WO2024102296A1 - Adherent cell culture systems with removable witness substrates for cell sampling - Google Patents

Abstract

A fixed bed bioreactor for culturing cells on a cell culture substrate is provided. The fixed bed bioreactor includes a cell culture vessel having a vessel body defining an interior reservoir, and a sample port disposed on an exterior of the cell culture vessel. The bioreactor further includes a cell culture substrate disposed in the interior reservoir; and a sample substrate at least partially disposed in the interior reservoir. The sample substrate is removable from the interior reservoir through the sample port without interfering with the cell culture occurring within the bioreactor.

Description

ADHERENT CELL CULTURE SYSTEMS WITH REMOVABLE WITNESS SUBSTRATES FOR CELL SAMPLING

CROSS-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. 63/423,665 filed on November 8, 2022, the content of which is relied upon and incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] This disclosure generally relates to bioreactor system with fixed bed cell culture substrates that enable sampling to monitor the cell culture. In particular, the present disclosure relates to cell culturing substrates and bioreactors incorporating such substrates that allow sampling without interrupting an ongoing cell culture within the bioreactor, and methods of performing such sampling, to monitor the health and progress of the culture and other processes.

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 nonuniformity 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 further population 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] Certain packed-bed bioreactors currently on the market uses small strips, disks, or pieces 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, there are drawbacks to this type of packed-bed substrate, including 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 bioreactor 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 that the 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, it can be difficult for customers to predict cell culture performance, since the substrate varies between cultures. Furthermore, the packed substrates of some existing bioreactors make efficiently harvesting cells very difficult or impossible, as it is believed that cells are entrapped by the packed bed. [0010] 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 greater numbers in order to reach late-stage commercial manufacturing scale. [0011] In addition, it is desirable to be able to monitor bioreactors used to culture cells or make AAV or to create a seed train to facilitate cell expansion for biochemical production. Since transient transfection, such as triple plasmid transfection, is commonly used for viral vector production, it is necessary to periodically examine cell confluency and status in order to achieve optimal performance. However, for both existing fixed bed bioreactors, cell sampling requires stopping cell culture, opening the bioreactor, followed up by removing microfibers or webs, which is not only inconvenient, but also can introduce contamination. Therefore, a removable sample substrate that can be removed without interfering with cell culture would be desired for periodic examination of cell confluency and status during the culture using fixed bed bioreactors. When using adherent cell reactors, samples of the growth media do not contain cells, or at least not to an extent that is useful for monitoring the state of the culture on the adherent substrate. Given the above-discussed potential for irregularities in existing bioreactor substrates, this monitoring can prove even more valuable if it can be performed at various positions within the bioreactor without disrupting the cell culture.

[0012] There is a need for bioreactors, cell culture substrates, systems, and methods that enable culturing of cells in a high-density format, with uniform cell distribution, and easily attainable and increased harvesting yields, while also enabling users to monitor the state of the cell culture process by examining the cells on the substrate during and/or after the cell culture process, including performing such monitoring aseptically and/or without disrupting an on-going cell culture.

SUMMARY

[0013] According to embodiments of this disclosure, fixed bed bioreactor systems and cell culture substrates are disclosed that allow for sampling of a cell culture using sample substrates located at predetermined locations within the cell culture substrate to monitor the status or health of the cell culture. Embodiments include a fixed bed bioreactor and/or cell culture substrate with one or more removable sample substrates specifically designed to enable this sampling by removing the sample substrates from the bioreactor without interfering with an ongoing cell culture. In embodiments, the removable sample substrate is made of the same material as the fixed bed cell culture substrate in the bioreactor, thus offering the possibility for reliable detection of cell culture quality at a specific time and/or place during the culture. As a further aspect of embodiments, the removable cell witness substrate can be removed without opening of the bioreactor and stopping the culture, thus avoiding potential contamination. In additional aspects of embodiments, the removable sample substrate can be placed at a specific location of the fixed bed, thus enabling the detection of cell culture quality at the predefined location. The use of multiple removable cell witness substrates, each at a specific location, can further allow examination of cell culture quality and homogeneity throughout the entire fixed bed.

[0014] To be able to estimate the number of cells and their distribution and health in an adherent cell bioreactor bed, embodiments include methods using removable sample substrates strategically placed in in, near, or among the cell culture substrate, and doing so in the midst of the cell culture or cell expansion process. By sampling during the process, information about the cell culture run can be used to assess the quality and performance of the process. Cell count can be estimated from the sample and growth can be monitored by sampling at different times and/or locations of the fixed bed cell culture substrate. This information can be used to develop and optimize performance of specific biological processes such as seed train and viral vector production. In production, runs that are contaminated or out of specification, can be terminated to reduce the cost of running the process to its end without a satisfactory result. Growth media and lost production time represent significant cost for typical biological processes. Embodiments of this disclosure allow removal of one or more sample substrates from the bioreactor to give users access to the cell health within the bed without destroying the bed or bioreactor vessel, and allowing the cell culture to continue during or after the sampling process. Embodiments enable any portion or select portions of the fixed bed to be assessed. The bed can also be accessed after the cell culture process or harvesting of the desired component is completed for a “post-mortem” analysis of the cell culture.

[0015] According to an embodiment of this disclosure, a fixed bed bioreactor system for culturing cells on a cell culture substrate is provided. The fixed bed bio reactor includes: a cell culture vessel having a vessel body defining an interior reservoir, and a sample port disposed on an exterior of the cell culture vessel. The bioreactor also includes a cell culture substrate disposed in the interior reservoir, and a sample substrate at least partially disposed in the interior reservoir. The sample substrate is able of being removed from the interior reservoir through the sample port. Removal of the sample substrate from the interior reservoir through the sample port is possible during an active cell culture and without interfering with the ongoing culture. The sample substrate is configured to be removable aseptically.

[0016] According to embodiments of this disclosure, a cell culture substrate bed for a fixed bed bioreactor is provided. The cell culture substrate bed has a plurality of layers of substrate material in a stacked arrangement, each layer of the substrate material having a structurally defined surface for culturing cells thereon and an ordered array of openings through a thickness of the layer. The cell culture substrate bed also includes a sample substrate with a first end and a second end opposite the first end, the first end being disposed between two consecutive layers of the plurality of layers of substrate material. The second end of the sample substrate has a pull configured to be pulled by an external force and thereby remove the first end from between the two consecutive layers.

[0017] According to embodiments herein, a method of sampling a cell culture is provided that includes providing a cell culture media to an interior of a bioreactor containing a cell culture substrate, the cell culture substrate being suitable for growing cells thereon and having a sample substrate with a first end disposed within the cell culture substrate. The method further includes, during the cell culture, removing the sample substrate from the bioreactor without interfering with the culture process.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows a schematic view of a fixed bed bioreactor system, according to one or more embodiments.

[0019] Figure 2 shows a cross-section schematic view of a fixed bed bioreactor system with a sample substrate, according to one or more embodiments.

[0020] Figure 3 shows a cross-section schematic view of the fixed bed bioreactor system of Figure 2 during the removal of a sample substrate, according to one or more embodiments.

[0021] Figure 4 shows a cross-section schematic view of the fixed bed bioreactor system of Figures 2 and 3 after removal of a sample substrate, according to one or more embodiments.

[0022] Figure 5 shows a cross-section schematic view of a fixed bed bioreactor system with multiple sample substrates at different locations in the cell culture substrate, according to one or more embodiments. DETAILED DESCRIPTION

[0023] 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.

[0024] Embodiments of this disclosure include cell culture substrates, as well as cell culture bioreactors incorporating such a substrate, that enabling sampling of the substrate or a portion of the substrate for monitoring cell culture.

[0025] A cell culture system is provided, according to one or more embodiments, in which the cell culture substrate is used within a culture chamber of a bioreactor vessel. Figure 1 shows an example of a cell culture system 100 that includes a bioreactor vessel 102 having an interior reservoir as a cell culture chamber 104 within the bioreactor vessel 102. Within the cell culture chamber 104 is a cell culture substrate 106. According to aspects of embodiments, the cell culture substrate 106 can take different forms and/or arrangements within the cell culture chamber 104, as discussed herein. For example, in Figure 1, the cell culture substrate 106 is made from a stack of substrate layers 108. The substrate layers 108 are stacked with the first or second side of a substrate layer facing a first or second side of an adjacent substrate layer. However, embodiments of this disclosure include other arrangements for the cell culture substrate 106, including, for example: one or more rolled sheets (the center longitudinal axis of the roll being parallel to the direction between the inlet 110 and outlet 112 of the bioreactor vessel 102); a three-dimensional moonlight substrate matrix; sheets of substrate material stacked such that their major faces are parallel to a direction between the inlet 110 and the outlet 112; etc. In Figure 1, the bulk flow direction is in a direction from the inlet 110 to the outlet 112, and, in this example, the first and second major sides of the substrate layers 108 are perpendicular to the bulk flow direction.

[0026] The bioreactor vessel 100 has an inlet 110 at one end for the input of media, cells, and/or nutrients into the culture chamber 104, and an outlet 112 at the opposite end for removing media, cells, or cell products from the culture chamber 104. 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 100 may generally be described as having an inlet 110 and an outlet 112, some embodiments may use one or both of the inlet 110 and outlet 112 for flowing media, cells, or other contents both into and out of the culture chamber 104. For example, inlet 110 may be used for flowing media or cells into the culture chamber 104 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 110 in a harvesting phase. Thus, the terms “inlet” and “outlet” are not intended to restrict the function of those openings. [0027] The cell culture substrate 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 substrate 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.

[0028] 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).

[0029] The “defined culture space,” as used herein, refers to a space within the culture chamber occupied by the cell culture substrate 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 substrate during the culturing of cells, and/or during the inflow or outflow of culture media to the culture chamber.

[0030] In one or more embodiments, the cell culture substrate is secured within the culture chamber by a fixing mechanism. The fixing mechanism may secure a portion of the cell culture substrate to a wall of the culture chamber that surrounds the substrate (e.g., the wall of the vessel 102 that forms the culture chamber 104), or to a chamber wall at one end of the culture chamber near the inlet 110 or the outlet 112. In some embodiments, the fixing mechanism adheres a portion of the cell culture substrate 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 substrate may be contained within the culture chamber without being fixedly attached to the wall of the chamber or bioreactor vessel. For example, the substrate may be contained by the boundaries of the culture chamber or other structural members within the chamber such that the substrate is held within a predetermined area of the bioreactor vessel without the substrate being fixedly secured to those boundaries or structural members.

[0031] To enable sampling on the bioreactor 100 of Figure 1 without disrupting the cell culture, embodiments herein provide a fixed bed bioreactor having at least one removable sample substrate. Removing the sample substrate from the bioreactor enables periodic sampling or monitoring of cell culture quality by the bioreactor user. As used herein, sampling or monitoring can refer to observing, measuring, or tracking any of a number of indicators related to the status, health, or success of the cell culture process, including, for example, confluency, cellular status, uniformity of cell coverage, homogeneity throughout the entire fixed bed, and/or harvesting efficiency.

[0032] As shown in Figure 2, the fixed bed bioreactor 200 has of a cell culture vessel that includes a vessel body 202 defining an interior reservoir 204. The vessel body 202 can be any suitable and biocompatible container capable of housing a cell culture. In embodiments, the vessel body 202 is an extruded or molded polymer tube, but may also be metallic (e.g., stainless steel, aluminum), glass, or ceramic. The ends of the vessel body 202 may be enclosed or capped with a lower end cap 214 and an upper end cap 216. The lower end cap 214 has an inlet 210 that may be connected to tubing or other fluid pathway able to supply fluid, such as cell media, nutrients, and cells to the interior reservoir 204. The upper end cap 216 includes an outlet 212 that may also be connected to tubing or other fluid pathway for fluid exiting the interior reservoir 204. Each of the inlet 210 and outlet 212 may be fluidly connected to an external container, such as a media conditioning vessel, such that conditioned media can be recirculated through the bioreactor for continuous long-term perfusion culture. However, the outlet 212 may be fluidly connected to a different external container than the inlet 210 such that the medium once passing through the fixed bed can be collected, which is particular useful during viral vector production phase. In embodiments, the bulk flow direction of media is from the bottom to the top (as shown in Figure 2) of the bioreactor, but the flow may also be in the reverse direction.

[0033] The cell culture vessel also includes a sample port 218 disposed on an exterior of the cell culture vessel and allowing access to an area within the bioreactor vessel. The sample port 218 may be formed in one of the end caps (such as upper end cap 216, as shown in Figure 2) or may be formed in a sidewall of the vessel body 202. Preferably, the sample port 218 includes a closure mechanism to preserve the integrity of the interior reservoir 204 and, by extension, protect the cell culture from contamination. The closure mechanism may be a cap 220, sealable tubing, air lock, or other suitable closure. The sample port 218 may include a passage 219 formed through the vessel body an into a suitable location within the bioreactor. The passage 219 may include additional features to aid in preventing contamination inside the bioreactor, such as gaskets, screens, brushes, flaps, or other means of minimizing the opening to the interior reservoir 204. In aspects of some embodiments, the second end 234 of the sample substrate 230 is coupled to the cap 220 of the sample port 218, such that removal of the cap 220 automatically removes the sample substrate 230 from the cell culture vessel.

[0034] A fixed bed substrate 206 is provided within the interior reservoir 204. The substrate 206 has a surface upon which adherent or semi-adherent cells can grow. The fixed bed substrate 206 may be a monolithic structure or may include multiple substrates arranged within the interior reservoir 204. In some embodiments, the substrate material is made of woven polymer fibers, as described herein. A removable sample substrate 230 is also provided at least partially within the interior reservoir 204. The sample substrate 230 is preferably in close proximity to, or touching, the cell culture substrate 206. As an aspect of embodiments, the first end 232 or first portion 233 of the sample substrate 230 is not separated from the cell culture substrate 206 by any physical barrier and/or is in direct physical contact with the cell culture substrate 206. In some embodiments, at least a first end 232 or a first portion 233 is placed in a space 208 provided within the fixed bed substrate 206. The space 208 can be a space between two separate pieces of substrate 206, or can be part of an opening formed in a substrate 206. The sample substrate 230 has a second portion 235 and/or a second end 234 that extends outward from the interior of the cell culture substrate 206, such that the second end 234 can be pulled from the vessel body 202 when a user desired to remove the sample substrate 230. In embodiments, the first portion 233 of the sample substrate extends toward the outside of the cell culture substrate 206 and a second portion 235 of the sample substrate 230 then extends towards the sample port 218. The second end 234 may be disposed in the vicinity of the external opening of the sample port 218 so that the second end 234 can be grasped to full the sample substrate 230 from the bioreactor when taking a sample. The second end 234 may be grasped directly via forceps, tweezers, or other mechanical implement in order to exert a pulling force (F in Figure 3) to remove the sample substrate 230. Alternatively, a pull may be coupled to the second end 234 of the sample substrate 230, where the pull may be any structure suitable to be pulled by an external source (e.g., string, fiber, tab, pull-tap, rod, stick, loop, handle, etc.).

[0035] According to embodiments, the sample substrate 230 can be removed through the sample port 218 without disrupting the cell culture within the bioreactor. The sample port 218 can be made to be aseptic by having a tightly closed cap or any other standard aseptic means, such that removal of the sample substrate 230 will require stopping the cell culture and opening the bioreactor, thus avoiding potential contamination.

[0036] The position of the space 208 can be predetermined based on a desired location from which a sample is desired. In embodiments, multiple sample substrates 230 and multiple spaces 208 are provided to enable sampling from different locations within the cell culture substrate 206. For example, sample substrates 230 and spaces 208 may be provided a low, middle, and top positions with a vertical fixed bed to provide sampling from those different regions, which may have very different cell culture performance. Alternatively, the removable sample substrate can be placed vertically along the inner wall of the vessel body 202 so that the cell quality data obtained along the length of the sample substrate can be used as an estimation of overall cell culture quality along the height of the fixed bed 206.

[0037] The removable sample substrate 206 is preferably made of the same material as the fixed bed cell culture substrate (e.g., PET woven mesh), so that cell culture quality data obtained on the sample substrate is representative to the one on the fixed bed cell culture substrate. The removable sample substrate 230 is preferably in the form of a string or a strip, such that cell sampling can be easily achieved by removing the sample substrate 230 via the sample port 218, and will not result in any significant loss of cells cultured and thus have limited impact on the productivity of the bioreactor. As an aspect of some embodiments, the removable sample substrates 230 preferably have the same surface area (e.g., 1 cm², 5 cm², 10 cm², 50 cm²) for cell attachment, so that cell confluency or other cell quality parameters obtained via sampling can be directly compared each other. After being removed from the cell culture, the sample substrate 230 can be subject to examination for any desired performance criteria, including, for example, cell confluency using microscopy, or cell counting after trypsin treatment, or cellular status using fluorescence staining.

[0038] Other bioreactors often require opening the entire vessel body with manually remove the cell culture substrate for sampling. Of course, such invasive measures disrupt or even destroy any ongoing culture and thus are often only done after the culture is completed. By enabling sampling using the removable sample substrate 230 through the dedicated and aseptic sample port 218 in a way that does not expose the cell culture substrate 206 and/or disrupt perfusion fluid flow through the cell culture vessel, sampling can be performed at any or multiple stages of the cell culture without negatively impacting the remaining culture process, and thus provide valuable insight into the current and evolving status of the cell culture.

[0039] The sample substrate 230 is preferably the same material as the cell culture substrate 206, so that the sample provided by the sample substrate 230 is representative to the culture on the cell culture substrate 206. The cell culture substrate 230 is a substrate material having a plurality openings arranged in a regular and uniform array. The cell culture substrate preferably has a physical structure that is regular and uniform. The physical structure includes a plurality of fibers in a predetermined and ordered arrangement. In some embodiments, the cell culture substrate is a mesh material, and may be a mesh material that is a woven mesh having a plurality of interwoven fibers. Accordingly, the sample substrate may also be a fibrous mesh material, such as a woven mesh. However, the sample substrate may have a different physical structure, such as one or more non-woven or unconnected strings of fiber that are the same material as the cell culture substrate.

[0040] Figure 3 shows the fixed bed bioreactor 200 of Figure 2 during the process of removing a sample substrate 230. After removing the cap 220 of the sample port 218, an upward force F is exerted on the second end 234 of the sample substrate to being withdrawing the sample substrate 230. As a result, the first end 232 of the sample substrate slides out from within the cell culture substrate 206. The sample substrate 230 is continued to be pulled until the entire sample substrate 230 is removed from the bioreactor 200, as shown in Figure 4, and the cap 220 is replaced.

[0041] As discussed herein, according to embodiments, the cell culture substrate can include a plurality of substrate layers 207, as shown in Figure 5. The plurality of substrate layers 207 can be arranged in a stacked configuration, with the stack height being in a direction of the height of the interior reservoir 204, or in the bulk flow direction for fluid flowing therethrough. As shown in Figure 5, multiple sample substrates (230a, 230b, and 230c) can be provided at different locations within the stacked substrate layers. Although three sample substrates are shown in Figure 5, embodiments are not limited to this number, and any number of sample substrate can be employed. The first ends 232a, 232b, 232c of the sample substrates 230a, 230b, 230c can be provided at desired locations within the stack. For example, the first ends 232a, 232b, 232c may be located at lower, middle, and upper regions of the stacked substrate layers 207 to provide samples representing different regions of the cell culture.

[0042] According to embodiments, individual layers of the plurality of substrate layers 207 in the stacked configuration are not separated from each other by any physical barrier. In this sense, the sample substrates 230a-230c themselves are not considered a physical barrier between the layers, but instead are considered part of the cell culture substrate, albeit removable parts of the substrate. Each first end 232a-232c of the sample substrates 230a-230c is disposed between two layers of the plurality of substrate layers. First portions of the sample substrates 230a-230c including the first ends 232a-232c are arranged parallel to the layers of the cell culture substrate 207. Second portions of the sample substrates 230a-230c are arranged perpendicular to the layers of the cell culture substrate 207 in Figure 5, for ease of pulling the sample substrates through the sample port 218 and, optionally, for provided sampling along a vertically oriented span of the sample substrates 230a-230c. The second portion is disposed between the cell culture substrate 207 and a wall of the interior reservoir of the vessel body 202. However, in embodiments, the vertical portions of the sample substrates 230a-230c may extend through the packed bed substrate 207 itself.

[0043] According to embodiments of this disclosure, the vessel body 202 has a first end, a second end, and a longitudinal axis extending in a direction from the first end to the second end, and fluid flows through the inlet 210 into the interior reservoir 204, through the cell culture substrate 207 in the interior reservoir 204, and out through the outlet 212 in a flow direction that is substantially parallel to the longitudinal axis. As an aspect of embodiments, the interior reservoir 204 is free from any flow channel, flow diverter, or flow recirculation path that would substantially deviate fluid flow through the cell culture vessel or interior reservoir 204 from a direction parallel to the longitudinal axis.

[0044] Embodiments of this disclosure also include a cell culture substrate bed for a fixed bed bioreactor that includes a plurality of layers of substrate material in a stacked arrangement. Each layer of the substrate material has a structurally defined surface for culturing cells thereon and an ordered array of openings through a thickness of the layer. The cell culture substrate bed further includes a sample substrate having a first end and a second end opposite the first end, the first end being disposed between two consecutive layers of the plurality of layers of substrate material. The second end of the sample substrate can be pulled by an external force and thereby remove the first end from between the two consecutive layers. The second end is disposed outside of the plurality of layers of substrate material. The sample substrate includes a first portion having the first end, and a second portion having the second end. The first portion runs parallel to the layers of the plurality of layers of substrate material, and the second portion runs perpendicular to the plurality of layers of substrate material. The pull of the sample substrate can be disposed above a top-most layer of the plurality of substrate layers in the stacked arrangement, or below the bottom-most layer, for ease of reaching the end of the sample substrate so that it can be pulled from the fixed bed cell culture substrate.

[0045] According to embodiments of this disclosure, a method of sampling a cell culture is provided that includes providing a cell culture media to an interior of a bioreactor as disclosed herein, the bioreactor containing a cell culture substrate, and the cell culture substrate being for growing cells thereon. The bioreactor also contains a sample substrate with a first end disposed within the cell culture substrate. During the cell culture, the sample substrate is removed from the bioreactor. Removing of the sample substrate can include removing a cap from the bioreactor to access a space from which the sample substrate can be pulled out of the bioreactor. The removing of the cap is performed without ending a cell culture process within the bioreactor, and can be performed aseptically. The method may include removing a plurality of sample substrates from different locations within the bioreactor.

[0046] 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 substrate. 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. [0047] 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 or metabolite 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.

[0048] 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 fixed 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 substrate 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 substrate 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 10¹⁶ to 10¹⁸ viral genomes (VG) per batch.

[0049] In one embodiment, a cell culture substrate or 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 fixed bed or other bioreactor. In particular embodiments, a mechanically stable, non-degradable woven mesh can be used as the substrate to support adherent cell production. The structurally defined, mechanically stable substrate has a predetermined arrangement of cell culture surface that enables uniform fluid flow therethrough, uniform seeding of cells, uniform cell growth, and efficient and uniform harvesting of cells from the substrate. The cell culture substrate disclosed herein supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. Uniform cell seeding of such a substrate 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 cell culture substrate, and can avoid formation of large and/or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the cell culture substrate eliminates diffusional limitations during operation of the bioreactor. In addition, the substrate enables easy and efficient cell harvest from the bioreactor. The structurally defined substrate of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.

[0050] According to some embodiments, a method of cell culturing is also provided using bioreactors with the cell culture substrate and sampling substrate(s) for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0051] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., non-woven substrates or other 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 fixed bed 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 can be 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 a polymer-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 substrate has a high surface-to-volume ratio for culturing anchorage dependent cells. According to various embodiments, the substrate 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.

[0052] In embodiments, the sampling substrate is made of the same material as the cell culture substrate. In embodiments, a physical structure of the sampling substrate is similar to that to the cell culture substrate. This physical structure can include properties such as fiber shape and diameter, opening shape and diameter, surface treatment or coating, material, and arrangement and/or method of manufacturing the substrate material (e.g., having a woven or other structure). It is believed that using a similar structure and/or material for the cell culture substrate and the sampling substrate will result in the sample substrates providing a relatively accurate representation of the health and status of the cells on the cell culture substrate. The sampling substrate can also be co-located with the cell culture substrate to give an accurate representation of the status of the cell culture at that location. In embodiments, at least part of the sample substrate may be embedded in or in contact with the cell culture substrate.

[0053] 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 10¹⁴ viral genomes per batch, greater than about 10¹⁵ viral genomes per batch, greater than about 10¹⁶ viral genomes per batch, greater than about 10¹⁷ viral genomes per batch, or up to or greater than about g 10¹⁶ viral genomes per batch. In some embodiments, production is about 10¹⁵ to about 10¹⁸ or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be about 10¹⁵ to about 10¹⁶ viral genomes or batch, or about 10¹⁶ to about 10¹⁹ viral genomes per batch, or about 10¹⁶- 10¹⁸ viral genomes per batch, or about 10¹⁷ to about 10¹⁹ viral genomes per batch, or about 10¹⁸ to about 10¹⁹ viral genomes per batch, or about 10¹⁸ or more viral genomes per batch.

[0054] 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.

[0055] Examples of cell culture substrates within embodiments of this disclosure can be found in U.S Patent No. 11,434,460, U.S. Patent Application No. 16/765722, and U.S. Provisional Patent Application No. 63/284153, which are incorporated herein by reference. According to embodiments, the cell culture substrate can be a woven mesh layer made of a first plurality of fibers running in a first direction and a second plurality of fibers running in a second direction. The woven fibers of the substrate form a plurality of openings, which can be defined by one or more widths or diameters. The openings in the cell culture substrate have a diameter defined as a distance between opposite fibers. 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. As used herein, the thickness 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 substrate structure that enables uniform fluid flow. According to some embodiments, the plurality of fibers of the cell culture substrate may all have the same thickness, or the substrate may include fibers having different thicknesses, either within a single woven substrate or among separate pieces of substrate.

[0056] In one or more embodiments, a fiber may have a diameter in a range of about 10 pm to about 1000 pm; about 20 pm to about 750 pm; about 25 pm to about 600 pm; about 30 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm; about 150 pm to about 300 pm; about 10 pm to 175 pm; about 15 pm to 150 pm; or about 15 pm to 100 pm. On a microscale level, due to the scale of the larger-diameter fibers compared to the cells (e.g., the fiber diameters being larger than the cells), the surface of monofilament fiber may approximate a 2D surface for adherent cells to attach and proliferate. Fibers can be woven into a mesh with openings ranging from about 20 pm x 20 pm to about 1000 pm x 1000 pm. In some embodiments, the opening may have a diameter of about 20 pm to about 100 pm; 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 substrate comprises a number of adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0057] Factors such as the fiber diameter, opening diameter, and weave type/pattem will determine the surface area available for cell attachment and growth. In addition, when the cell culture substrate includes a stack, roll, or other arrangement of overlapping substrate, the packing density of the cell culture substrate will impact the surface area of the fixed bed substrate. 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 substrate 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. For instance, 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 substrate 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. [0058] The above structural factors can determine the surface area of a cell culture substrate, 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 cm² to about 90 cm²; about 53 cm² to 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 substrate can also be characterized in terms of porosity, as discussed in the Examples herein.

[0059] 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 substrates may 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).

[0060] 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. [0061] By using a structurally defined culture substrate of sufficient rigidity, high-flow- resistance uniformity across the substrate or fixed bed is achieved. According to various embodiments, the substrate 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 substrate. In addition, the open substrate lacks any cell entrapment regions in the packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The substrate 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 substrate 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 substrate yields high bioprocess productivity in volumes manageable at the industrial scale.

[0062] As used herein, “structurally defined” means that the structure of the substrate follows a predetermined design and is not random. The structurally defined substrate can thus be a woven design, 3D printed, molded, or formed by some other technique known in the art that allows the structure to follow a predetermined planned structure.

[0063] The geometry of the mesh substrate layers, according to embodiments, is designed to allow efficient and uniform flow through one or multiple substrate layers. In addition, the structure of the substrate can accommodate fluid flow through the substrate in multiple orientations. For example, the direction of bulk fluid flow through the fixed bed substrate can be perpendicular to the major side surfaces of the substrate layers. However, the substrate can also be oriented with respect to the flow such that the sides of the substrate layers are parallel to the bulk flow direction. In addition to fluid flow being perpendicular or parallel to the first and second sides of the mesh layers, the fixed bed substrate can be arranged with multiple pieces of substrate at intermediate angles, or even in random arrangements with respect to fluid flow. This flexibility in orientation is enabled by the essentially isotropic flow behavior of the uniform woven substrate. In contrast, substrates for adherent cells in existing bioreactors with randomly-oriented fibers or randomly-packed substrate material do not exhibit this behavior and instead their packed beds tend to create preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the substrate of the current disclosure allows for its use in various applications and bioreactor or container designs while enabling better and more uniform permeability throughout the bioreactor vessel.

[0064] 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 fixed 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.

[0065] 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.

[0066] The fixed bed cell culture substrate 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 substrate. 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.

[0067] 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., 10¹⁶ to 10¹⁸ AAV VG per batch) can be greatly reduced compared to other cell culture solutions. [0068] This disclosure describes substrates and methods to cut and perforate layers of cell culture substrate, including polymer mesh substrates, to create a detachable sample piece. The disclosure also describes methods and apparatus to aseptically remove the sample from a bioreactor. By sampling during the cell culture process, information about the run can be used to assess the quality and performance of the culture process. Cell count can be estimated from the sample and growth can be monitored by sampling at different times or at different places within the bioreactor. This information can be used to develop and optimize parameters for specific biological processes such as seed train and viral vector production. In production, processes that are contaminated or out of specification, can be terminated to reduce the cost of running the process to its end without a satisfactory result. Growth media and lost production time represent significant cost for typical biological processes.

[0069] In embodiments herein, substrate sample portions are separable from a remainder of the cell culture substrate with a low force to allow sampling to be accomplished ideally by hand and without disturbing the main mesh body during the sampling process. For example, a relatively low (e.g., applied by hand) force can cause the sample portion to separate from the remainder of the substrate via the tension between the sample portion to which the force is applied and the remainder of the substrate. To keep the removal force low, the separation boundary can be applied between the sample portion and the remainder of the substrate. This separation boundary can be formed, for example, by scoring, perforation, laser cutting, or other cutting means such as die cutting, and can be used to create a layer of substrate that includes separable pieces of the substrate that can be removed from the fixed bed.

[0070] Some embodiments use woven polymer mesh substrates that have woven fibers defining an ordered array of pores or openings. Because each of the fibers in the mesh is very strong, it is desirable to have no fibers that run between the detachable sample and the main body of the mesh to facilitate the sample being removed with a low force. It is also desirable to have the mesh layer be robust when handled during the manufacturing and assembly process used to create a mesh stack bioreactor bed. To accomplish this, some fibers can be cut in such a way to leave a woven portion of the mesh that connects the sample to the main mesh body. Due to the relative stiffness of the fibers, which may be formed from a variety of polymers disclosed herein (including PET), the interwoven fibers may remain attached even though individual fibers are severed to create the separation boundary of the sample portion. [0071] Illustrative Implementations

[0072] The following is a description of various aspects of implementations of 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 a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations. [0073] Aspect 1 pertains to a fixed bed bioreactor for culturing cells on a cell culture substrate, the fixed bed bioreactor comprising: a cell culture vessel comprising a vessel body defining an interior reservoir, and a sample port disposed on an exterior of the cell culture vessel; a cell culture substrate disposed in the interior reservoir; and a sample substrate at least partially disposed in the interior reservoir, wherein the sample substrate is configured to be removable from the interior reservoir through the sample port.

[0074] Aspect 2 pertains to the fixed bed bioreactor of Aspect 1, wherein the sample substrate is configured to be removable from the interior reservoir through the sample port during an active cell culture.

[0075] Aspect 3 pertains to the fixed bed bioreactor of Aspect 1 or 2, wherein the sample substrate is configured to be removable aseptically.

[0076] Aspect 4 pertains to the fixed bed bioreactor of Aspects 1-3, wherein the sample substrate is configured to be removable from the interior reservoir without opening the cell culture vessel in a way that would expose the cell culture substrate or disrupt perfusion fluid flow through the cell culture vessel.

[0077] Aspect 5 pertains to the fixed bed bioreactor of Aspects 1-4, wherein the sample port comprises a removable cap or closure.

[0078] Aspect 6 pertains to the fixed bed bioreactor of Aspects 1-5, wherein the sample substrate comprises a first end disposed in the interior reservoir, the first end being close proximity to the cell culture substrate.

[0079] Aspect 7 pertains to the fixed bed bioreactor of Aspect 6, wherein the first end of the sample substrate is not separated from the cell culture substrate by any physical barrier. [0080] Aspect 8 pertains to the fixed bed bioreactor of Aspect 6 or 7, wherein the first end of the sample substrate is in physical contact with the cell culture substrate. [0081] Aspect 9 pertains to the fixed bed bioreactor of Aspects 6-8, wherein the sample substrate comprises a second end configured to be accessible through the sample port from an exterior of the cell culture vessel.

[0082] Aspect 10 pertains to the fixed bed bioreactor of Aspect 9, wherein the second end is disposed adjacent to the sample port such that the second end can be grasped by a physical implement when the sample port is open and pulled thereby from the sample port.

[0083] Aspect 11 pertains to the fixed bed bioreactor of Aspect 9 or 10, wherein the second end is connected to a string, pull-tap, rod, stick, loop, or handle that is disposed adjacent to the sample port such that the string, pull-tap, rod, stick, loop, or handle can be grasped by a physical implement when the sample port is open and pulled thereby from the sample port. [0084] Aspect 12 pertains to the fixed bed bioreactor of Aspects 9-11, wherein the second end is coupled to the cover of the sample port, wherein removal of the cover is configured to remove the sample substrate from the cell culture vessel.

[0085] Aspect 13 pertains to the fixed bed bioreactor of Aspects 1-12, wherein the sample substrate comprises the same material as the cell culture substrate.

[0086] Aspect 14 pertains to the fixed bed bioreactor of Aspects 1-13, wherein the cell culture substrate comprises a substrate material comprising a plurality openings arranged in a regular and uniform array.

[0087] Aspect 15 pertains to the fixed bed bioreactor of Aspects 1-14, wherein the cell culture substrate comprises a physical structure that is regular and uniform.

[0088] Aspect 16 pertains to the fixed bed bioreactor of Aspect 15, wherein the physical structure comprises a plurality of fibers in a predetermined and ordered arrangement.

[0089] Aspect 17 pertains to the fixed bed bioreactor of Aspects 1-16, wherein the cell culture substrate comprises a mesh material.

[0090] Aspect 18 pertains to the fixed bed bioreactor of Aspect 17, wherein the mesh material is a woven mesh comprising a plurality of fibers.

[0091] Aspect 19 pertains to the fixed bed bioreactor of Aspects 1-18, wherein the sample substrate comprises a fibrous mesh material.

[0092] Aspect 20 pertains to the fixed bed bioreactor of Aspect 19, wherein the fibrous mesh material is a woven mesh. [0093] Aspect 21 pertains to the fixed bed bioreactor of Aspects 1-19, wherein the sample substrate comprises one or more strings of fiber comprising the same material as the cell culture substrate.

[0094] Aspect 22 pertains to the fixed bed bioreactor of Aspects 1-21, wherein the cell culture substrate comprises a plurality of substrate layers.

[0095] Aspect 23 pertains to the fixed bed bioreactor of Aspect 22, wherein the plurality of substrate layers is arranged in a stacked configuration.

[0096] Aspect 24 pertains to the fixed bed bioreactor of Aspect 23, wherein individual layers of the plurality of substrate layers in the stacked configuration are not separated from each other by any physical barrier.

[0097] Aspect 25 pertains to the fixed bed bioreactor of Aspects 22- 24, wherein the first end of the sample substrate is disposed between two layers of the plurality of substrate layers. [0098] Aspect 26 pertains to the fixed bed bioreactor of Aspect 25, wherein a first portion of the sample substrate comprising the first end is arranged parallel to the layers of the cell culture substrate.

[0099] Aspect 27 pertains to the fixed bed bioreactor of Aspect 25 or 26, wherein a second portion of the sample substrate is arranged perpendicular to the layers of the cell culture substrate.

[00100] Aspect 28 pertains to the fixed bed bioreactor of Aspect 27, wherein the second portion is disposed between the cell culture substrate and a wall of the interior reservoir. [00101] Aspect 29 pertains to the fixed bed bioreactor of Aspects 1-28, wherein the sample substrate comprises a plurality of sample substrates.

[00102] Aspect 30 pertains to the fixed bed bioreactor of Aspect 29, wherein the plurality of substrates comprises sample substrates at different positions at different locations of the cell culture substrate.

[00103] Aspect 31 pertains to the fixed bed bioreactor of Aspect 30, wherein the different locations correspond to different heights of the cell culture substrate along a direction of fluid flow through the cell culture substrate.

[00104] Aspect 32 pertains to the fixed bed bioreactor of Aspects 1-28, wherein the cell culture vessel further comprises an inlet fluidly connected to the interior reservoir and configured for flowing fluid into the cell culture vessel, and an outlet fluidly connected to the interior reservoir and configured for flowing fluid out of the cell culture vessel. [00105] Aspect 33 pertains to the fixed bed bioreactor of Aspect 32, wherein the vessel body comprises a first end, a second end, and a longitudinal axis extending in a direction from the first end to the second end, and wherein the cell culture vessel is configured to flow fluid through the inlet into the interior reservoir, through the cell culture substrate in the interior reservoir, and out through the outlet in a flow direction that is substantially parallel to the longitudinal axis.

[00106] Aspect 34 pertains to the fixed bed bioreactor of Aspect 32 or 33, wherein the interior reservoir is free from any flow channel, flow diverter, or flow recirculation path configured to substantially deviate fluid flow through the cell culture vessel from a direction parallel to the longitudinal axis.

[00107] Aspect 35 pertains to a cell culture substrate bed for a fixed bed bioreactor comprising: a plurality of layers of substrate material in a stacked arrangement, each layer of the substrate material comprising a structurally defined surface for culturing cells thereon and an ordered array of openings through a thickness of the layer; and a sample substrate comprising a first end and a second end opposite the first end, the first end being disposed between two consecutive layers of the plurality of layers of substrate material, wherein the second end of the sample substrate comprises a pull configured to be pulled by an external force and thereby remove the first end from between the two consecutive layers.

[00108] Aspect 36 pertains to the cell culture substrate bed of Aspect 35, wherein the second end is disposed outside of the plurality of layers of substrate material.

[00109] Aspect 37 pertains to the cell culture substrate bed of Aspect 36, wherein the sample substrate comprises a first portion comprising the first end, and a second portion comprising the second end, wherein the first portion runs parallel to the layers of the plurality of layers of substrate material, and the second portion runs perpendicular to the plurality of layers of substrate material.

[00110] Aspect 38 pertains to the cell culture substrate bed of Aspects 35-37, wherein the pull is disposed above a top-most layer of the plurality of substrate layers in the stacked arrangement, or below the bottom-most layer.

[00111] Aspect 39 pertains to a method of sampling a cell culture comprising: providing a cell culture media to an interior of a bioreactor containing a cell culture substrate, the cell culture substrate configured for growing cells thereon and comprising a sample substrate with a first end disposed within the cell culture substrate; during the cell culture, removing the sample substrate from the bioreactor.

[00112] Aspect 40 pertains to the method of Aspect 39, wherein the cell culture substrate comprising a plurality of layers of substrate material in a stacked arrangement, each layer of the substrate material comprising a structurally defined surface for culturing cells thereon and an ordered array of openings through a thickness of the layer.

[00113] Aspect 41 pertains to the method of Aspect 40, wherein the first end is disposed between two consecutive layers of the plurality of layers of substrate material.

[00114] Aspect 42 pertains to the method of Aspects 39-41, further comprising removing a cap from the bioreactor to access a space from which the sample substrate can be pulled out of the bioreactor.

[00115] Aspect 43 pertains to the method of Aspect 42, wherein the removing of the cap is performed without ending a cell culture process within the bioreactor.

[00116] Aspect 44 pertains to the method of Aspect 42 or 43, wherein the removing of the cap is performed aseptically.

[00117] Aspect 45 pertains to the method of Aspects 39-44, further comprising removing a plurality of sample substrates from different locations within the bioreactor.

Definitions

[00118] “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.

[00119] ‘ ‘Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.

[00120] ‘ ‘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.

[00121] “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.

[00122] “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.

[00123] 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.

[00124] 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).

[00125] 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.

[00126] 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.

[00127] 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 fixed bed bioreactor for culturing cells on a cell culture substrate, the fixed bed bioreactor comprising: a cell culture vessel comprising a vessel body defining an interior reservoir, and a sample port disposed on an exterior of the cell culture vessel; a cell culture substrate disposed in the interior reservoir; and a sample substrate at least partially disposed in the interior reservoir, wherein the sample substrate is configured to be removable from the interior reservoir through the sample port.

2. The fixed bed bioreactor of claim 1, wherein the sample substrate is configured to be removable from the interior reservoir through the sample port during an active cell culture.

3. The fixed bed bioreactor of claim 1 or claim 2, wherein the sample substrate is configured to be removable aseptically.

4. The fixed bed bioreactor of any of claims 1-3, wherein the sample substrate is configured to be removable from the interior reservoir without opening the cell culture vessel in a way that would expose the cell culture substrate or disrupt perfusion fluid flow through the cell culture vessel.

5. The fixed bed bioreactor of any of claims 1-4, wherein the sample port comprises a removable cap or closure.

6. The fixed bed bioreactor of any of claims 1-5, wherein the sample substrate comprises a first end disposed in the interior reservoir, the first end being close proximity to the cell culture substrate.

7. The fixed bed bioreactor of claim 6, wherein the first end of the sample substrate is not separated from the cell culture substrate by any physical barrier.

8. The fixed bed bioreactor of claim 6 or claim 7, wherein the first end of the sample substrate is in physical contact with the cell culture substrate.

9. The fixed bed bioreactor of any of claims 6-8, wherein the sample substrate comprises a second end configured to be accessible through the sample port from an exterior of the cell culture vessel.

10. The fixed bed bioreactor of claim 9, wherein the second end is disposed adjacent to the sample port such that the second end can be grasped by a physical implement when the sample port is open and pulled thereby from the sample port.

11. The fixed bed bioreactor of claim 9 or claim 10, wherein the second end is connected to a string, pull-tap, rod, stick, loop, or handle that is disposed adjacent to the sample port such that the string, pull-tap, rod, stick, loop, or handle can be grasped by a physical implement when the sample port is open and pulled thereby from the sample port.

12. The fixed bed bioreactor of any of claims 9-11, wherein the second end is coupled to the cover of the sample port, wherein removal of the cover is configured to remove the sample substrate from the cell culture vessel.

13. The fixed bed bioreactor of any of claims 1-12, wherein the sample substrate comprises the same material as the cell culture substrate.

14. The fixed bed bioreactor of any of claims 1-13, wherein the cell culture substrate comprises a substrate material comprising a plurality openings arranged in a regular and uniform array.

15. The fixed bed bioreactor of any of claims 1-14, wherein the cell culture substrate comprises a physical structure that is regular and uniform.

16. The fixed bed bioreactor of claim 15, wherein the physical structure comprises a plurality of fibers in a predetermined and ordered arrangement.

17. The fixed bed bioreactor of any of claims 1-16, wherein the cell culture substrate comprises a mesh material.

18. The fixed bed bioreactor of claim 17, wherein the mesh material is a woven mesh comprising a plurality of fibers.

19. The fixed bed bioreactor of any of claims 1-18, wherein the sample substrate comprises a fibrous mesh material.

20. The fixed bed bioreactor of claim 19, wherein the fibrous mesh material is a woven mesh.

21. The fixed bed bioreactor of any of claims 1-19, wherein the sample substrate comprises one or more strings of fiber comprising the same material as the cell culture substrate.

22. The fixed bed bioreactor of any of claims 1-21, wherein the cell culture substrate comprises a plurality of substrate layers.

23. The fixed bed bioreactor of claim 22, wherein the plurality of substrate layers is arranged in a stacked configuration.

24. The fixed bed bioreactor of claim 23, wherein individual layers of the plurality of substrate layers in the stacked configuration are not separated from each other by any physical barrier.

25. The fixed bed bioreactor of any of claims 22- 24, wherein the first end of the sample substrate is disposed between two layers of the plurality of substrate layers.

26. The fixed bed bioreactor of claim 25, wherein a first portion of the sample substrate comprising the first end is arranged parallel to the layers of the cell culture substrate.

27. The fixed bed bioreactor of claim 25 or claim 26, wherein a second portion of the sample substrate is arranged perpendicular to the layers of the cell culture substrate.

28. The fixed bed bioreactor of claim 27, wherein the second portion is disposed between the cell culture substrate and a wall of the interior reservoir.

29. The fixed bed bioreactor of any of claims 1-28, wherein the sample substrate comprises a plurality of sample substrates.

30. The fixed bed bioreactor of claim 29, wherein the plurality of substrates comprises sample substrates at different positions at different locations of the cell culture substrate.

31. The fixed bed bioreactor of claim 30, wherein the different locations correspond to different heights of the cell culture substrate along a direction of fluid flow through the cell culture substrate.

32. The fixed bed bioreactor of any of claims 1-28, wherein the cell culture vessel further comprises an inlet fluidly connected to the interior reservoir and configured for flowing fluid into the cell culture vessel, and an outlet fluidly connected to the interior reservoir and configured for flowing fluid out of the cell culture vessel.

33. The fixed bed bioreactor of claim 32, wherein the vessel body comprises a first end, a second end, and a longitudinal axis extending in a direction from the first end to the second end, and wherein the cell culture vessel is configured to flow fluid through the inlet into the interior reservoir, through the cell culture substrate in the interior reservoir, and out through the outlet in a flow direction that is substantially parallel to the longitudinal axis.

34. The fixed bed bioreactor of claim 32 or claim 33, wherein the interior reservoir is free from any flow channel, flow diverter, or flow recirculation path configured to substantially deviate fluid flow through the cell culture vessel from a direction parallel to the longitudinal axis.

35. A cell culture substrate bed for a fixed bed bioreactor comprising: a plurality of layers of substrate material in a stacked arrangement, each layer of the substrate material comprising a structurally defined surface for culturing cells thereon and an ordered array of openings through a thickness of the layer; and a sample substrate comprising a first end and a second end opposite the first end, the first end being disposed between two consecutive layers of the plurality of layers of substrate material, wherein the second end of the sample substrate comprises a pull configured to be pulled by an external force and thereby remove the first end from between the two consecutive layers.

36. The cell culture substrate of claim 35, wherein the second end is disposed outside of the plurality of layers of substrate material.

37. The cell culture substrate of claim 36, wherein the sample substrate comprises a first portion comprising the first end, and a second portion comprising the second end, wherein the first portion runs parallel to the layers of the plurality of layers of substrate material, and the second portion runs perpendicular to the plurality of layers of substrate material.

38. The cell culture substrate of any of claims 35-37, wherein the pull is disposed above a top-most layer of the plurality of substrate layers in the stacked arrangement, or below the bottom-most layer.

39. A method of sampling a cell culture comprising: providing a cell culture media to an interior of a bioreactor containing a cell culture substrate, the cell culture substrate configured for growing cells thereon and comprising a sample substrate with a first end disposed within the cell culture substrate; during the cell culture, removing the sample substrate from the bioreactor.

40. The method of claim 39, wherein the cell culture substrate comprising a plurality of layers of substrate material in a stacked arrangement, each layer of the substrate material comprising a structurally defined surface for culturing cells thereon and an ordered array of openings through a thickness of the layer.

41. The method of claim 40, wherein the first end is disposed between two consecutive layers of the plurality of layers of substrate material.

42. The method of any of claims 39-41, further comprising removing a cap from the bioreactor to access a space from which the sample substrate can be pulled out of the bioreactor.

43. The method of claim 42, wherein the removing of the cap is performed without ending a cell culture process within the bioreactor.

44. The method of claim 42 or claim 43, wherein the removing of the cap is performed aseptically.

45. The method of any of claims 39-44, further comprising removing a plurality of sample substrates from different locations within the bioreactor.