Cell culture bioreactor system with bidirectional perfusion and related methods

Abstract

A cell culture bioreactor system and method of using the same is disclosed. The method includes providing a fluid pathway for conveying cell culture media through the bioreactor system; providing a bioreactor vessel with an interior reservoir for housing a cell culture, and a first bioreactor inlet and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; flowing cell culture media in a first direction from the fluid pathway into the interior reservoir through the first bioreactor inlet and out of the reservoir to the fluid pathway through the second bioreactor inlet; and after maintaining flow in the first direction, reversing the flow of cell culture media to a second direction, the second direction being from the fluid pathway into the interior reservoir through the second bioreactor inlet and out of the interior reservoir to the fluid pathway through the first bioreactor inlet.

Description

CELL CULTURE BIOREACTOR SYSTEM WITH BIDIRECTIONAL PERFUSION AND RELATED METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 604,534 filed on November 30, 2023, the content of which are relied upon and incorporated herein by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] This disclosure general relates to bioreactor systems for culturing cells, as well as methods for culturing cells. In particular, the present disclosure relates to cell culture bioreactors systems and methods employing reversible fluid flow for improved cell culture uniformity and viability.BACKGROUND

[0003] In the bioprocessing industry, large-scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, vaccines, and cell therapies. Cell and gene therapy markets are growing rapidly, with promising treatments moving into clinical trials and quickly toward commercialization. However, one cell therapy dose can require billions of cells or trillions of viruses. As such, being able to provide a large quantity of cell products in a short amount of time is critical for clinical success.

[0004] A significant portion of the cells used in bioprocessing are anchorage dependent, meaning the cells need a surface to adhere to for growth and functioning. Traditionally, the culturing of adherent cells is performed on two-dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and HYPERStack® vessels. These approaches can have significant drawbacks, including the difficulty in achieving cellular density high enough to make it feasible for large scale production of therapies or cells.

[0005] Alternative methods have been suggested to increase volumetric density of cultured cells. These include microcarrier cultures performed in stir tanks. In this approach, cells that are attached to the surface of microcarriers are subject to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space. However, the cells growth and performance are significantly inhibited by the lack nutrients. To mitigate this problem, these bioreactors are made small and are not suitable for large scale manufacturing.

[0006] Another example of a high-density culture system for anchorage dependent cells is a packed-bed bioreactor system. In this this type of bioreactor, a cell substrate is used to provide a surface for the attachment of adherent cells. Medium is perfused along the surface or through the semi-porous substrate to provide nutrients and oxygen needed for the cell growth. For example, packed bed bioreactor systems that contain a packed bed of support or matrix systems to entrap the cells have been previously disclosed U.S. Patent Nos. 4,833,083; 5,501,971; and 5,510,262. Packed bed matrices usually are made of porous particles as substrates or non-woven microfibers of polymer. Such bioreactors function as recirculation flow-through bioreactors. One of the significant issues with such bioreactors is the non-uniformity of cell distribution inside the packed bed. For example, the packed bed functions as depth filter with cells predominantly trapped at the inlet regions, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packaging, flow resistance and cell trapping efficiency of cross sections of the packed bed are not uniform. For example, medium flows fast though the regions with low cell packing density and flows slowly through the regions where resistance is higher due to higher number of entrapped cells. This creates a channeling effect where nutrients and oxygen are delivered more efficiently to regions with lower volumetric cells densities and regions with higher cell densities are being maintained in suboptimal culture conditions.

[0007] Another significant drawback of packed bed systems disclosed in a prior art is the inability to efficiently harvest intact viable cells at the end of culture process. Harvesting of cells is important if the end product is cells, or if the bioreactor is being used as part of a “seed train,” where a cell population is grown in one vessel and then transferred to another vessel for furtherpopulation growth. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during cells harvesting step. It is based on loosening the packed bed matrix and agitation or stirring of packed bed particles to allow porous matrices to collide and thus detach the cells. However, this approach is laborious and may cause significant cells damage, thus reducing overall cell viability.

[0008] Some preexisting packed-bed bioreactors on the market use small strips of cell substrate material consisting of randomly oriented fibers in a non-woven arrangement. These strips are packed into a vessel to create a packed bed. However, as with similar solutions on the market, there are drawbacks to this type of packed-bed substrate. Specifically, non-uniform packing of the substrate strips creates visible channels within the packed bed, leading to preferential and non-uniform media flow and nutrient distribution through the packed bed. Studies of such systems have noted a “systemic inhomogeneous distribution of cells, with their number increasing from top to bottom of fixed bed,” as well as a “nutrient gradient... leading to restricted cell growth and production,” all of which lead to the “unequal distribution of cells [that] may impair transfection efficiency.” (Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.Biotechnol. J. 2016, 11, 290-297). Studies have noted that agitation of the packed bed may improve dispersion, but would have other drawbacks (i.e., “necessary agitation for better dispersion during inoculation and transfection would induce increased shear stress, in turn leading to reduced cell viability.” Id.). Another study noted of that uneven distribution of cells makes monitoring of the cell population using biomass sensors difficult (“... if the cells are unevenly distributed, the biomass signal from the cells on the top carriers may not show the general view of the entire bioreactor.” Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale. Human Gene Therapy, Vol. 26, No. 8, 2015).

[0009] In addition, because of the random arrangement of fibers in the substrate strips and the variation in packing of strips between one packed bed and another in such systems, it can be difficult for customers to predict cell culture performance, since the substrate varies betweencultures. Furthermore, such packed beds can make efficiently harvesting cells very difficult or impossible, as it is believed that cells are entrapped by the packed bed.

[0010] Roller bottles have several advantages such as ease of handling, and ability to monitor cells on the attachment surface. However, from a production standpoint, the main disadvantage is the low surface area to volume ratio while the roller bottle configuration occupies a large area of manufacturing floor space. Various approaches have been used to increase the surface area available for adherent cells in a roller bottle format. Some solutions have been implemented in commercially available products, but there remains room for improvement to increase roller bottle productivity even further. Traditionally, a roller bottle is produced as a single structure by a blow-molding process. Such manufacturing simplicity enables economic viability of roller bottles in bioprocessing industry. Some roller bottle modifications to increase the available surface area for cell culturing can be achieved without changing manufacturing process, however only marginal increase of modified roller bottle surface area is obtained. Other modifications of the roller bottle design add significant complexity to manufacturing processes making it economically unviable in the bioprocessing industry. It is desirable therefore to provide roller bottle with increased surface area and bioprocessing productivity, while using the same blowmolding process for its manufacturing.

[0011] Corning Incorporated has developed a bioreactor technology that overcomes many of the above shortcomings, including providing uniform fluid flow through a fixed bed, and more uniform cell growth. Examples of embodiments these technologies can be found, for example, in U.S. Patent No. 11,434,460; 11,118,151; 11,661,576; and 11,692,161. Those solutions include using a fixed bed with a uniform and defined structure that allows for uniform fluid flow. However, due to the unidirectional nature of fluid flow through the fixed bed, it is possible that a nutrient gradient in the cell culture media can develop from the bioreactor inlet to the outlet as cells within the fixed bed consume nutrients in the media as it flows through the reactor. As a result, cells near the outlet of the bioreactor may be exposed to lower nutrient and / or oxygen levels in the media than those near the inlet of the bioreactor.

[0012] While manufacturing of viral vectors for early-phase clinical trials is possible with existing platforms, there is a need for a platform that can produce high-quality product in greaternumbers in order to reach late-stage commercial manufacturing scale, while allowing uniformity in cell growth and cell viability through the bioreactor.SUMMARY

[0013] According to an embodiment of this disclosure, a method of operating a bioreactor system for culturing cells is provided. The method comprises providing a fluid pathway for conveying cell culture media through the bioreactor system; providing a bioreactor vessel comprising an interior reservoir for housing a cell culture, a first bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway, and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; flowing cell culture media in a first direction from the fluid pathway into the interior reservoir through the first bioreactor inlet and out of the interior reservoir to the fluid pathway through the second bioreactor inlet; and after maintaining the flow of cell culture media in the first direction for a predetermined time, reversing the flow of cell culture media to a second direction, the second direction being from the fluid pathway into the interior reservoir through the second bioreactor inlet and out of the interior reservoir to the fluid pathway through the first bioreactor inlet.

[0014] In embodiments, the method comprises providing a fixed bed within the interior reservoir, the fixed bed comprising a cell culture substrate, the cell culture substrate comprising a surface configured for adhering cells of the cell culture thereto. The method can further include providing a media conditioning vessel (MCV) configured to control one or more conditions of the cell culture media in the bioreactor system, the media conditioning vessel comprising a first MCV inlet and a second MCV inlet that each fluidly connects the MCV to the fluid pathway.

[0015] In aspects of embodiments, the method includes reversing the flow of the cell culture media back to the first direction. In further aspects, the method includes reversing the flow of the cell culture media one or more additional times. The reversing of the flow of the cell culture media can performed manually by an operator of the bioreactor system. The reversing of the flow of the cell culture media can also be performed automatically according to a schedule. The schedule can be predetermined based on at least one of a type of cell in the cell culture, a rate of flow of cell culture media, a total culture time of the cell culture, a maximum time for flowing cell culture media in one direction, a stage of the cell culture, and a monitored propertyindicative of the health of the cell culture. The monitored property comprises at least one of a rate of growth of the cell culture, a nutrient level in the cell culture media, a dissolved gas level in the cell culture media, a rate of change of the nutrient level or the dissolved gas in the cell culture media, a temperature of the cell culture media, and a pH of the cell culture media. In aspects of embodiments, the monitored property can be at least one of the nutrient level in the cell culture media, the dissolved gas level in the cell culture media, and the rate of change of the nutrient level or the dissolved gas in the cell culture media, and the monitored property is measured at or near at least one of the first and second bioreactor inlets. The monitored property can be measured by an inline sensor in the fluid pathway.

[0016] In aspects of embodiments, the first bioreactor inlet is on a bottom of the bioreactor vessel and the second bioreactor inlet is on a top of the bioreactor vessel. In other embodiments, the first bioreactor inlet is on a top of the bioreactor vessel and the second bioreactor inlet is on a bottom of the bioreactor vessel.

[0017] In embodiments, the method may include maintaining and reversing the flow in the first and second directions to achieve at least one of a uniform cell number distribution and a uniform cell viability distribution along a direction parallel to the flow of cell culture media through the fixed bed. In embodiments, the method also includes seeding cells into the bioreactor vessel. The method can include alternating the flow of cell culture media through the fixed bed to maintain cells within the bioreactor vessel during seeding. The alternating of the flow of cell culture media during seeding causes the cells to be tumbled up and down within the fixed bed until the cells adhere to the fixed bed. Further aspects of embodiments include alternating the direction of flow of the cell culture media during a transfection step of the cell culture.

[0018] In embodiments, the method includes setting a dissolved oxygen level of the cell culture media in the MCV in a range from about 40% to about 60%.

[0019] In embodiments, the method includes alternating the direction of flow of the cell culture media during a harvesting step of the cell culture, wherein the alternating of the direction of flow is configured to loosen and / or detach cells from the fixed bed.

[0020] The method of any of claims 2-20, wherein a cell density (as measured in cells / cm2) of cells in the cell culture at any point in the fixed bed is less than or equal to 2x the cell density at any other point in the fixed bed.

[0021] In aspects of embodiments of the method, the cell density at any point in the fixed bed is less than or equal to 1.5x the cell density at any other point in the fixed bed, or less than or equal to 1.25x the cell density at any other point in the fixed bed. The cell viability (expressed as a percentage of cells that are viable) of cells in the cell culture at any level in the fixed bed is less than or equal to 20% the cell viability at any other level in the fixed bed, or is less than or equal to 10% the cell viability at any other level in the fixed bed, or is less than or equal to 5% the cell viability at any other level in the fixed bed, or is less than or equal to 2% the cell viability at any other level in the fixed bed.

[0022] According to embodiments of this disclosure, a bioreactor system for culturing cells is provided. The bioreactor system includes a fluid pathway for conveying cell culture media through the bioreactor system; a bioreactor vessel comprising an interior reservoir for housing a cell culture, a first bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway, and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; a media conditioning vessel (MCV) configured to control one or more conditions of the cell culture media in the bioreactor system, the media conditioning vessel comprising a first MCV inlet and a second MCV inlet that each fluidly connects the MCV to the fluid pathway; and at least one pump fluidly attached to the fluid pathway and configured to flow fluid through the bioreactor system. The bioreactor system is configured to alternative the flow of fluid through the bioreactor vessel between a first direction and a second direction, the first direction being from the first bioreactor inlet, through the interior reservoir, and to the second bioreactor inlet, and the second direction being from the second bioreactor inlet, through the interior reservoir, and to the first bioreactor inlet.

[0023] In aspects of embodiments, the bioreactor system further includes a control system comprising a computer with a processor and a computer-readable medium storing instructions that, when executed by the processor, cause the bioreactor system to reverse a direction of flow of fluid through the bioreactor system. The bioreactor system can include one or more sensorsconfigured to measure at least one of a rate of growth of the cell culture, a nutrient level in the cell culture media, a dissolved gas level in the cell culture media, a rate of change of the nutrient level or the dissolved gas in the cell culture media, a temperature of the cell culture media, a biomass measurement of the cell culture, and a pH of the cell culture media. In aspects of embodiments, the control system receives data from the one or more sensors and controls a direction of flow of fluid through the bioreactor system based on the data.

[0024] According to embodiments, the bioreactor system further includes a fixed bed within the interior reservoir, the fixed bed comprising a cell culture substrate comprising a surface configured for adhering cells thereto.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows schematic of an embodiment of a bioreactor system using unidirectional flow.

[0026] Figure 2 is a graph of cell density at three locations along a height of a bioreactor subjected to unidirectional flow.

[0027] Figure 3 shows a schematic of a bioreactor system capable of bidirectional flow, according to embodiments of this disclosure.

[0028] Figure 4 is a graph of cell viability for bioreactor system subjected to unidirectional and bidirectional flow, according to embodiments of this disclosure.

[0029] Figure 5 is a graph of cell density for bioreactor systems subjected to unidirectional and bidirectional flow, according to embodiments of this disclosure.DETAILED DESCRIPTION

[0030] Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0031] Embodiments of this disclosure relate to methods of operating a bioreactor system for culturing cells, as well as a bioreactor system for culturing cells. In particular, the methods andsystem disclosed herein relate to the use and configuration of bidirectional flow through the bioreactor system. As used herein, “bidirectional flow” means that the direction of flow of fluid through the system is flowed in at least two directions during the course of operating a cell culture. The fluid can be cell culture media, reagents, water, washing fluid, enzymes, or any other fluid used in the course of operating the cell culture system. At times herein, the fluid may be referred to as “cell culture media,” but this descriptor should not be considered limiting on any of the embodiments, as “cell culture media,” as used herein, can be considered any type of fluid used in the operation of a cell culture system.

[0032] Figure 1 shows a schematic of a bioreactor system capable of unidirectional flow. Fixed-bed bioreactor (FBR) systems typically rely on one or more recirculation pumps to perfuse conditioned cell culture media through a bioreactor to provide dissolved oxygen (DO) and nutrients to cells during a cell culture. Most FBR systems are designed to perfuse media through the bioreactor in one direction (e.g., from bottom of the reactor (inlet) to top of the reactor (outlet)). Corning Incorporated’s Ascent® FBR system has used such a configuration. However, the unidirectional bottom-to-top flow design can have several drawbacks. First, cells can grow unevenly due to gradients of DO from inlet (which may have, e.g., 100% DO) to the outlet (where the level may only be -20% DO) of the reactor. This DO gradient is depicted in Figure 1. Also, cells may grow faster in the middle of reactor since optimal DO ranges (40-60%) may be found in the middle section of the reactor. This leads to variations in cell density across the bottom, middle, and top of the bioreactor, as shown in Figure 2. The data in Figure 2 was collected from an Ascent® FBR system with a substrate surface area of 2.5 m2(from Corning Incorporated) using HEK293 cells cultured in the reactor for 6 days. Cells were harvested with Accutase from mesh substate layers removed from the top, middle, and bottom sections of the bioreactor. Higher cell density was seen in the middle of the bioreactor.

[0033] Second, lower cell viability or variations in cell viability throughout the bioreactor may result due to low oxygen level (e.g., < 10% DO) at or near the top of the reactor when culturing high-cell-density cells on large and / or long reactor beds. Third, there can be an uneven distribution of cells in the bioreactor during cell seeding processes, including, for example, more cells seeded in the lower part of the bioreactor. Fourth, there may be lower transfectionefficiency at or near the top of the bioreactor. Fifth, shear-sensitive cell types can be damaged due to the high fluid flowrates sometimes needed to compensate for low oxygen levels at or near the top of the bioreactor. Sixth, there may be entrapment of air in the bioreactor causing nonuniformities due to unidirectional flow during priming processes and / or outgassing during perfusion upstream of the bioreactor. However, embodiments disclosed herein overcome these challenges.

[0034] To overcome the above challenges, embodiments of this disclosure include methods of operating bioreactor systems and the bioreactor systems themselves that employ or are configured for bidirectional flow of fluid (e.g., cell culture media) through the bioreactor system, as shown in Figure 3. The flow directions can be controlled by the bioreactor system with software and one or more control algorithms, and / or with modifications of the perfusion recirculation flow path and addition of valves (e.g., pinch valves, etc). The flow direction can be changed automatically to allow flow of freshly conditioned media to either inlet or outlet in an alternating manner, as shown in Figure 3. Because embodiments herein allow for flow into multiple openings in the bioreactor due to the bidirectional flow, the terms “inlet” and “outlet” can be considered interchangeable. Thus, the bioreactor can be considered to have an inlet and an outlet, or a first inlet and a second inlet, for example.

[0035] This bidirectional flow will ensure cells cultured inside the bioreactor are exposed to cell culture media with similar concentrations of dissolved oxygen (DO), nutrients, reagents, and other components used in cell culture. In addition, the percentage of DO in a media conditioning vessel (MCV) can be set at an optimal cell culture level (e.g., 40-60%), as shown in Figure 3 — instead of the typical 100% DO — to minimize the DO gradient across the bioreactor. Thus, cells are cultured in an optimal environment in terms of % DO in the cell culture media. As shown in Figure 3, the DO gradient in the bioreactor can be eliminated.

[0036] Another advantage of embodiments disclosed herein is related to uniform cell seeding and transfection. The alternating flow direction will allow even distribution of cells or transfection complexes across the entire fixed bed in the bioreactor. Additionally, for shearsensitive cell types, alternating the flow direction will allow sufficient media perfusion to be performed under much lower pump flow without a risk of low levels of DO and nutrients in oneend, especially in the case of large and / or long fixed-bed bioreactors. A further advantage is accomplished by removing air pockets during initial priming of a bioreactor system using the bidirectional flow. This results in improved growth uniformity as well as the clearing of outgassed bubbles formed upstream of the bioreactor in a uni-directional system during perfusion by returning them to the media conditioner before they can build up and be carried downstream to the bioreactor.

[0037] To demonstrate these advantages, two Ascent® FBR systems (from Corning Incorporated) were compared under unidirectional and bidirectional flow conditions. Both systems used an effective substrate surface area of 2.5 m2, with the fixed bed comprising stacked layers of mesh disks. HEK293 cells were cultured in both reactors for 6 days and harvested with Accustase from mesh disks removed from the top, middle, and bottom section of the bioreactors. The bidirectional control of flow was performed by changing the direction of the recirculation pump manually. As shown in Figure 4, the cell viability of the unidirectional flow system drops significantly in the top section of the bioreactor. In contrast, the bidirectional flow system exhibits remarkably consistent cell viability across all three sections of the bioreactor.

[0038] Figure 5 compares the distribution of cell density (in cells / cm2) of two Ascent® FBR systems (from Corning Incorporated) with effective substrate surface areas of 1 m2Ad-MSC cells were seeded into the bioreactors by perfusing cells in suspension either from the bottom of the bioreactor or from the top of the bioreactor. After 100 hours of culture time, the fixed bed was sampled to estimate the uniformity of cell distribution. As shown in Figure 5, the bioreactor that was seeded from the top demonstrated significant improvement in cell distribution uniformity. Thus, by enabling a reverse flow direction during seeding, the system was able to achieve better cell density uniformity throughout the fixed bed.

[0039] According to an embodiment of this disclosure, a method of operating a bioreactor system for culturing cells is provided. The method comprises providing a fluid pathway for conveying cell culture media through the bioreactor system; providing a bioreactor vessel comprising an interior reservoir for housing a cell culture, a first bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway, and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; flowing cell culture media in a firstdirection from the fluid pathway into the interior reservoir through the first bioreactor inlet and out of the interior reservoir to the fluid pathway through the second bioreactor inlet; and after maintaining the flow of cell culture media in the first direction for a predetermined time, reversing the flow of cell culture media to a second direction, the second direction being from the fluid pathway into the interior reservoir through the second bioreactor inlet and out of the interior reservoir to the fluid pathway through the first bioreactor inlet.

[0040] In embodiments, the method comprises providing a fixed bed within the interior reservoir, the fixed bed comprising a cell culture substrate, the cell culture substrate comprising a surface configured for adhering cells of the cell culture thereto. The method can further include providing a media conditioning vessel (MCV) configured to control one or more conditions of the cell culture media in the bioreactor system, the media conditioning vessel comprising a first MCV inlet and a second MCV inlet that each fluidly connects the MCV to the fluid pathway.

[0041] In aspects of embodiments, the method includes reversing the flow of the cell culture media back to the first direction. In further aspects, the method includes reversing the flow of the cell culture media one or more additional times. The reversing of the flow of the cell culture media can performed manually by an operator of the bioreactor system. The reversing of the flow of the cell culture media can also be performed automatically according to a schedule. The schedule can be predetermined based on at least one of a type of cell in the cell culture, a rate of flow of cell culture media, a total culture time of the cell culture, a maximum time for flowing cell culture media in one direction, a stage of the cell culture, and a monitored property indicative of the health of the cell culture. The monitored property comprises at least one of a rate of growth of the cell culture, a nutrient level in the cell culture media, a dissolved gas level in the cell culture media, a rate of change of the nutrient level or the dissolved gas in the cell culture media, a temperature of the cell culture media, and a pH of the cell culture media. In aspects of embodiments, the monitored property can be at least one of the nutrient level in the cell culture media, the dissolved gas level in the cell culture media, and the rate of change of the nutrient level or the dissolved gas in the cell culture media, and the monitored property is measured at or near at least one of the first and second bioreactor inlets. The monitored property can be measured by an inline sensor in the fluid pathway.

[0042] In aspects of embodiments, the first bioreactor inlet is on a bottom of the bioreactor vessel and the second bioreactor inlet is on a top of the bioreactor vessel. In other embodiments, the first bioreactor inlet is on a top of the bioreactor vessel and the second bioreactor inlet is on a bottom of the bioreactor vessel.

[0043] In embodiments, the method may include maintaining and reversing the flow in the first and second directions to achieve at least one of a uniform cell number distribution and a uniform cell viability distribution along a direction parallel to the flow of cell culture media through the fixed bed. In embodiments, the method also includes seeding cells into the bioreactor vessel. The method can include alternating the flow of cell culture media through the fixed bed to maintain cells within the bioreactor vessel during seeding. The alternating of the flow of cell culture media during seeding causes the cells to be tumbled up and down within the fixed bed until the cells adhere to the fixed bed. Further aspects of embodiments include alternating the direction of flow of the cell culture media during a transfection step of the cell culture.

[0044] In embodiments, the method includes setting a dissolved oxygen level of the cell culture media in the MCV in a range from about 40% to about 60%.

[0045] In embodiments, the method includes alternating the direction of flow of the cell culture media during a harvesting step of the cell culture, wherein the alternating of the direction of flow is configured to loosen and / or detach cells from the fixed bed.

[0046] The method of any of claims 2-20, wherein a cell density (as measured in cells / cm2) of cells in the cell culture at any point in the fixed bed is less than or equal to 2x the cell density at any other point in the fixed bed.

[0047] In aspects of embodiments of the method, the cell density at any point in the fixed bed is less than or equal to 1.5x the cell density at any other point in the fixed bed, or less than or equal to 1.25x the cell density at any other point in the fixed bed. The cell viability (expressed as a percentage of cells that are viable) of cells in the cell culture at any level in the fixed bed is less than or equal to 20% the cell viability at any other level in the fixed bed, or is less than or equal to 10% the cell viability at any other level in the fixed bed, or is less than or equal to 5% the cell viability at any other level in the fixed bed, or is less than or equal to 2% the cell viability at any other level in the fixed bed.

[0048] According to embodiments of this disclosure, a bioreactor system for culturing cells is provided. The bioreactor system includes a fluid pathway for conveying cell culture media through the bioreactor system; a bioreactor vessel comprising an interior reservoir for housing a cell culture, a first bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway, and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; a media conditioning vessel (MCV) configured to control one or more conditions of the cell culture media in the bioreactor system, the media conditioning vessel comprising a first MCV inlet and a second MCV inlet that each fluidly connects the MCV to the fluid pathway; and at least one pump fluidly attached to the fluid pathway and configured to flow fluid through the bioreactor system. The bioreactor system is configured to alternative the flow of fluid through the bioreactor vessel between a first direction and a second direction, the first direction being from the first bioreactor inlet, through the interior reservoir, and to the second bioreactor inlet, and the second direction being from the second bioreactor inlet, through the interior reservoir, and to the first bioreactor inlet.

[0049] In aspects of embodiments, the bioreactor system further includes a control system comprising a computer with a processor and a computer-readable medium storing instructions that, when executed by the processor, cause the bioreactor system to reverse a direction of flow of fluid through the bioreactor system. The bioreactor system can include one or more sensors configured to measure at least one of a rate of growth of the cell culture, a nutrient level in the cell culture media, a dissolved gas level in the cell culture media, a rate of change of the nutrient level or the dissolved gas in the cell culture media, a temperature of the cell culture media, a biomass measurement of the cell culture, and a pH of the cell culture media. In aspects of embodiments, the control system receives data from the one or more sensors and controls a direction of flow of fluid through the bioreactor system based on the data.

[0050] According to embodiments, the bioreactor system further includes a fixed bed within the interior reservoir, the fixed bed comprising a cell culture substrate comprising a surface configured for adhering cells thereto.

[0051] Embodiments of this disclosure can use a fixed bed made of a cell culture substrate material that has a structurally defined surface area. In embodiments, the substrate is highly ordered and uniform, leading to structural and flow uniformity through the fixed bed. Examples of suitable substrate include those disclosed in in U.S. Patent No. 11,434,460; 11,118,151;11,661,576; and 11,692,161, which are incorporated herein by reference. However, it should be appreciated that the embodiments of this disclosure are not limited to such ordered fixed beds. Indeed, the systems and methods of this disclosure can benefit bioreactor systems using any substrate type.

[0052] In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors have been used. Usually these packed beds contain porous matrices to retain adherent or suspension cells, and to support growth and proliferation. Packed-bed matrices provide high surface area to volume ratios, so cell density can be higher than in the other systems. However, the packed bed often functions as a depth filter, where cells are physically trapped or entangled in fibers of the matrix. Thus, because of linear flow of the cell inoculum through the packed bed, cells are subject to heterogeneous distribution inside the packed-bed, leading to variations in cell density through the depth or width of the packed bed. For example, cell density may be higher at the inlet region of a bioreactor and significantly lower nearer to the outlet of the bioreactor. This non-uniform distribution of the cells inside of the packed-bed significantly hinders scalability and predictability of such bioreactors in bioprocess manufacturing, and can even lead to reduced efficiency in terms of growth of cells or viral vector production per unit surface area or volume of the packed bed.

[0053] Another problem encountered in packed bed bioreactors disclosed in prior art is the channeling effect. Due to random nature of packed nonwoven fibers, the local fiber density at any given cross section of the packed bed is not uniform. Medium flows quickly in the regions with low fiber density (high bed permeability) and much slower in the regions of high fiber density (lower bed permeability). The resulting non-uniform media perfusion across the packed bed creates the channeling effect, which manifests itself as significant nutrient and metabolite gradients that negatively impact overall cell culture and bioreactor performance. Cells located in the regions of low media perfusion will starve and very often die from the lack of nutrients ormetabolite poisoning. Cell harvesting is yet another problem encountered when bioreactors packed with non-woven fibrous scaffolds are used. Due to packed-bed functions as depth filter, cells that are released at the end of cell culture process are entrapped inside the packed bed, and cell recovery is very low. This significantly limits utilization of such bioreactors in bioprocesses where live cells are the products. Thus, the non-uniformity leads to areas with different exposure to flow and shear, effectively reducing the usable cell culture area, causing non-uniform culture, and interfering with transfection efficiency and cell release.

[0054] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed- bed systems using such substrates that enable efficient and high-yield cell culturing for anchorage-dependent cells and production of cell products (e.g., proteins, antibodies, viral particles). Embodiments include a porous cell-culture matrix made from an ordered and regular array of porous substrate material that enables uniform cell seeding and media / nutrient perfusion, as well as efficient cell harvesting. Embodiments also enable scalable cell-culture solutions with substrates and bioreactors capable of seeding and growing cells and / or harvesting cell products from a process development scale to a full production size scale, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, a bioreactor can be easily scaled from process development scale to product scale with comparable viral genome per unit surface area of substrate (VG / cm2) across the production scale. The harvestability and scalability of the embodiments herein enable their use in efficient seed trains for growing cell populations at multiple scales on the same cell substrate. In addition, the embodiments herein provide a cell culture matrix having a high surface area that, in combination with the other features described, enables a high yield cell culture solution. In some embodiments, for example, the cell culture substrate and / or bioreactors discussed herein can produce 1016to 1018viral genomes (VG) per batch.

[0055] In one embodiment, a matrix is provided with a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, a mechanically stable, non-degradable woven mesh canbe used as the substrate to support adherent cell production. The cell culture matrix disclosed herein supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. Uniform cell seeding of such a matrix is achievable, as well as efficient harvesting of cells or other products of the bioreactor. In addition, the embodiments of this disclosure support cell culturing to provide uniform cell distribution during the inoculation step and achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, and can avoid formation of large and / or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the matrix eliminates diffusional limitations during operation of the bioreactor. In addition, the matrix enables easy and efficient cell harvest from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.

[0056] According to some embodiments, a method of cell culturing is also provided using bioreactors with the matrix for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0057] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., nonwoven substrates of randomly ordered fibers), embodiments of this disclosure include a cell culture substrate having a defined and ordered structure. The defined and order structure allows for consistent and predictable cell culture results. In addition, the substrate has an open porous structure that prevents cell entrapment and enables uniform flow through the packed bed. This construction enables improved cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more particular embodiments, the matrix is formed with a substrate material having a thin, sheet-like construction having first and second sides separated by a relatively small thickness, such that the thickness of the sheet is small relative to the width and / or length of the first and second sides 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 of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were approximately a two-dimensional (2D) surface, while also allowing adequate fluid flow around the substrate material and through the openings. In some embodiments, the substrate is apolymer-based material, and can be formed as a molded polymer sheet; a polymer sheet with openings punched through the thickness; a number of filaments that are fused into a mesh-like layer; a 3D-printed substrate; or a plurality of filaments that are woven into a mesh layer. The physical structure of the matrix has a high surface-to-volume ratio for culturing anchorage dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in certain ways discussed here for uniform cell seeding and growth, uniform media perfusion, and efficient cell harvest.

[0058] Embodiments of this disclosure can achieve viral vector platforms of a practical size that can produce viral genomes on the scale of greater than about 1014viral genomes per batch, greater than about 1015viral genomes per batch, greater than about 1016viral genomes per batch, greater than about 1017viral genomes per batch, or up to or greater than about g 1016viral genomes per batch. In some embodiments, productions is about 1015to about 1018or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be about 1015to about 1016viral genomes or batch, or about 1016to about 1019viral genomes per batch, or about 1016-l 018viral genomes per batch, or about 1017to about 1019viral genomes per batch, or about 1018to about 1019viral genomes per batch, or about 1018or more viral genomes per batch.

[0059] In addition, the embodiments disclosed herein enable not only cell attachment and growth to a cell culture substrate, but also the viable harvest of cultured cells. The inability to harvest viable cells is a significant drawback in current platforms, and it leads to difficulty in building and sustaining a sufficient number of cells for production capacity. According to an aspect of embodiments of this disclosure, it is possible to harvest viable cells from the cell culture substrate, including between 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the cells that are harvested, at least 80% 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. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0060] According to embodiments, the cell culture substrate is a woven mesh layer made of a first plurality of fibers running in a first direction and a second plurality of fibers running in asecond direction. The woven fibers of the substrate form a plurality of openings, which can be defined by one or more widths or diameters (e.g., Di, D2). The size and shape of the openings can vary based on the type of weave (e.g., number, shape and size of filaments; angle between intersecting filaments, etc.). A woven mesh may be characterized as, on a macro-scale, a two- dimensional sheet or layer. However, a close inspection of a woven mesh reveals a three- dimensional structure due to the rising and falling of intersecting fibers of the mesh. Thus, a thickness of the woven mesh may be thicker than the thickness of a single fiber (e.g., ti). As used herein, the thickness T is the maximum thickness between a first side and a second side of the woven mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate is advantageous as it provides a large surface area for culturing adherent cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure that enables uniform fluid flow.

[0061] The openings in the mesh can have a diameter Di, defined as a distance between opposite fibers, and a diameter D2, defined as a distance between a second set of opposite fibers defining the opening. Di and D2 can be equal or unequal, depending on the weave geometry. Where Di and D2 are unequal, the larger can be referred to as the major diameter, and the smaller as the minor diameter. In some embodiments, the diameter of an opening may refer to the widest part of the opening. Unless otherwise specified, the opening diameter, as used herein, will refer to a distance between parallel fibers on opposite sides of an opening.

[0062] A given fiber of the plurality of fibers has a thickness ti, and a given fiber of the plurality of fibers has a thickness t2. In the case of fibers of round cross-section, or other three- dimensional cross-sections, the thicknesses ti and t2 are the maximum diameters or thicknesses of the fiber cross-section. According to some embodiments, the plurality of fibers all have the same thickness ti, and the plurality of fiber all have the same thickness t2. In addition, ti and t2 may be equal. However, in one or more embodiments, ti and t2 are not equal such as when the plurality of fibers are different from the plurality of fiber. In addition, each of the plurality of fibers and plurality of fibers may contain fibers of two or more different thicknesses (e.g., tia, tib, etc., and t2a, t2b, etc.). According to embodiments, the thicknesses ti and t2 are large relative to the size of the cells cultured thereon, so that the fibers provide an approximation of a flat surfacefrom the perspective of the cell, which can enable better cell attachment and growth as compared to some other solutions in which the fiber size is small (e.g., on the scale of the cell diameter). Due to three-dimensional nature of woven mesh, the 2D surface area of the fibers available for cell attachment and proliferation exceeds the surface area for attachment on an equivalent planar 2D surface.

[0063] In one or more embodiments, a fiber may have a diameter in a range of about 10 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm; or about 150 pm to about 300 pm. On a microscale level, due to the scale of the fiber compared to the cells (e.g., the fiber diameters being larger than the cells), the surface of monofilament fiber is presented as an approximation of a 2D surface for adherent cells to attach and proliferate. Fibers can be woven into a mesh with openings ranging from about 100 pm x 100 pm to about 1000 pm x 1000 pm. In some embodiments, the opening may have a diameter o about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; or about 200 pm to about 300 pm. These ranges of the filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is chosen to provide efficient and uniform fluid flow through the substrate when, for example, the cell culture matrix is comprises a number of adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0064] Factors such as the fiber diameter, opening diameter, and weave type / pattern will determine the surface area available for cell attachment and growth. In addition, when the cell culture matrix includes a stack, roll, or other arrangement of overlapping substrate, the packing density of the cell culture matrix will impact the surface area of the packed bed matrix. Packing density can vary with the packing thickness of the substrate material (e.g., the space needed for a layer of the substrate). For example, if a stack of cell culture matrix has a certain height, each layer of the stack can be said to have a packing thickness determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness will vary based on fiber diameter and weave, but can also vary based the alignment of adjacent layers in the stack. Forinstance, due to the three-dimensional nature of a woven layer, there is a certain amount of interlocking or overlapping that adjacent layers can accommodate based on their alignment with one another. In a first alignment, the adjacent layers can be tightly nestled together, but in a second alignment, the adjacent layers can have zero overlap, such as when the lower-most point of the upper layer is in direct contact with the upper-most point of the lower layer. It may be desirable for certain applications to provide a cell culture matrix with a lower density packing of layers (e.g., when higher permeability is a priority) or a higher density of packing (e.g., when maximizing substrate surface area is a priority). According to one or more embodiments, the packing thickness can be from about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm.

[0065] The above structural factors can determine the surface area of a cell culture matrix, whether of a single layer of cell culture substrate or of a cell culture matrix having multiple layers of substrate). For example, in a particular embodiment, a single layer of woven mesh substrate having a circular shape and diameter of 6 cm can have an effective surface area of about 68 cm2. The “effective surface area,” as used herein, is the total surface area of fibers in a portion of substrate material that is available for cell attachment and growth. Unless stated otherwise, references to “surface area” refer to this effective surface area. According to one or more embodiments, a single woven mesh substrate layer with a diameter of 6 cm may have an effective surface area of about 50 cm2to about 90 cm2; about 53 cm2to about 81 cm2; about 68 cm2; about 75 cm2; or about 81 cm2. These ranges of effective surface area are provided for example only, and some embodiments may have different effective surface areas. The cell culture matrix can also be characterized in terms of porosity, as discussed in the Examples herein.

[0066] The substrate mesh can be fabricated from monofilament or multifilament fibers of polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. Mesh substratesmay have a different patterns or weaves, including, for example knitted, warp-knitted, or woven (e.g., plain weave, twilled weave, dutch weave, five needle weave).

[0067] The surface chemistry of the mesh filaments may need to be modified to provide desired cell adhesion properties. Such modifications can be made through the chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules to the filament surface. Alternatively, meshes can be coated with thin layer of biocompatible hydrogels that demonstrate cell adherence properties, including, for example, collagen or Matrigel®. Alternatively, surfaces of filament fibers of the mesh can be rendered with cell adhesive properties through the treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry. In one or more embodiments, however, the mesh is capable of providing an efficient cell growth surface without surface treatment.

[0068] By using a structurally defined culture matrix of sufficient rigidity, high-flow- resistance uniformity across the matrix or packed bed is achieved. According to various embodiments, the matrix can be deployed in monolayer or multilayer formats. This flexibility eliminates diffusional limitations and provides uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the open matrix lacks any cell entrapment regions in the packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The matrix also delivers packaging uniformity for the packed bed, and enables direct scalability from process development units to large-scale industrial bioprocessing unit. The ability to directly harvest cells from the packed bed eliminates the need of resuspending a matrix in a stirred or mechanically shaken vessel, which would add complexity and can inflict harmful shear stresses on the cells. Further, the high packing density of the cell culture matrix yields high bioprocess productivity in volumes manageable at the industrial scale.

[0069] As discussed herein, the cell culture substrate can be used within a bioreactor vessel, according to one or more embodiments. For example, the substrate can be used in a packed bed bioreactor configuration, or in other configurations within a three-dimensional culture chamber. However, embodiments are not limited to a three-dimensional culture space, and it is contemplated that the substrate can be used in what may be considered a two-dimensional culture surface configuration, where the one or more layers of the substrate lay flat, such as within a flat-bottomed culture dish, to provide a culture substrate for cells. Due to contamination concerns, the vessel can be a single-use vessel that can be disposed of after use.

[0070] A cell culture system is provided, according to one or more embodiments, in which the cell culture matrix is used within a culture chamber of a bioreactor vessel. A cell culture system can include a bioreactor vessel having a cell culture chamber in the interior of the bioreactor vessel. Within the cell culture chamber is a fixed bed of a cell culture matrix that is made from a stack of substrate layers. The substrate layers are stacked with the first or second side of a substrate layer facing a first or second side of an adjacent substrate layer. The bioreactor vessel has an inlet at one end for the input of media, cells, and / or nutrients into the culture chamber, and an outlet at the opposite end for removing media, cells, or cell products from the culture chamber. By allowing stacking of substrate layers in this way, the system can be easily scaled up without negative impacts on cell attachment and proliferation, due to the defined structure and efficient fluid flow through the stacked substrates. While the vessel may generally be described as having an inlet and an outlet, some embodiments may use one or both of the inlet and outlet for flowing media, cells, or other contents both into and out of the culture chamber. For example, inlet may be used for flowing media or cells into the culture chamber during cell seeding, perfusion, or culturing phases, but may also be used for removing one or more of media, cells, or cell products through the inlet in a harvesting phase. Thus, the terms “inlet” and “outlet” are not intended to restrict the function of those openings.

[0071] According to embodiments, a cell culture system includes a bioreactor housing the cell culture matrix of one or more embodiments disclosed herein. The bioreactor can be fluidly connected to a media conditioning vessel, and the system is capable of supplying a cell culture media within the conditioning vessel to the bioreactor. The media conditioning vessel can include sensors and control components found in typical bioreactor used in the bioprocessing industry for a suspension batch, fed-batch or perfusion culture. These include but are not limited to DO oxygen sensors, pH sensors, oxygenator / gas sparging unit, temperature probes, and nutrient addition and base addition ports. A gas mixture supplied to sparging unit can be controlled by a gas flow controller for N2, O2, and CO2 gasses. The media conditioning vessel also contains an impeller for media mixing. All media parameters measured by sensors listedabove can be controlled by a media conditioning control unit in communication with the media conditioning vessel, and capable of measuring and / or adjusting the conditions of the cell culture media to the desired levels. The media conditioning vessel is provided as a vessel that is separate from the bioreactor vessel. This can have advantages in terms of being able to condition the media separate from where the cells are cultured, and then supplying the conditioned media to the cell culture space. However, in some embodiments, media conditioning can be performed within the bioreactor vessel.

[0072] The media from the media conditioning vessel is delivered to the bioreactor via an inlet, which may also include an injection port for cell inoculum to seed and begin culturing of cells. The bioreactor vessel may also include on or more outlets through which the cell culture media exits the vessel. In addition, cells or cell products may be output through the outlet. To analyze the contents of the outflow from the bioreactor, one or more sensors may be provided in the line. In some embodiments, the system includes a flow control unit for controlling the flow into the bioreactor. For example, the flow control unit may receive a signal from the one or more sensors (e.g., an O2 sensor) and, based on the signal, adjust the flow into the bioreactor by sending a signal to a pump (e.g., peristaltic pump) upstream of the inlet to the bioreactor. Thus, based on one or a combination of factors measured by the sensors, the pump can control the flow into the bioreactor to obtain the desired cell culturing conditions.

[0073] The media perfusion rate is controlled by the signal processing unit that collects and compares sensors signals from media conditioning vessel and sensors located at the packed bed bioreactor outlet. Because of the pack flow nature of media perfusion through the packed bed bioreactor, nutrients, pH and oxygen gradients are developed along the packed bed. The perfusion flow rate of the bioreactor can be automatically controlled by the flow control unit operably connected to the peristaltic pump.

[0074] One or more embodiments of this disclosure offer a cell inoculation step that is different from conventional methods. In conventional methods, a pack bed with a conventional matrix is filled with culture media and concentrated inoculum is injected into the media circulation loop. The cell suspension is pumped through the bioreactor at increased flow rate to reduce nonuniformity of cell seeding via capture on the conventional packed bed matrix. In suchconventional methods, the pumping of cells in the circulation loop at an elevated flow rate continues for perhaps several hours until the majority of the cells are captured in packed bed bioreactor. However, because of the nonuniform deep bed filtration nature of conventional packed bed bioreactors, cells are distributed nonuniformly inside the packed bed with the higher cell density at the inlet region of the bioreactor and lower cell density at the outlet region of the bioreactor.

[0075] In contrast, according to embodiments of the present disclosure, cell inoculum of equal volume to the void volume of the culture chamber in the bioreactor is directly injected into the packed bed through a cell inoculum injection port at the inlet of the bioreactor. The cell suspension is then uniformly distributed inside the packed bed because of uniform and continuous fluidic passages present in the cell culture matrix described herein. To prevent cells sedimentation due to gravity forces at the initial seeding stage, media perfusion can be started immediately after the inoculum injection. The perfusion flow rate is maintained below a preprogrammed threshold to balance the force of gravity and to avoid cells being washed from the packed bed bioreactor. Thus, at the initial cell attachment stage, cells are gently tumbled inside the packed bed and uniform cells distribution and attachment on available substrate surface is achieved.

[0076] The cell culture matrix can be arranged in multiple configurations within the culture chamber depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of the substrate with a width extending across the width of a defined cell culture space in the culture chamber. Multiple layers of the substrate may be stacked in this way to a predetermined height. As discussed above, the substrate layers may be arranged such that the first and second sides of one or more layers are perpendicular to a bulk flow direction of culture media through the defined culture space within the culture chamber, or the first and second sides of one or more layers may be parallel to the bulk flow direction. In one or more embodiments, the cell culture matrix includes one or more substrate layers at a first orientation with respect to the bulk flow, and one or more other layers at a second orientation that is different from the first orientation. For example, various layers may have first and second sides that are parallel or perpendicular to the bulk flow direction, or at some angle in between.

[0077] In one or more embodiments, the cell culture system includes a plurality of discrete pieces of the cell culture substrate in a packed bed configuration, where the length and or width of the pieces of substrate are small relative to the culture chamber. As used herein, the pieces of substrate are considered to have a length and / or width that is small relative to the culture chamber when the length and / or width of the piece of substrate is about 50% or less of the length and / or width of the culture space. Thus, the cell culture system may include a plurality of pieces of substrate packed into the culture space in a desired arrangement. The arrangement of substrate pieces may be random or semi-random, or may have a predetermined order or alignment, such as the pieces being oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° relative to the bulk flow direction).

[0078] The “defined culture space,” as used herein, refers to a space within the culture chamber occupied by the cell culture matrix and in which cell seeding and / or culturing is to occur. The defined culture space can fill approximately the entirety of the culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, the “bulk flow direction” is defined as a direction of bulk mass flow of fluid or culture media through or over the cell culture matrix during the culturing of cells, and / or during the inflow or outflow of culture media to the culture chamber.

[0079] In one or more embodiments, the cell culture matrix is secured within the culture chamber by a fixing mechanism. The fixing mechanism may secure a portion of the cell culture matrix to a wall of the culture chamber that surrounds the matrix, or to a chamber wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture matrix to a member running through the culture chamber, such as member running parallel to the longitudinal axis of the culture chamber, or to a member running perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture matrix may be contained within the culture chamber without being fixedly attached to the wall of the chamber or bioreactor vessel. For example, the matrix may be contained by the boundaries of the culture chamber or other structural members within the chamber such that the matrix is held within a predetermined area of the bioreactor vessel without the matrix being fixedly secured to those boundaries or structural members.

[0080] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. The culture chamber is capable of containing a cell culture matrix and substrate according to one or more of the embodiments described in this disclosure. In the roller bottle configuration, the bioreactor vessel may be operably attached to a means for moving the bioreactor vessel about a central longitudinal axis of the vessel. For example, the bioreactor vessel may be rotated about the central longitudinal axis. The rotation may be continuous (e.g., continuing in one direction) or discontinuous (e.g., an intermittent rotation in a single direction or alternating directions, or oscillating in back and forth rotational directions). In operation, the rotation of the bioreactor vessel causes movement of cells and / or fluid within the chamber. This movement can be considered relative with respect to the walls of the chamber. For example, as the bioreactor vessel rotates about its central longitudinal axis, gravity may cause the fluid, culture media, and / or unadhered cells to remain toward a lower portion of the chamber.However, in one or more embodiments, the cell culture matrix is essentially fixed with respect to the vessel, and thus rotates with the vessel. In one or more other embodiments, the cell culture matrix can be unattached and free to move to a desired degree relative to the vessel as the vessel rotates. The cells may adhere to the cell culture matrix, while the movement of the vessel allows the cells to receive exposure to both the cell culture media or liquid, and to oxygen or other gases within the culture chamber.

[0081] By using a cell culture matrix according to embodiments of this disclosure, such as a matrix including a woven or mesh substrate, the roller bottle vessel is provided with an increased surface area available for adherent cells to attach, proliferate, and function. In particular, using a substrate of a woven mesh of monofilament polymer material within the roller bottle, the surface area may increase by of about 2.4 to about 4.8 times, or to about 10 times that of a standard roller bottle. As discussed herein, each monofilament strand of the mesh substrate is capable of presenting itself as 2D surface for adherent cells to attach. In addition, multiple layers of mesh can we arranged in roller bottle, resulting in increases of total available surface area ranging from about 2 to 20 times that of a standard roller bottle. Thus, existing roller bottle facilities and processing, including cell seeding, media exchange, and cell harvesting, can be modified by theaddition of the improved cell culture matrix disclosed herein, with minimal impact on existing operation infrastructure and processing steps.

[0082] The bioreactor vessel optionally includes one or more outlets capable of being attached to inlet and / or outlet means. Through the one or more outlets, liquid, media, or cells can be supplied to or removed from the chamber. A single port in the vessel may act as both the inlet and outlet, or multiple ports may be provided for dedicated inlets and outlets.

[0083] The packed bed cell culture matrix of one or more embodiments can consist of the woven cell culture mesh substrate without any other form of cell culture substrate disposed in or interspersed with the cell culture matrix. That is, the woven cell culture mesh substrate of embodiments of this disclosure are effective cell culture substrates without requiring the type of irregular, non-woven substrates used in existing solution. This enables cell culture systems of simplified design and construction, while providing a high-density cell culture substrate with the other advantages discussed herein related to flow uniformity, harvestability, etc.

[0084] As discussed herein, the cell culture substrates and bioreactor systems provided offer numerous advantages. For example, the embodiments of this disclosure can support the production of any of a number of viral vectors, such as AAV (all serotypes) and lentivirus, and can be applied toward in vivo and ex vivo gene therapy applications. The uniform cell seeding and distribution maximizes viral vector yield per vessel, and the designs enable harvesting of viable cells, which can be useful for seed trains consisting of multiple expansion periods using the same platform. In addition, the embodiments herein are scalable from process development scale to production scale, which ultimately saves development time and cost. The methods and systems disclosed herein also allow for automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, the number of vessels needed to reach production-level scales of viral vectors (e.g., 1016to 1018AAV VG per batch) can be greatly reduced compared to other cell culture solutions.

[0085] Embodiments are not limited to the vessel rotation about a central longitudinal axis. For example, the vessel may rotate about an axis that is not centrally located with respect to the vessel. In addition, the axis of rotation may be a horizonal or vertical axis.Definitions

[0086] “Wholly synthetic” or “fully synthetic” refers to a cell culture article, such as a microcarrier or surface of a culture vessel, that is composed entirely of synthetic source materials and is devoid of any animal derived or animal sourced materials. The disclosed wholly synthetic cell culture article eliminates the risk of xenogeneic contamination.

[0087] ‘ ‘Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.

[0088] ‘ ‘Users” refers to those who use the systems, methods, articles, or kits disclosed herein, and include those who are culturing cells for harvesting of cells or cell products, or those who are using cells or cell products cultured and / or harvested according to embodiments herein.

[0089] ‘ ‘About” modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, or a dimension of a component, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, component parts, articles of manufacture, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.

[0090] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0091] The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.

[0092] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” for gram(s), “mL” for milliliters, and “rt” for room temperature, “nm” for nanometers, and like abbreviations).

[0093] Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The systems, kits, and methods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.

[0094] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that any particular order be inferred.

[0095] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the embodiments may occur to persons skilled in the art, the disclosed embodiments should be construed to include everything within the scope of the appended claims and their equivalents.

Claims

What is claimed:

1. A method of operating a bioreactor system for culturing cells, the method comprising: providing a fluid pathway for conveying cell culture media through the bioreactor system; providing a bioreactor vessel comprising an interior reservoir for housing a cell culture, a first bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway, and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; flowing cell culture media in a first direction from the fluid pathway into the interior reservoir through the first bioreactor inlet and out of the interior reservoir to the fluid pathway through the second bioreactor inlet; and after maintaining the flow of cell culture media in the first direction for a predetermined time, reversing the flow of cell culture media to a second direction, the second direction being from the fluid pathway into the interior reservoir through the second bioreactor inlet and out of the interior reservoir to the fluid pathway through the first bioreactor inlet.

2. The method of claim 1 , further comprising providing a fixed bed within the interior reservoir, the fixed bed comprising a cell culture substrate, the cell culture substrate comprising a surface configured for adhering cells of the cell culture thereto.

3. The method of claim 1 or claim 2, further comprising providing a media conditioning vessel (MCV) configured to control one or more conditions of the cell culture media in the bioreactor system, the media conditioning vessel comprising a first MCV inlet and a second MCV inlet that each fluidly connects the MCV to the fluid pathway.

4. The method of any one of claims 1-3, further comprising reversing the flow of the cell culture media back to the first direction.

5. The method of claim 4, further comprising reversing the flow of the cell culture media one or more additional times.

6. The method of any one of claims 1-5, wherein the reversing of the flow of the cell culture media is performed manually by an operator of the bioreactor system.

7. The method of any one of claims 1 -6, wherein the reversing of the flow of the cell culture media is performed automatically according to a schedule.

8. The method of claim 7, wherein the schedule is predetermined based on at least one of a type of cell in the cell culture, a rate of flow of cell culture media, a total culture time of the cell culture, a maximum time for flowing cell culture media in one direction, a stage of the cell culture, and a monitored property indicative of the health of the cell culture.

9. The method of claim 8, wherein the monitored property comprises at least one of a rate of growth of the cell culture, a nutrient level in the cell culture media, a dissolved gas level in the cell culture media, a rate of change of the nutrient level or the dissolved gas in the cell culture media, a temperature of the cell culture media, and a pH of the cell culture media.

10. The method of claim 9, wherein the monitored property is at least one of the nutrient level in the cell culture media, the dissolved gas level in the cell culture media, and the rate of change of the nutrient level or the dissolved gas in the cell culture media, and wherein the monitored property is measured at or near at least one of the first and second bioreactor inlets.

11. The method of claim 10, wherein the monitored property is measured by an inline sensor in the fluid pathway.

12. The method of any one of claims 1-11, wherein the first bioreactor inlet is on a bottom of the bioreactor vessel and the second bioreactor inlet is on a top of the bioreactor vessel.

13. The method of any one of claims 1-11, wherein the first bioreactor inlet is on a top of the bioreactor vessel and the second bioreactor inlet is on a bottom of the bioreactor vessel.

14. The method of any one of claims 1-13, further comprising maintaining and reversing the flow in the first and second directions to achieve at least one of a uniform cell number distribution and a uniform cell viability distribution along a direction parallel to the flow of cell culture media through the fixed bed.

15. The method of any one of claims 1-14, further comprising seeding cells into the bioreactor vessel.

16. The method of claim 15, further comprising alternating the flow of cell culture media through the fixed bed to maintain cells within the bioreactor vessel during seeding.

17. The method of claim 15 or claim 16, wherein alternating the flow of cell culture media during seeding causes the cells to be tumbled up and down within the fixed bed until the cells adhere to the fixed bed.

18. The method of any one of claims 1-17, further comprising alternating the direction of flow of the cell culture media during a transfection step of the cell culture.

19. The method of any one of claims 3-18, further comprising setting a dissolved oxygen level of the cell culture media in the MCV in a range from about 40% to about 60%.

20. The method of any one of claims 2-19, further comprising alternating the direction of flow of the cell culture media during a harvesting step of the cell culture, wherein the alternating of the direction of flow is configured to loosen and / or detach cells from the fixed bed.

21. A bioreactor system for culturing cells comprising:a fluid pathway for conveying cell culture media through the bioreactor system; a bioreactor vessel comprising an interior reservoir for housing a cell culture, a first bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway, and a second bioreactor inlet fluidly connecting the interior reservoir to the fluid pathway; a media conditioning vessel (MCV) configured to control one or more conditions of the cell culture media in the bioreactor system, the media conditioning vessel comprising a first MCV inlet and a second MCV inlet that each fluidly connects the MCV to the fluid pathway; and at least one pump fluidly attached to the fluid pathway and configured to flow fluid through the bioreactor system, wherein the bioreactor system is configured to alternative the flow of fluid through the bioreactor vessel between a first direction and a second direction, the first direction being from the first bioreactor inlet, through the interior reservoir, and to the second bioreactor inlet, and the second direction being from the second bioreactor inlet, through the interior reservoir, and to the first bioreactor inlet.

22. The bioreactor system of claim 21, further comprising a control system comprising a computer with a processor and a computer-readable medium storing instructions that, when executed by the processor, cause the bioreactor system to reverse a direction of flow of fluid through the bioreactor system.

23. The bioreactor system of claim 21 or claim 22, further comprising one or more sensors configured to measure at least one of a rate of growth of the cell culture, a nutrient level in the cell culture media, a dissolved gas level in the cell culture media, a rate of change of the nutrient level or the dissolved gas in the cell culture media, a temperature of the cell culture media, a biomass measurement of the cell culture, and a pH of the cell culture media.

24. The bioreactor system of claim 23, wherein the control system receives data from the one or more sensors and controls a direction of flow of fluid through the bioreactor system based on the data.

25. The bioreactor system of any one of claims 21-24, further comprising a fixed bed within the interior reservoir, the fixed bed comprising a cell culture substrate comprising a surface configured for adhering cells thereto.

26. The method of any of claims 2-20, wherein a cell density (as measured in cells / cm2) of cells in the cell culture at any point in the fixed bed is less than or equal to 2x the cell density at any other point in the fixed bed.

27. The method of claim 26, wherein the cell density at any point in the fixed bed is less than or equal to 1.5x the cell density at any other point in the fixed bed.

28. The method of claim 26, wherein the cell density at any point in the fixed bed is less than or equal to 1.25x the cell density at any other point in the fixed bed.

29. The method of any of claims 2-20 and 26-28, wherein a cell viability (expressed as a percentage of cells that are viable) of cells in the cell culture at any level in the fixed bed is less than or equal to 20% the cell viability at any other level in the fixed bed.

30. The method of claim 29, wherein the cell viability of cells in the cell culture at any level in the fixed bed is less than or equal to 10% the cell viability at any other level in the fixed bed.

31. The method of claim 29, wherein the cell viability of cells in the cell culture at any level in the fixed bed is less than or equal to 5% the cell viability at any other level in the fixed bed.

32. The method of claim 29, wherein the cell viability of cells in the cell culture at any level in the fixed bed is less than or equal to 2% the cell viability at any other level in the fixed bed.

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