Method and apparatus for sampling fixed bed bioreactors during cell culture

Abstract

A fixed-bed bioreactor for culturing cells on a cell culture substrate and allowing in- process sampling is provided. The bioreactor includes a cell culture vessel with an interior reservoir in which the fixed bed is disposed. A fixed bed disposed in the interior reservoir includes multiple layers of a cell culture substrate, and one or more sample substrates at least partially disposed between at least some of the multiple layers of the cell culture substrate. The vessel has an opening to the interior reservoir that can be closed during a perfusion cell culture process, but is openable by a user to access the sample substrate during a sampling process, and re-closeable after the sampling process. The sample substrate is removable from the interior reservoir during the sampling process without destroying a remainder of the fixed bed so that the cell culture can continue.

Description

Attorney Docket No.: SP24-305METHOD AND APPARATUS FOR SAMPLING FIXED BED BIOREACTORS DURING CELL CULTURECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application Serial No. 63 / 723,435 filed on November 21, 2024, 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 while allowing for the cell culture to continue within the bioreactor after sampling, 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.Attorney Docket No.: SP24-305

[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 or fixed-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 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. ItAttorney Docket No.: SP24-305 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 that 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.” / ri.). 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 users of these systems 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.Attorney Docket No.: SP24-305

[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. 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. Previous methods of sampling substrates for adherent cells have been destructive, inefficient, and / or inconsistent, and were not optimized for reassembly of the bioreactor and resumption of the cell culture process after sampling.

[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. According to embodiments, a fixed-bed bioreactor for culturing cells on a cell culture substrate is provided that has a cell culture vessel having a vessel body defining an interior reservoir and a fixed bed disposed in the interior reservoir. The cell culture vessel has an opening to the interior reservoir, allowing access to the fixed bed in the interior reservoir to performing sampling of the fixed bed and cells thereon. The fixed bed includes multiple layers of a cell culture substrate having a surface for cells to adhere thereto. The fixed bed also includes one or more sample substrates at least partially disposed between at least some of the multiple layers of the cell culture substrate. The opening is configured to be closed during a perfusion cell culture process, openable by a user to access the sample substrate during a sampling process, and re-closeable after the sampling process. The sample substrate removable from the interior reservoir during the samplingAttorney Docket No.: SP24-305 process without destroying a remainder of the fixed bed, so that cell culture can optionally be continued after one or more samples are taken from the fixed bed.

[0014] In aspects of embodiments, the multiple layers of the cell culture substrate include a plurality of rolled substrate sheets arranged as a plurality of cylindrical substrate sections in a stacked arrangement. The one or more sample substrates can be disposed between consecutive cylindrical substrate sections in the stacked arrangement.

[0015] In aspects of embodiments, the multiple layers of the cell culture substrate include a plurality of substrate disks in a stacked arrangement. The one or more sample substrates are disposed between consecutive substrate disks of the plurality of substrate disks.

[0016] In embodiments, the fixed-bed bioreactor further includes a guide rod disposed in the interior reservoir, wherein the fixed bed has a bed opening sized and shaped to fit around the guide rod. The guide rod can, as an aspect of embodiments, include a plurality of guide rod sections moveably connected to one another such that the guide rod can be extended or contracted in a telescoping manner. The fixed bed can include a plurality of bed sections, at least a portion of which are coupled to at least a portion of the plurality of guide rod sections, and the portion of the plurality of bed sections that are coupled to the portion of the plurality of guide rod sections can move with the portion of the plurality of guide rod sections when extended or contracted. At least a portion of the one or more sample substrates is disposed between at least two of the plurality of bed sections. The one or more sample substrates can have a cutout comprising a length extending from the guide rod to an outer edge of the one or more sample substrates.Embodiments of this disclosure include a method of sampling a cell culture. The method includes providing a bioreactor containing an interior reservoir and a fixed bed disposed in the interior reservoir. The fixed bed includes a cell culture substrate and one or more sample substrates. The cell culture substrate have a surface for growing cells thereon. The method further includes culturing cells in the fixed bed while perfusing cell culture media through the interior reservoir; sampling the cell culture by removing at least a portion of the one or more sample substrate; and after sampling, continuing the culturing of the cells in the fixed bed.

[0017] Additional aspects of the present disclosure will be set forth, in part, in the detailed description, figures and any claims which follow, and in part will be derived from the detailed description, or can be learned by practice of the disclosure. It is to be understoodAttorney Docket No.: SP24-305 that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure as disclosed.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] A more complete understanding of the present disclosure may be had by reference to the following detailed description when taken in conjunction with the accompanying drawings, which are described in brief below.

[0019] Figure 1 shows an exploded perspective view of a fixed bed bioreactor system, according to embodiments of this disclosure.

[0020] Figure 2A shows a perspective view of a fixed bed bioreactor system with a stacked rolls of substrate of a first size, according to one or more embodiments.

[0021] Figure 2B shows a perspective view of a fixed bed bioreactor system with a stacked rolls of substrate of a second, larger size, according to one or more embodiments.

[0022] Figure 2C shows a perspective view of a fixed bed bioreactor system with a stacked rolls of substrate of a third, larger size, according to one or more embodiments.

[0023] Figure 3 shows a perspective view of a fixed bed bioreactor similar to Figures 2A- 2C that is opened to reveal a sampling substrate disk, according to one or more embodiments.

[0024] Figure 4 shows a perspective view of a fixed bed bioreactor similar to Figures 2A-3 that is opened to reveal multiple sampling substrate disks and expanded alignment rod, according to one or more embodiments.

[0025] Figure 5 shows a perspective view of a fixed bed bioreactor having stacked substrate disks that is opened to reveal multiple sampling substrate disks and expanded alignment rod, according to one or more embodiments.

[0026] Figure 6 shows a cross-section view of a telescoping alignment rod of a bioreactor, according to embodiments.

[0027] Figure 7 shows a cross-section view of a tension-loaded telescoping alignment rod, according to embodiments.

[0028] Figure 8 shows a cross-section view of a telescoping alignment rod with tapered side wall section, according to embodiments.

[0029] Figure 9A shows a perspective view of a substrate sampling disk with pull tab, according to embodiments.Attorney Docket No.: SP24-305

[0030] Figure 9B shows a perspective view of a substrate sampling disk with multiple pull tabs, according to embodiments.

[0031] Figure 9C shows a perspective view of multiple substrate sampling disks with pull tabs in a stack, according to embodiments.DETAILED DESCRIPTION

[0032] 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.

[0033] Given the drawbacks and limitations of many adherent-cell-based bioreactors, a removable sample substrate that can be removed without interfering with cell culture is desirable for periodic examination of cell confluency and status during the culture using fixed bed bioreactors. Given the above-discussed potential for irregularities in existing bioreactor substrates, this sampling and monitoring can prove even more valuable if it can be performed at various positions within the bioreactor without disrupting the cell culture. Accordingly, embodiments of this disclosure include fixed-bed bioreactors and / or fixed-bed substrate assemblies for cell culture that enable non-destructive sampling of sampling substrate material from the fixed bed in a way that allows for the cell culture to continue after the sample has been collected, as well as methods of performing the same. In addition, embodiments include the aforementioned bioreactors, substrate assemblies, and methods that allow such sampling with reduced negative impact to the surrounding cell culture substate and the ongoing cell culture process. As such, embodiments disclosed herein allow for simple and effective sampling and resumption of the cell culture process for adherent cells in a fixed-bed bioreactor.

[0034] 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 104 as a cell culture chamber within the bioreactor vessel 102. Within the cell culture chamber is a fixed-bed substrate 106. According to aspects of embodiments, theAttorney Docket No.: SP24-305 fixed-bed substrate 106 can take different forms and / or arrangements within the cell culture chamber 104, as discussed herein. For example, in Figure 1, the fixed-bed substrate 106 is made from a stack of substrate layers or disks 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 fixed-bed 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 monolithic 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.

[0035] The bioreactor vessel 100 has an inlet 110 at one end for the input of media, cells, and / or nutrients into the interior reservoir 104, and an outlet 112 at the opposite end for removing media, cells, or cell products from the interior reservoir 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 interior reservoir 104. For example, inlet 110 may be used for flowing media or cells into the interior reservoir 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.

[0036] 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 spaceAttorney Docket No.: SP24-305 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.

[0037] In 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).

[0038] 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.

[0039] 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 interior reservoir 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. In embodiments,Attorney Docket No.: SP24-305 such member can be a guide rod. The guide rod can be used to support and / or align layers of the substrate within the bioreactor during a cell culture. Aspects of embodiments include the substrate fixed bed having a bed opening through which the guide rod is disposed. 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.

[0040] To enable sampling on the bioreactor 100 of Figure 1 without disrupting the cell culture, embodiments herein provide substrate assemblies and fixed-bed bioreactors 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.

[0041] Figures 2A-2C show an aspect of embodiments of bioreactors 200A, 200B, 200C according to this disclosure that enable a scalable cell culture platform. An aspect of the embodiments as depicted is the use of a fixed bed 202A, 202B, 202C made of cylindrical rolled substrates 204 where the rolled substrates 204 are rolled about a central axis that is parallel to or aligned with the longitudinal axis A of the bioreactor vessel. Different cell culture scales can be achieved by varying the height h of the fixed bed 202A-202C, even with the diameter D of the cell culture vessel remains the same. Aspects of embodiments of this disclosure include bioreactors with uniform fluid flow through the fixed bed. In embodiments, the height h of the fixed bed can be varied by rolling substrates of different widths (where the width of the substrate equates to the height of the fixed bed). However, when using rolled substrates for the fixed bed 202A-202C, as shown in Figures 2A-2C, tension of the rolled substrate can have an impact on the geometric consistency, and thus fluid flow uniformity, in the fixed bed. It was discovered that using substrates of different widths to achieve different heights h of the fixed bed could easily result in different tensions in the rolled fixed bed, or that achieving consistent tension is difficult. Thus, in embodiments,Attorney Docket No.: SP24-305 the different heights h of the fixed bed 202A-202C can be achieved by using one or more rolled substates in a stacked arrangement, as shown in Figures 2A-2C, where the width w of each substrate is the same. To vary the height of the fixed bed, different numbers of rolled substrates (each having the same width w) can be stacked until the desired fixed bed height h is reached. This has the advantage of simplified manufacturing and assembly processes. Alternatively, substrates of different widths can be used, ideally with the tension in the rolls controlled to achieve consistent geometries in the fixed beds. However, an additional advantage of using stacks of rolled substrates with the same width w is that the space 206 between the individual rolled substrates can be used to store sample substrates, according to embodiments of this disclosure. The cell culture vessel body can be any suitable and biocompatible container capable of housing a cell culture. In embodiments, the cell culture vessel is an extruded or molded polymer tube, but may also be metallic (e.g., stainless steel, aluminum), glass, or ceramic. The ends of the cell culture vessel may be enclosed or capped with an upper end cap 210 and a lower end cap 212A, 212B, 212C. The lower end cap 212A- C has an inlet 214 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. The upper end cap 210 includes an outlet 216 that may also be connected to tubing or other fluid pathway for fluid exiting the interior reservoir. Each of the inlet 214 and outlet 216 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 216 may be fluidly connected to a different external container than the inlet 214 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 F of media is from the bottom to the top (as shown in Figure 2A) of the bioreactor, but the flow may also be in the reverse direction.

[0042] The upper end cap 210 and the lower end cap 212A-C may have similar heights or different. In embodiments, one of them (e.g., the upper end cap 210 in the examples shown in Figures 2A-2C) can be the same height regardless of the scale of the fixed bed, while the other (e.g., the lower end cap 212A-212C in the examples of Figures 2A-2C) may have a variable height to account for different fixed bed heights. The upper and lower end caps are separated by a line of separation 218 around a periphery (e.g., circumference) of the bioreactor vessel. The upper and lower end caps may be releasably sealed to preventAttorney Docket No.: SP24-305 contamination from entering the interior reservoir and to prevent fluid from exiting the interior reservoir through the line of separation 218. The upper and lower end caps may have mating faces (e.g., flanges 220) that can be pressed together to ensure a seal. For example, clamps, nuts and bolts, press fittings, or other known solutions can be used to hold the two halves together.

[0043] According to embodiments, having two or more of these rolled substrates are stacked in the fixed bed to allow for multiple sample substrates in the packed bed, either in the space between consecutive rolled substrates or on the top of the topmost or bottom of the bottommost rolled substrate. Having multiple sample substrates in different vertical locations throughout the fixed bed can provide valuable insight to users in terms of providing sampling locations from different heights of the bed. Embodiments also include fixed beds made of multiple sections or rolls of substrate of different heights. The heights of each fixed bed section (or the width of the substrate in each roll) can be customized so that the openings between the fixed bed sections, and thus the sample substrates, are located at certain desired heights within the bioreactor where samples are desired for a given cell culture.

[0044] Figure 3 shows an embodiment of a bioreactor 300 that is open to reveal a sample substrate 302 in the space 304 between consecutive rolled substrates 306A, 306B. A guide rod 308 inserted through one or more rolled substrates (e.g., rolled substrate 306A) can assist in lifting the rolled substate above the sample substrate 302 to ease removal of the sample substate 302. In the example of Figure 3, tweezers 310 are shown to indicate the removal of the sample substrate 302. Sampling substates, such as the sampling substrate 302 in disk form in Figure 3, can provide both radial and vertical variability indications without the need to unroll the rolled substrate. Unrolling the substrate can be effective for end-of-run destructive sampling, but the embodiments of this disclosure, such as that shown in Figure 3, allow for the remaining substate to be undisturbed and that substrate can continue to be used in further cell culture after sampling. This helps avoid the difficulties and uncertainties inherent in more involved disassembly (e.g., unrolling the substrate) that would otherwise be required, and simplifies re-assembly of the bioreactor for further cell culture processing, if desired.

[0045] As shown in Figure 4, embodiments of this disclosure include bioreactors 400 that use a guide rod 402 to assist in sampling the cell culture. In particular, the guide rod 402 can be a telescoping rod having multiple guide rod sections 404 moveably coupled to each otherAttorney Docket No.: SP24-305 and able to move relative to each other in a telescoping fashion. For example, when the top of the guide rod 402 is pulled in the direction P, the guide rod 402 extends or telescopes out in the direction shown (P). Embodiments include aspects where sections of the fixed bed are coupled to the guide rod sections 404 such that the fixed bed sections move with the guide rod section. Upon applying a load, the core of both the guide rod and the fixed bed can elongate exposing the areas between the rolled substrates where the sample substrates can be positioned for easy removal. Thus, as the guide rod 402 extends, the space between consecutive substrates of the fixed bed can increase, easing removal of the sample substrates from those spaces. In addition, one or more sample substrates 406 can have a cutout 408 sized and shaped to allow the guide rod 402 to easily pass through the cutout 408 when the sample substrate is pulled in a direction R. That is, the cutout 408 can have a width greater than or equal to a diameter of the guide rod 402 where it passes through the sample substrate. A length of the cutout 408 can extend from the guide rod 402 to an outer edge of the sample substrate 406.

[0046] Embodiments of this disclosure are not limited to using the guide rod or the telescoping guide rod of Figure 4 with a rolled substrate fixed bed. As shown in Figure 5, the fixed bed can include fixed bed sections 502 made of stacked layers 504 of substrate material with sample substrates 506 therebetween. The examples as shown in Figures 4 and 5 are otherwise the same and so additional features of Figure 5 are not labeled or discussed here for brevity.

[0047] Figures 6, 7, and 8 show additional features of the telescoping guide rod, according to embodiments. With reference to Figure 6, the guide rod 600 may have a plurality of guide rod sections 602 that have hollow cores 604 with nesting features to enable vertical sliding. In embodiments, the guide rod sections are prevented from accidental separation by stops 606 or other features built into the guide rod sections that prevent relative motion of the sections past a maximum extension point. As the guide rod sections 602 are separated, the sample substrate 608 is revealed between the consecutive fixed bed substrate sections 610. As shown in Figure 7, embodiments can include the telescoping guide rod 700 can have a retraction force built in to contract the guide rod when an external force is not acting on it. For example, a spring 702 can connected two guide rod sections. The spring can be in an extended position when the guide rod sections are extended or moved apart from one another. As shown in Figure 8,Attorney Docket No.: SP24-305 embodiments also include a telescoping guide rod 800 with a tapered interface 802. In embodiments, the guide rod 800 can use plastic deformation to drive retraction.

[0048] With reference now to Figures 9A, 9B, and 9C, according to embodiments, the sample substrates 900 can include one or more pull tabs 902, which are designed to be gripped and pulled by a user or an implement controlled by a user or computerized controller, to remove the sample substate 900 from the fixed bed. The pull tabs 902 extend past an edge of the fixed bed so that they can be easily gripped. As shown, the sample substrates 900 can have a cutout 904 or slit on at one side of the sample substrate 900 to enable easy removal of the sample substrate from around a guide rod that is inserted through the guide rod opening 906 when the sample substrate 900 is in the fixed bed. In embodiments, as shown in Figure 9A, for example, the cutout 904 can be on an opposite side of the sample substrate 900 from the pull tab 902 so that, as the sample substrate 900 is pulled in the direction in which the pull tab 902 protrudes, the guide rod can easily move relatively in the opposite direction through the cutout 904. In embodiments, as shown in Figure 9B, the sample substrate 900 can have multiple pull tabs 902. Multiple pull tabs 902 can be included for redundancy or be used for identification within the vertical stack. Pull tabs can also be laser marked or otherwise labeled for identification. For example, the sample substrates can be labeled to indicate their relative position within the fixed bed so that the position from which they were taken is easily discerned after removal. For example, the sample substrates can be labeled with a descriptive name (e.g., “top,” “middle,” and “bottom”) or an alphanumeric code that indicates relative positions (e.g., “I,” “2,” “3,” “4,” etc.) or a barcode or QR code. As shown in Figure 9C, according to an aspect of embodiments, multiple sample substrates 900 can be stacked together in a sample stack 901 at one location with the fixed bed. An advantage of three or more stacked disks for sampling is that the “exterior” substate disks (e.g., the topmost and bottommost substrate layers) can be discarded if the cells on them might have been affected by the sample removal process or contact with the rolled fixed bed, such that the remaining layers in the interior of the sample stack 901 are more representative of the cell culture within the fixed bed.

[0049] As described herein, according to embodiments, the fixed bed containing a cell culture substrate is provided within the interior reservoir of the bioreactor vessel. The substrate has a surface upon which adherent or semi-adherent cells can grow. The fixed bed substrate may be a monolithic structure (e.g., a foam, 3D-printed substate, or fused substrate)Attorney Docket No.: SP24-305 or may include multiple substrates arranged within the interior reservoir, such as multiple layers of thin substrate material. In some embodiments, the substrate material is made of woven polymer fibers, as described herein. According to embodiments, the sample substrate is preferably in close proximity to, or touching, the cell culture substrate. As an aspect of embodiments, the sample substrate is not separated from the cell culture substrate by any physical barrier and / or is in direct physical contact with the cell culture substrate.

[0050] According to embodiments, the sample substrate can be removed from the fixed bed through the sample port in a side wall of the bioreactor vessel without disrupting the cell culture within the bioreactor. The sample port 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 will require stopping the cell culture and opening the bioreactor, thus avoiding potential contamination.

[0051] The position of the sample substrate can be predetermined based on a desired location from which a sample is desired. In embodiments, multiple sample substrates are provided to enable sampling from different locations within the fixed bed. For example, sample substrates may be provided in at least 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.

[0052] The removable sample substrate 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 is, according to embodiments, of the same general shape and dimensions as a layer of the cell culture substrate. As an aspect of embodiments, removal of the sample substrate will not result in a noticeable gap in the remaining fixed bed so that cell culture can continue after sampling with the same level of uniformity in the fixed bed structure and fluid flow performance. As an aspect of some embodiments, the removable sample substrates preferably have the same surface area (e.g., 1 cm2, 5 cm2, 10 cm2, 50 cm2) for cell attachment as each other and / or as a similarly sized substrate layer, 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 can be subject to examination for any desired performance criteria, including, for example, cell confluencyAttorney Docket No.: SP24-305 using microscopy, or cell counting after trypsin treatment, or cellular status using fluorescence staining.

[0053] As discussed herein, the sample substrate is preferably the same material as the cell culture substrate, so that the sample provided by the sample substrate is representative to the culture on the cell culture substrate. The cell culture substrate can be, in embodiments, a substrate material having a plurality of openings arranged in a regular and uniform array. The cell culture substrate can have a physical structure that is regular and uniform. The physical structure can include 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, or a material where the physical structure is not uniform and regular.

[0054] As discussed herein, according to embodiments, the cell culture substrate can include a plurality of substrate layers. The plurality of substrate layers can be arranged in a stacked configuration, with the stack height being in a direction of the height of the interior reservoir, or in the bulk flow direction for fluid flowing therethrough. Multiple sample substrates can be provided at different locations within the stacked substrate layers. Any number of sample substrates can be employed in a single fixed bed, depending on how many samples need to be collected.

[0055] According to embodiments, individual layers of the plurality of substrate layers in the stacked configuration are not separated from each other by any physical barrier. In this sense, the sample substrates 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.

[0056] According to embodiments of this disclosure, the vessel body 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 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. As an aspect of embodiments, the interior reservoir is free from any flow channel, flow diverter, or flow recirculation path that wouldAttorney Docket No.: SP24-305 substantially deviate fluid flow through the cell culture vessel or interior reservoir 204 from a direction parallel to the longitudinal axis.

[0057] 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.

[0058] 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.Attorney Docket No.: SP24-305

[0059] 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 1016to 1018viral genomes (VG) per batch.

[0060] 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 ofAttorney Docket No.: SP24-305 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.

[0061] 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.

[0062] 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.Attorney Docket No.: SP24-305

[0063] In embodiments, the sample substrate is made of the same material as the cell culture substrate. In embodiments, a physical structure of the sample 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 sample 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 sample 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.

[0064] 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 IO14viral genomes per batch, greater than about IO15viral genomes per batch, greater than about IO16viral 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, production is about 1015to about 1018or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be about I015to about IO16viral genomes or batch, or about IO16to about IO19viral genomes per batch, or about 1016- 1018viral genomes per batch, or about IO17to about IO19viral genomes per batch, or about I018to about IO19viral genomes per batch, or about I018or more viral genomes per batch.

[0065] 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% areAttorney Docket No.: SP24-305 viable. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0066] 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.

[0067] 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 ofAttorney Docket No.: SP24-305 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).

[0068] 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.

[0069] 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 andAttorney Docket No.: SP24-305 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 substrate can also be characterized in terms of porosity, as discussed in the Examples herein.

[0070] 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).

[0071] 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.

[0072] 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,Attorney Docket No.: SP24-305 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.

[0073] 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.

[0074] 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.

[0075] 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. DueAttorney Docket No.: SP24-305 to contamination concerns, the vessel can be a single-use vessel that can be disposed of after use.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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 fromAttorney Docket No.: SP24-305 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.

[0080] 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.

[0081] 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.

[0082] Illustrative Implementations

[0083] 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 areAttorney Docket No.: SP24-305 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.

[0084] 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 comprising an opening to the interior reservoir; a fixed bed disposed in the interior reservoir, the fixed bed comprising: multiple layers of a cell culture substrate, the cell culture substrate comprising a surface configured for cells to adhere thereto; and one or more sample substrates at least partially disposed between at least some of the multiple layers of the cell culture substrate, wherein the opening is configured to be closed during a perfusion cell culture process, openable by a user to access the sample substrate during a sampling process, and re-closeable after the sampling process, and wherein the sample substrate is configured to be removable from the interior reservoir during the sampling process without destroying a remainder of the fixed bed.

[0085] Aspect 2 pertains to the fixed bed bioreactor of Aspect 1, wherein the opening comprises at least one of: a removable cap on a top side or a bottom side of the cell culture vessel; a port in a sidewall of the cell culture vessel; and a line of separation in at least one of the bottom side, the top side, and the sidewall of the cell culture vessel separating the cell culture vessel into at least two vessel components.

[0086] Aspect 3 pertains to the fixed bed bioreactor of Aspect 2, wherein the opening comprises the line of separation separating the cell culture vessel into at least two vessel components, and wherein the at least two vessel components comprise mating closure faces configured to seal the interior reservoir when the opening is closed.

[0087] Aspect 4 pertains to the fixed bed bioreactor of Aspect 3, wherein the mating closure faces comprise flanges on the at least two vessel components.

[0088] Aspect 5 pertains to the fixed bed bioreactor of any one of Aspects 1 -4, wherein the one or more sample substrates comprises a maximum diameter that is 90% or more of a maximum diameter of the cell culture substrate.

[0089] Aspect 6 pertains to the fixed bed bioreactor of Aspect 5, wherein the maximum diameter of the one or more sample substrates is equal to the maximum diameter of the cell culture substrate.Attorney Docket No.: SP24-305

[0090] Aspect 7 pertains to the fixed bed bioreactor of any one of Aspects 1 -6, wherein the multiple layers of the cell culture substrate comprise a plurality of rolled substrate sheets arranged as a plurality of cylindrical substrate sections in a stacked arrangement.

[0091] Aspect 8 pertains to the fixed bed bioreactor of Aspect 7, wherein the one or more sample substrates are disposed between consecutive cylindrical substrate sections in the stacked arrangement.

[0092] Aspect 9 pertains to the fixed bed bioreactor of Aspect 8, wherein the one or more sample substrates comprises a plurality of sample substrates, with a least some of the plurality of sample substrate disposed between different consecutive cylindrical substrate sections.

[0093] Aspect 10 pertains to the fixed bed bioreactor of any one of Aspects 1-6, wherein the multiple layers of the cell culture substrate comprise a plurality of substrate disks in a stacked arrangement.

[0094] Aspect 11 pertains to the fixed bed bioreactor of Aspect 10, wherein the one or more sample substrates are disposed between consecutive substrate disks of the plurality of substrate disks.

[0095] Aspect 12 pertains to the fixed bed bioreactor of Aspect 11, wherein the one or more sample substrates comprises a plurality of sample substrates, with at least some of the plurality of sample substrates disposed between different consecutive substrate disks of the plurality of substrate disks.

[0096] Aspect 13 pertains to the fixed bed bioreactor of any one of Aspects 1-12, further comprising a guide rod disposed in the interior reservoir, wherein the fixed bed comprises a bed opening sized and shaped to fit around the guide rod.

[0097] Aspect 14 pertains to the fixed bed bioreactor of Aspect 13, wherein the guide rod is configured to restrain at least one of rotational and translational motion of the fixed bed within the interior reservoir during the cell culture process.

[0098] Aspect 15 pertains to the fixed bed bioreactor of Aspect 13 or Aspect 14, wherein the guide rod has a cross section that is a circle, a semi-circle, a rhombus, and a cross.

[0099] Aspect 16 pertains to the fixed bed bioreactor of Aspects 13-15, wherein the guide rod extends in a longitudinal direction of the cell culture vessel.Attorney Docket No.: SP24-305

[0100] Aspect 17 pertains to the fixed bed bioreactor of Aspect 16, wherein the longitudinal direction is parallel to a direction running from an inlet to an outlet of the interior reservoir.

[0101] Aspect 18 pertains to the fixed bed bioreactor of Aspects 13-17, wherein the guide rod is disposed in a center of the fixed bed.

[0102] Aspect 19 pertains to the fixed bed bioreactor of Aspects 13-18, wherein the guide rod comprises a plurality of guide rod sections moveably connected to one another such that the guide rod can be extended or contracted in a telescoping manner.

[0103] Aspect 20 pertains to the fixed bed bioreactor of Aspect 19, wherein the plurality of guide rod sections comprises a locking feature that limits a maximum displacement of the guide rod sections when extended.

[0104] Aspect 21 pertains to the fixed bed bioreactor of Aspect 19 or Aspect 20, wherein the fixed bed comprises a plurality of bed sections, wherein at least a portion of the plurality of bed sections are coupled to at least a portion of the plurality of guide rod sections, and wherein the portion of the plurality of bed sections that are coupled to the portion of the plurality of guide rod sections move with the portion of the plurality of guide rod sections when extended or contracted.

[0105] Aspect 22 pertains to the fixed bed bioreactor of Aspect 21, wherein at least a portion of the one or more sample substrates is disposed between at least two of the plurality of bed sections.

[0106] Aspect 23 pertains to the fixed bed bioreactor of any one of Aspects 19-22, wherein the guide rod is put in tension when at least a portion of plurality of guide rod sections are extended.

[0107] Aspect 24 pertains to the fixed bed bioreactor of Aspect 23, wherein at least a portion of the plurality of guide rod sections are connected by a spring, the spring being configured to be in tension when the guide rod is extended.

[0108] Aspect 25 pertains to the fixed bed bioreactor of any one of Aspects 13-24, wherein the one or more sample substrates comprises a cutout comprising a length extending from the guide rod to an outer edge of the one or more sample substrates.

[0109] Aspect 26 pertains to the fixed bed bioreactor of Aspect 25, wherein the cutout further comprises a width equal to or greater than a diameter of the guide rod.Attorney Docket No.: SP24-305

[0110] Aspect 27 pertains to the fixed bed bioreactor of any one of Aspects 1-26, wherein the one or more sample substrates comprises a pull tab that protrudes past a periphery of the multiple layers of the cell culture substrate in the fixed bed, the pull tab comprising a grip surface configured to be gripped when pulling the one or more sample substrates from the fixed bed.

[0111] Aspect 28 pertains to the fixed bed bioreactor of any one of Aspects 1-27, wherein the one or more sample substrates comprises a sample substrate stack comprising at least three consecutive sample substrates.

[0112] Aspect 29 pertains to the fixed bed bioreactor of any one of Aspects 1-28, wherein the one or more sample substrates are not separated from the cell culture substrate by any physical barrier.

[0113] Aspect 30 pertains to the fixed bed bioreactor of any one of Aspects 1-29, wherein the one or more sample substrates are in physical contact with the cell culture substrate.

[0114] Aspect 31 pertains to the fixed bed bioreactor of any one of Aspects 1-30, wherein the one or more sample substrates comprise a same material as the cell culture substrate.

[0115] Aspect 32 pertains to the fixed bed bioreactor of any one of Aspects 1-31, wherein the cell culture substrate comprises a substrate material comprising a plurality openings arranged in a regular and uniform array.

[0116] Aspect 33 pertains to the fixed bed bioreactor of any one of Aspects 1-32, wherein the cell culture substrate comprises a physical structure that is regular and uniform.

[0117] Aspect 34 pertains to the fixed bed bioreactor of Aspect 33, wherein the physical structure comprises a plurality of fibers in a predetermined and ordered arrangement.

[0118] Aspect 35 pertains to the fixed bed bioreactor of any one of Aspects 1-34, wherein the cell culture substrate comprises a mesh material.

[0119] Aspect 36 pertains to the fixed bed bioreactor of Aspect 35, wherein the mesh material is a woven mesh comprising a plurality of fibers.

[0120] Aspect 37 pertains to the fixed bed bioreactor of any one of Aspects 1-36, wherein the sample substrate comprises a fibrous mesh material.

[0121] Aspect 38 pertains to the fixed bed bioreactor of Aspect 37, wherein the fibrous mesh material is a woven mesh.

[0122] Aspect 39 pertains to the fixed bed bioreactor of any one of Aspects 1-38, wherein the cell culture vessel further comprises an inlet fluidly connected to the interior reservoir andAttorney Docket No.: SP24-305 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.

[0123] Aspect 40 pertains to the fixed bed bioreactor of Aspect 39, 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 fixed bed in the interior reservoir, and out through the outlet in a flow direction that is substantially parallel to the longitudinal axis.

[0124] Aspect 41 pertains to the fixed bed bioreactor of Aspect 39 or Aspect 40, 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.

[0125] Aspect 42 pertains to a method of sampling a cell culture comprising: providing a bioreactor containing an interior reservoir and a fixed bed disposed in the interior reservoir, the fixed bed comprising a cell culture substrate and one or more sample substrates, the cell culture substrate comprising a surface configured for growing cells; culturing cells in the fixed bed while perfusing cell culture media through the interior reservoir; sampling the cell culture by removing at least a portion of the one or more sample substrate; and after sampling, continuing the culturing of the cells in the fixed bed.

[0126] Aspect 43 pertains to a method of sampling a cell culture comprising: providing a fixed-bed bioreactor according to any one of Aspects 1-41; culturing cells in the fixed bed while perfusing cell culture media through the interior reservoir; sampling the cell culture by removing at least a portion of the one or more sample substrate; and after sampling, continuing the culturing of the cells in the fixed bed.Definitions

[0127] “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.

[0128] ‘ ‘Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.Attorney Docket No.: SP24-305

[0129] ‘ ‘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.

[0130] “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.

[0131] “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.

[0132] 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.

[0133] 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).

[0134] 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.Attorney Docket No.: SP24-305

[0135] 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.

[0136] 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

Attorney Docket No.: SP24-305What 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 comprising an opening to the interior reservoir; a fixed bed disposed in the interior reservoir, the fixed bed comprising: multiple layers of a cell culture substrate, the cell culture substrate comprising a surface configured for cells to adhere thereto; and one or more sample substrates at least partially disposed between at least some of the multiple layers of the cell culture substrate, wherein the opening is configured to be closed during a perfusion cell culture process, openable by a user to access the sample substrate during a sampling process, and re-closeable after the sampling process, and wherein the sample substrate is configured to be removable from the interior reservoir during the sampling process without destroying a remainder of the fixed bed.

2. The fixed-bed bioreactor of claim 1, wherein the opening comprises at least one of: a removable cap on a top side or a bottom side of the cell culture vessel; a port in a side wall of the cell culture vessel; and a line of separation in at least one of the bottom side, the top side, and the sidewall of the cell culture vessel separating the cell culture vessel into at least two vessel components.

3. The fixed-bed bioreactor of claim 2, wherein the opening comprises the line of separation separating the cell culture vessel into at least two vessel components, and wherein the at least two vessel components comprise mating closure faces configured to seal the interior reservoir when the opening is closed.

4. The fixed-bed bioreactor of claim 3, wherein the mating closure faces comprise flanges on the at least two vessel components.Attorney Docket No.: SP24-3055. The fixed-bed bioreactor of any one of claims 1-4, wherein the one or more sample substrates comprises a maximum diameter that is 90% or more of a maximum diameter of the cell culture substrate.

6. The fixed-bed bioreactor of claim 5, wherein the maximum diameter of the one or more sample substrates is equal to the maximum diameter of the cell culture substrate.

7. The fixed-bed bioreactor of any one of claims 1-6, wherein the multiple layers of the cell culture substrate comprise a plurality of rolled substrate sheets arranged as a plurality of cylindrical substrate sections in a stacked arrangement.

8. The fixed-bed bioreactor of claim 7, wherein the one or more sample substrates are disposed between consecutive cylindrical substrate sections in the stacked arrangement.

9. The fixed-bed bioreactor of claim 8, wherein the one or more sample substrates comprises a plurality of sample substrates, with a least some of the plurality of sample substrate disposed between different consecutive cylindrical substrate sections.

10. The fixed-bed bioreactor of any one of claims 1-6, wherein the multiple layers of the cell culture substrate comprise a plurality of substrate disks in a stacked arrangement.

11. The fixed-bed bioreactor of claim 10, wherein the one or more sample substrates are disposed between consecutive substrate disks of the plurality of substrate disks.

12. The fixed-bed bioreactor of claim 11, wherein the one or more sample substrates comprises a plurality of sample substrates, with at least some of the plurality of sample substrates disposed between different consecutive substrate disks of the plurality of substrate disks.

13. The fixed-bed bioreactor of any one of claims 1-12, further comprising a guide rod disposed in the interior reservoir,Attorney Docket No.: SP24-305 wherein the fixed bed comprises a bed opening sized and shaped to fit around the guide rod.

14. The fixed-bed bioreactor of claim 13, wherein the guide rod is configured to restrain at least one of rotational and translational motion of the fixed bed within the interior reservoir during the cell culture process.

15. The fixed-bed bioreactor of claim 13 or claim 14, wherein the guide rod has a cross section that is a circle, a semi-circle, a rhombus, and a cross.

16. The fixed-bed bioreactor of any one of claims 13-15, wherein the guide rod extends in a longitudinal direction of the cell culture vessel.

17. The fixed-bed bioreactor of claim 16, wherein the longitudinal direction is parallel to a direction running from an inlet to an outlet of the interior reservoir.

18. The fixed-bed bioreactor of any one of claims 13-17, wherein the guide rod is disposed in a center of the fixed bed.

19. The fixed-bed bioreactor of any one of claims 13-18, wherein the guide rod comprises a plurality of guide rod sections moveably connected to one another such that the guide rod can be extended or contracted in a telescoping manner.

20. The fixed-bed bioreactor of claim 19, wherein the plurality of guide rod sections comprises a locking feature that limits a maximum displacement of the guide rod sections when extended.

21. The fixed-bed bioreactor of claim 19 or claim 20, wherein the fixed bed comprises a plurality of bed sections, wherein at least a portion of the plurality of bed sections are coupled to at least a portion of the plurality of guide rod sections, andAttorney Docket No.: SP24-305 wherein the portion of the plurality of bed sections that are coupled to the portion of the plurality of guide rod sections move with the portion of the plurality of guide rod sections when extended or contracted.

22. The fixed-bed bio reactor of claim 21, wherein at least a portion of the one or more sample substrates is disposed between at least two of the plurality of bed sections.

23. The fixed-bed bioreactor of any one of claims 19-22, wherein the guide rod is put in tension when at least a portion of plurality of guide rod sections are extended.

24. The fixed-bed bioreactor of claim 23, wherein at least a portion of the plurality of guide rod sections are connected by a spring, the spring being configured to be in tension when the guide rod is extended.

25. The fixed-bed bioreactor of any one of claims 13-24, wherein the one or more sample substrates comprises a cutout comprising a length extending from the guide rod to an outer edge of the one or more sample substrates.

26. The fixed-bed bioreactor of claim 25, wherein the cutout further comprises a width equal to or greater than a diameter of the guide rod.

27. The fixed-bed bioreactor of any one of claims 1-26, wherein the one or more sample substrates comprises a pull tab that protrudes past a periphery of the multiple layers of the cell culture substrate in the fixed bed, the pull tab comprising a grip surface configured to be gripped when pulling the one or more sample substrates from the fixed bed.

28. The fixed-bed bioreactor of any one of claims 1-27, wherein the one or more sample substrates comprises a sample substrate stack comprising at least three consecutive sample substrates.

29. The fixed-bed bioreactor of any one of claims 1-28, wherein the one or more sample substrates are not separated from the cell culture substrate by any physical barrier.Attorney Docket No.: SP24-30530. The fixed-bed bioreactor of any one of claims 1-29, wherein the one or more sample substrates are in physical contact with the cell culture substrate.

31. The fixed-bed bioreactor of any one of claims 1-30, wherein the one or more sample substrates comprise a same material as the cell culture substrate.

32. The fixed bed bioreactor of any of claims 1-31, wherein the cell culture substrate comprises a substrate material comprising a plurality openings arranged in a regular and uniform array.

33. The fixed bed bioreactor of any of claims 1-32, wherein the cell culture substrate comprises a physical structure that is regular and uniform.

34. The fixed bed bioreactor of claim 33, wherein the physical structure comprises a plurality of fibers in a predetermined and ordered arrangement.

35. The fixed bed bioreactor of any of claims 1-34, wherein the cell culture substrate comprises a mesh material.

36. The fixed bed bioreactor of claim 35, wherein the mesh material is a woven mesh comprising a plurality of fibers.

37. The fixed bed bioreactor of any of claims 1-36, wherein the sample substrate comprises a fibrous mesh material.

38. The fixed bed bioreactor of claim 37, wherein the fibrous mesh material is a woven mesh.

39. The fixed bed bioreactor of any of claims 1-38, wherein the cell culture vessel further comprises an inlet fluidly connected to the interior reservoir and configured for flowing fluidAttorney Docket No.: SP24-305 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.

40. The fixed bed bioreactor of claim 39, 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 fixed bed in the interior reservoir, and out through the outlet in a flow direction that is substantially parallel to the longitudinal axis.

41. The fixed bed bioreactor of claim 39 or claim 40, 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.

42. A method of sampling a cell culture comprising: providing a bioreactor containing an interior reservoir and a fixed bed disposed in the interior reservoir, the fixed bed comprising a cell culture substrate and one or more sample substrates, the cell culture substrate comprising a surface configured for growing cells; culturing cells in the fixed bed while perfusing cell culture media through the interior reservoir; sampling the cell culture by removing at least a portion of the one or more sample substrate; and after sampling, continuing the culturing of the cells in the fixed bed.

43. A method of sampling a cell culture comprising: providing a fixed-bed bioreactor according to any one of claims 1-41; culturing cells in the fixed bed while perfusing cell culture media through the interior reservoir; sampling the cell culture by removing at least a portion of the one or more sample substrate; and after sampling, continuing the culturing of the cells in the fixed bed.

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