056] Aspect 12 relates to a cell culture system according to Aspect 11, further comprising one or more sensors configured to measure one or more conditions within the container.

[0057] Aspect 13 relates to a cell culture system according to Aspect 12, wherein the one or more conditions include pH, dissolved oxygen, temperature, composition, analyte concentration, and spectral characteristics.

[0058] Aspect 14 relates to a cell culture system according to any of Aspects 1-13, wherein the cell culture substrate is made of a porous material.

[0059] Aspect 15 relates to a cell culture system according to Aspect 14, wherein the cell culture substrate is made of at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0060] Aspect 16 relates to the cell culture system of Aspect 14 or Aspect 15, wherein the cell culture substrate is made from at least one of a molded polymer lattice, a 3D printed polymer lattice sheet, and a woven mesh sheet.

[0061] Aspect 17 relates to the cell culture system of any one of Aspects 14 - 16, wherein the cell culture substrate is made from a woven mesh made from one or more fibers.

[0062] Aspect 18 relates to the cell culture system of Aspect 17, wherein one or more fibers have a fiber diameter of from about 50 μm to about 1000 μm, from about 50 μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μm to about 325 μm, or from about 150 μm to about 275 μm.

[0063] Aspect 19 relates to the cell culture system of Aspect 17 or Aspect 18, wherein the woven mesh includes a plurality of openings between the grids of one or more fibers, and the plurality of openings have a diameter of from about 100 μm to about 1000 μm, from about 200 μm to about 900 μm, or from about 225 μm to about 800 μm.

[0064] Aspect 20 relates to the cell culture system of Aspect 14, wherein the cell culture substrate is a soluble foam scaffold.

[0065] Aspect 21 relates to the cell culture system of Aspect 20, wherein the soluble foam scaffold is made from pectic acid; an ion channel crosslinked polygalacturonic acid compound selected from at least one of partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and at least one first water-soluble polymer having surface activity.

[0066] Aspect 22 relates to the cell culture system of Aspect 20 or Aspect 21, wherein the soluble foam scaffold includes an adhesive polymer coating.

[0067] Aspect 23 relates to the cell culture system of Aspect 22, wherein the adhesive polymer coating contains a peptide.

[0068] Aspect 24 relates to the cell culture system of Aspect 23, wherein the adhesive polymer coating contains a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

[0069] Aspect 25 relates to the cell culture system of Aspect 24, wherein the adhesive polymer coating is made from Synthemax® II-SC.

[0070] Aspect 26 relates to a method of culturing cells in a packed bed bioreactor, comprising the steps of: providing a cell culture vessel having an inlet, an outlet, and an internal cavity for containing a cell culture substrate, wherein the cell culture substrate has a surface adapted to culture cells thereon, and the inlet and the outlet are in fluid connection with the internal cavity; seeding cells onto the cell culture substrate; providing a cell culture medium to the internal cavity to culture the cells; and harvesting from the internal cavity at least one of cells, the cell culture medium, and one or more cell by-products, wherein the harvesting step includes filling the internal cavity with a pressurized fluid to extrude from the internal cavity at least one of cells, the cell culture medium, and one or more cell by-products.

[0071] Aspect 27 relates to the method of Aspect 26, wherein the cell culture medium is provided to the internal cavity through the inlet.

[0072] Aspect 28 relates to the method of Aspect 26 or Aspect 27, further comprising the step of removing the cell culture medium from the outlet during the culturing of the cells.

[0073] Aspect 29 relates to the method of Aspect 28, wherein the cell culture vessel is a perfusion bioreactor.

[0074] Aspect 30 relates to any of the methods of aspects 26 - 29, wherein pressurized fluid is provided to the internal cavity through one of the inlet and outlet, and at least one of cells, cell culture medium, and one or more cell by-products is removed from the internal cavity by the other of the inlet and outlet.

[0075] Aspect 31 relates to any of the methods of aspects 26 - 30, further comprising the step of supplying to the internal cavity a harvesting solution configured to separate cells from the surface of the cell culture substrate, at least in part, prior to the harvesting step.

[0076] Aspect 32 relates to the method of aspect 31, wherein the harvesting step further comprises the step of filling the internal cavity with pressurized fluid to extrude the harvesting solution from the internal cavity.

[0077] Aspect 33 relates to any of the methods of aspects 26 - 32, wherein the step of filling the internal cavity with pressurized fluid comprises at least one of the steps of pressurizing the fluid within the internal cavity using a pump, a piston, or a moving wall of the cell culture vessel.

[0078] As used herein, it should also be understood that a noun, unless otherwise expressly indicated, refers to an object of "at least one" and should not be limited to an object of "only one". Therefore, for example, a reference to an "opening" includes examples having two or more such "openings", unless the context clearly dictates otherwise.

[0079] Ranges can be expressed here as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, the examples include from one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by use of the antecedent "about", it will be understood that the particular value forms another aspect. Further, it will be understood that each endpoint of each range is significant both in relation to the other endpoint and independently of the other endpoint.

[0080] All numerical values presented herein are to be construed as being accompanied by the term "about", whether or not so stated, unless otherwise explicitly indicated. However, it will be further understood that each recited numerical value is also precisely intended, whether or not the term "about" is associated with that value. Accordingly, both "dimensions less than 10 mm" and "dimensions of less than about 10 mm" include embodiments of "dimensions of less than about 10 mm" and "dimensions less than 10 mm".

[0081] Unless otherwise specified, it is never intended that any of the methods described herein be construed as requiring that the steps be performed in a particular order. Thus, when a method claim does not actually recite an order for the steps to follow, or when the steps are not otherwise specifically recited in the claim or the specification as being limited to a particular order, no particular order is ever intended to be implied.

[0082] It should be understood that alternative embodiments are implied, including those in which various features, elements, or steps of a particular embodiment may be disclosed using the transitional phrase "comprising", but may be described using the transitional phrases "consisting of" or "consisting essentially of". Thus, for example, alternative embodiments implied for a method comprising A + B + C include embodiments where the method consists of A + B + C and embodiments where the method consists essentially of A + B + C.

[0083] Although numerous embodiments of the present disclosure have been shown in the accompanying drawings and described in the foregoing detailed description, it is to be understood that the present disclosure is not limited to the disclosed embodiments, and that many rearrangements, modifications, and substitutions are possible without departing from the present disclosure as defined by the following claims.

[0084] Hereinafter, preferred embodiments of the present invention will be described item by item.

[0085] Embodiment 1 In a cell culture system, A cell culture container, an internal cavity configured to contain a cell culture substrate within a cell culture space, at least one port configured for at least one of a fluid inlet to the internal cavity and a fluid outlet from the internal cavity, and a cell culture container having the same, a piston having a distal end disposed within the cell culture container above the cell culture space, the distal end of the piston being sealed by an airtight seal within the internal cavity, and a drive device coupled to the piston and configured to move the piston to increase or decrease a distance between the distal end and the cell culture space, comprising the same, wherein the drive device is configured to pressurize the internal cavity by actuation of the piston to harvest cells from the cell culture space through the at least one port. A cell culture system.

[0086] Embodiment 2 The cell culture system according to Embodiment 1, further comprising a cell harvest chamber fluidly connected to the at least one port and configured to capture the cells harvested from the cell culture space.

[0087] Embodiment 3 The cell culture system according to Embodiment 1 or Embodiment 2, further comprising a dissociation solution chamber fluidly connected to the at least one port and configured to contain a dissociation solution.

[0088] Embodiment 4 The cell culture system according to Embodiment 3, wherein the dissociation solution is configured to release cells from a cell substrate within the cell culture space.

[0089] Embodiment 5 The cell culture system according to Embodiment 3 or Embodiment 4, wherein the system is configured to increase a distance between the distal end of the piston and the cell culture space to draw the dissociation solution into the internal cavity.

[0090] Embodiment 6 The cell culture system according to embodiment 5, wherein the system is configured to reduce the distance between the distal end of the piston and the cell culture space in order to discharge the dissociation solution from the internal cavity.

[0091] Embodiment 7 The cell culture system according to any one of embodiments 1 to 6, further comprising a washing chamber fluidly connected to the at least one port and configured to contain a washing solution.

[0092] Embodiment 8 The cell culture system according to embodiment 7, wherein the washing solution is DPBS.

[0093] Embodiment 9 The cell culture system according to embodiment 7 or embodiment 8, wherein the system is configured to pump the washing solution into the internal cavity through the at least one port.

[0094] Embodiment 10 The cell culture system according to any one of embodiments 1 to 9, further comprising a cell culture substrate disposed within the cell culture space.

[0095] Embodiment 11 The cell culture system according to any one of embodiments 1 to 10, further comprising a control device configured to control at least one of the drive device, the medium temperature, the medium flow rate, and the supply of at least one of the medium, oxygen, caustic alkali, and cell nutrients to the internal cavity.

[0096] Embodiment 12 The cell culture system according to embodiment 11, further comprising one or more sensors configured to measure one or more conditions within the container.

[0097] Embodiment 13 The cell culture system according to Embodiment 12, wherein the one or more conditions include pH, dissolved oxygen, temperature, composition, analyte concentration, and spectral characteristics.

[0098] Embodiment 14 The cell culture system according to any one of Embodiments 1 to 13, wherein the cell culture substrate is made of a porous material.

[0099] Embodiment 15 The cell culture system according to Embodiment 14, wherein the cell culture substrate is made of at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0100] Embodiment 16 The cell culture system according to Embodiment 14 or Embodiment 15, wherein the cell culture substrate is made of at least one of a molded polymer lattice, a 3D printed polymer lattice sheet, and a woven mesh sheet.

[0101] Embodiment 17 The cell culture system according to any one of Embodiments 14 to 16, wherein the cell culture substrate is made of a woven mesh made of one or more fibers.

[0102] Embodiment 18 The cell culture system according to Embodiment 17, wherein the one or more fibers have a fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0103] Embodiment 19 The cell culture system according to Embodiment 17 or Embodiment 18, wherein the woven mesh includes a plurality of openings between the grids of the one or more fibers, and the plurality of openings have a diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0104] Embodiment 20 The cell culture system according to Embodiment 14, wherein the cell culture substrate is a soluble foam scaffold.

[0105] Embodiment 21 The soluble foam scaffold is pectinic acid; an ion channel cross-linked polygalacturonic acid compound selected from at least one of partially esterified pectinic acid, partially amidated pectinic acid, and salts thereof; and at least one first water-soluble polymer having surface activity, The cell culture system according to Embodiment 20, which is made from.

[0106] Embodiment 22 The cell culture system according to Embodiment 20 or Embodiment 21, wherein the soluble foam scaffold includes an adhesive polymer coating.

[0107] Embodiment 23 The cell culture system according to Embodiment 22, wherein the adhesive polymer coating contains a peptide.

[0108] Embodiment 24 The cell culture system according to Embodiment 23, wherein the adhesive polymer coating contains a peptide selected from the group consisting of BSP, vitronectin, fibronectin, laminin, type I collagen, type IV collagen, denatured collagen, and mixtures thereof.

[0109] Embodiment 25 The cell culture system according to Embodiment 24, wherein the adhesive polymer coating is made from "Synthemax" II-SC.

[0110] Embodiment 26 In a method for culturing cells in a packed bed bioreactor, Providing a cell culture vessel having an inlet, an outlet, and an internal cavity containing a cell culture substrate, the cell culture substrate having a surface adapted to culture cells thereon, the inlet and the outlet being in fluid connection with the internal cavity; Seeding cells onto the cell culture substrate; Providing a cell culture medium to the internal cavity to culture the cells, and Harvesting at least one of the cells, the cell culture medium, and one or more cell by-products from the internal cavity, comprising: The harvesting step includes filling the internal cavity with a pressurized fluid and extruding at least one of the cells, the cell culture medium, and the one or more cell by-products from the internal cavity. A method.

[0111] Embodiment 27 The method according to embodiment 26, wherein the cell culture medium is provided to the internal cavity through the inlet.

[0112] Embodiment 28 The method according to embodiment 26 or 27, further comprising removing the cell culture medium from the outlet during the culturing of the cells.

[0113] Embodiment 29 The method according to embodiment 28, wherein the cell culture vessel is a perfusion bioreactor.

[0114] Embodiment 30 The pressurized fluid is provided to the internal cavity through one of the inlet and the outlet, The method according to any one of embodiments 26 to 29, wherein at least one of the cells, the cell culture medium, and the one or more cell by-products is removed from the internal cavity by the other of the inlet and the outlet.

[0115] Embodiment 31 The method according to any one of Embodiments 26 to 30, further comprising, before the step of harvesting, supplying, at least in part, a harvesting solution configured to separate the cells from the surface of the cell culture substrate into the internal cavity.

[0116] Embodiment 32 The method according to Embodiment 31, wherein the step of harvesting further comprises filling the internal cavity with the pressurized fluid to extrude the harvesting solution from the internal cavity.

[0117] Embodiment 33 The method according to any one of Embodiments 26 to 32, wherein the step of filling the internal cavity with the pressurized fluid comprises at least one of the steps of pressurizing the fluid in the internal cavity using a pump, a piston, or a moving wall of the cell culture container.

Explanation of Reference Numerals

[0118] 100 Packed Bed Bioreactor System 102, 212 Containers 103 Substrate Material 104, 204 Cell Culture Substrate 106 Plunger 108, 110 Sources 200 Cell Culture System 202 Cell Culture Container 205 Driving Device 206 Piston 207 Connection 208 Harvest Collection Container 214 Control Device 216 Driving Device Control Unit 218 Pressure Regulator 220 Inlet 222 Outlet 400 Bioprocess System 402 Bioreactor (Container) 406 Cell Culture Medium 409 Pump 411 Medium Conditioning Container 412 Sensor Inlet of the 413 bioreactor 418 Medium acclimation control device

Claims

1. In a cell culture system, a cell culture vessel, an internal cavity configured to contain a cell culture substrate within a cell culture space, at least one port created for at least one of a fluid inlet to the internal cavity and a fluid outlet from the internal cavity, a cell culture vessel having the above, a piston having a distal end disposed within the cell culture vessel above the cell culture space, the distal end of the piston being sealed by an airtight seal within the internal cavity, and a drive device coupled to the piston and configured to move the piston to increase or decrease the distance between the distal end and the cell culture space, comprising, the drive device is configured to pressurize the fluid within the internal cavity by the operation of the piston, the pressurized fluid applies shear stress to the cells and is configured to harvest the cells from the cell culture substrate within the cell culture space through the at least one port. A cell culture system.

2. The cell culture system according to claim 1, further comprising a cell harvest chamber fluidly connected to the at least one port and configured to capture the cells harvested from the cell culture space.

3. The cell culture system according to claim 1 or 2, further comprising a dissociation solution chamber fluidly connected to the at least one port and configured to contain a dissociation solution.

4. The cell culture system according to claim 3, wherein the dissociation solution is configured to release cells from the cell substrate within the cell culture space.

5. The cell culture system according to claim 3 or 4, wherein the system is configured to increase the distance between the distal end of the piston and the cell culture space to draw the dissociation solution into the internal cavity.

6. The cell culture system according to claim 5, wherein the system is configured to decrease the distance between the distal end of the piston and the cell culture space to discharge the dissociation solution from the internal cavity.

7. The cell culture system according to any one of claims 1 to 6, further comprising a wash chamber fluidly connected to the at least one port and configured to contain a wash solution.

8. The cell culture system according to any one of claims 1 to 7, further comprising a control device configured to control at least one of the drive device, the medium temperature, the medium flow rate, and the supply of at least one of the medium, oxygen, caustic alkali, and cell nutrients to the internal cavity.

9. The cell culture system according to claim 8, further comprising one or more sensors configured to measure one or more conditions within the container, the one or more conditions including pH, dissolved oxygen, temperature, composition, analyte concentration, and spectral characteristics.

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