Method for improving cell harvesting efficiency in fixed-bed bioreactors

Abstract

A method is provided for recovering cells from a bioreactor, the bioreactor comprising cells for recovery, at least one inlet and / or outlet, and a culture chamber on which the cells are adhered to a substrate. The method comprises providing a washing solution having a cell dissociation reagent (CDR) to the culture chamber, and, after providing the washing solution, removing and collecting the cells separated from the culture chamber via at least one inlet and / or outlet. The cell dissociation reagent comprises an endonuclease, in particular a deoxyribonuclease such as deoxyribonuclease I (DNase I). The washing solution is Mg +2 Ca +2 , or Mn +2 It may further contain divalent metal ions such as those mentioned above.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under § 119 of U.S. Patent Act to U.S. Provisional Application No. 63 / 470,173, filed on 31 May 2023, the contents of which this Provisional Application is relied upon and incorporated herein by reference in its entirety.

[0002] This disclosure generally relates to the fields of cell culture and bioprocesses, and more specifically to fixed-bed bioreactors and methods for culturing cells using them and for harvesting cells from bioreactors. [Background technology]

[0003] In the bioprocess industry, large-scale cell cultures are performed for the purpose of producing hormones, enzymes, antibodies, vaccines, and cell therapies. Cell and gene therapy (CGT) is an innovative technique in modern medicine for treating diseases caused by genetic disorders. A significant portion of the cells used in bioprocesses are adhesion-dependent, meaning that the cells require a surface to adhere to in order to grow and function. Traditionally, adherent cell culture has been performed on two-dimensional (2D) cell adhesion surfaces incorporated into one of numerous container formats such as T flasks, Petri dishes, cell factories, cell stacking vessels, roller bottles, and HYPERStack® containers. These approaches can have significant drawbacks, including the difficulty in achieving cell densities high enough to enable the large-scale production of therapies or cells. With over 2,600 CGT clinical trials underway (see The Alliance for Regenerative Medicine: Regenerative Medicine in 2021: A Year of Firsts and Records, https: / / alliancerm.org / sector-report / h1-2021-report / ), there is an unmet need for a robust manufacturing platform to meet the rapidly increasing clinical demand.

[0004] The methods suggest increasing the volume density of cultured cells. These methods include microcarrier culture carried out in agitated tanks. In this approach, cells attached to the surface of microcarriers are subjected to constant shear stress, which significantly affects their growth and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, where cells can form large three-dimensional aggregates as they grow in the interfiber space. However, cell growth and performance are significantly inhibited by the lack of nutrients. To mitigate this problem, these bioreactors are made small and are not suitable for large-scale production.

[0005] Another example of a high-density culture system for colonization-dependent cells is a packed-bed bioreactor system. For example, packed-bed bioreactor systems including a packed bed of support or substrate systems for capturing cells have been previously disclosed in U.S. Patents 4,833,083, 5,501,971, and 5,510,262. The packed-bed substrate is typically made from porous particles, such as a polymer substrate or nonwoven microfiber. Such bioreactors function as recirculating flow-through bioreactors. One of the key problems with such bioreactors is the heterogeneity of cell distribution within the packed bed. For example, the packed bed acts as a depth filter with cells primarily captured in the inlet region, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packing, the flow resistance and cell capture efficiency across the packed bed cross-section are not uniform. For example, the culture medium flows more rapidly through areas of low cell packing density and more slowly through areas of higher resistance due to a larger number of captured cells. This results in a channeling effect, where nutrients and oxygen are more efficiently delivered to areas with lower volume cell density, while areas with higher cell density are maintained under suboptimal culture conditions.

[0006] Another significant drawback of packed bed systems disclosed in the prior art is the inability to efficiently recover intact, viable cells at the end of the culture process. U.S. Patent No. 9,273,278 discloses a bioreactor design for improving the efficiency of cell removal from a packed bed during the cell recovery step. This is based on loosening the packed bed substrate, agitating or stirring the packed bed particles to allow the porous substrate to collide and thus separate the cells. However, this approach is laborious and can cause significant cell damage, thus reducing overall cell viability. Often, the cause of the recovery inefficiency is the complex flow design in the packed bed bioreactor, where the complex flow channels make it difficult to efficiently remove cells from the reactor without manual disassembly and cell recovery. The channeling and cell capture effects described above also reduce the possibility of efficient cell recovery.

[0007] In other current solutions available on the market, cells cannot be easily recovered from perfusion bioreactors using standard enzymatic methods. Instead, virus extraction via cell lysis with detergent is used, which adds time and complexity to the downstream purification of the virus.

[0008] Corning® has developed the Corning® Ascent® FBR System, a novel fixed-bed reactor (FBR) platform that combines the advantages of an adhesive bioproduction platform with the scalability and automation of a suspension manufacturing system. The system is designed for use in CGT workflows, including those for adeno-associated viruses (AAV), lentiviruses (LV), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs), as well as, among other, viral vaccines and other biological production applications.

[0009] One of the main advantages of the Ascent® system is its ability to aseptically recover viable cells from fixed-bed substrates. This ability to recover viable cells allows the Ascent® FBR system to be used as a seed train for manufacturing scale, as well as a generation platform for cell therapy and regenerative medicine purposes. However, compared to cells cultured in a two-dimensional environment, cell recovery from fixed-bed bioreactors is challenging. While the Corning® Ascent® system has demonstrated the ability to extract viable cells from fixed-bed reactor substrates, recovery efficiency in fixed-bed reactor systems can generally be improved, including in terms of consistency, and especially with respect to high-density cell culture. Reduced cell recovery efficiency results in higher costs for both manufacturing and patients.

[0010] Therefore, the need remains for systems and methods that enable consistent and efficient recovery of adherent cells in fixed-bed bioreactors. [Overview of the project]

[0011] Embodiments of the present disclosure provide a method for recovering cells from a bioreactor. The method comprises providing a bioreactor having cells for recovery. The bioreactor has at least one inlet and / or outlet, as well as a culture chamber to which cells are adhered to a substrate. The method further comprises providing a washing solution containing a cell dissociation reagent (CDR) into the culture chamber. After providing the washing solution, the method further comprises removing and collecting the separated cells from the culture chamber via at least one inlet and / or outlet. The cell dissociation reagent may include an endonuclease. The endonuclease may be a deoxyribonuclease such as deoxyribonuclease I (DNase I). The washing solution contains Mg +2 Ca +2 , or Mn +2 It can also contain divalent metal ions such as those mentioned above.

[0012] The method may further include providing a pre-recovery solution into a culture chamber; providing a recovery solution into the culture chamber for a predetermined incubation time after providing the pre-recovery solution; flushing the recovery solution out of the culture chamber following the predetermined incubation time; and providing a post-recovery solution into the culture chamber following the flushing of the recovery solution. At least one of the pre-recovery solution, recovery solution, and post-recovery solution may include a washing solution. The separated cells are recovered during the flushing of the recovery solution. The method may further include removing the post-recovery solution from the culture chamber and collecting any residual cells removed by the post-recovery solution.

[0013] An additional embodiment of the present disclosure provides a method for recovering cells from a bioreactor. The method includes providing a bioreactor containing cells for recovery, wherein the bioreactor comprises an inlet, an outlet, and a culture chamber to which the cells are attached to a substrate; providing a pre-recovery solution into the culture chamber; providing a recovery solution into the culture chamber for a predetermined incubation time; flushing the recovery solution out of the culture chamber following the predetermined incubation time; and providing a post-recovery solution into the culture chamber following the flushing of the recovery solution. The separated cells are recovered during the flushing of the recovery solution.

[0014] Providing a pre-recovery solution into the culture chamber may include removing the cell culture medium from the culture chamber. In one embodiment, the pre-recovery solution includes a washing solution. The washing solution includes phosphate-buffered saline (PBS). The recovery solution may include a cell dissociation reagent (CDR). The cell dissociation reagent includes at least one of a calcium chelating agent and a proteolytic enzyme. In an embodiment, flushing the recovery solution from the culture chamber includes replacing the recovery solution using pressurized gas flowing into the culture chamber. Flushing may also include removing cells separated from the substrate. The method may include collecting the cells separated from the culture chamber and flushed in a recovery collection container. Providing a post-recovery solution into the culture chamber includes flowing the post-recovery solution through the culture chamber. The method may further include collecting residual cells removed from the culture chamber by the post-recovery solution. The post-recovery solution may include PBS. At least one of the pre-recovery solution, recovery solution, and post-recovery solution contains an endonuclease, such as a deoxyribonuclease. Deoxyribonuclease may include deoxyribonuclease I (DNase I). At least one of the pre-recovery solution, recovery solution, and post-recovery solution, which contains an endonuclease, is Mg +2 Ca +2 , or Mn +2 It can also contain divalent metal ions such as those mentioned above.

[0015] Aspects of the embodiment include mechanically agitating the culture chamber during recovery. The mechanical agitation of the culture chamber occurs in at least one of the pre-recovery solution, the recovery solution, and the post-recovery solution present in the culture chamber. The mechanical agitation includes at least one of the following: stirring the contents of the culture chamber, shaking or vibrating the contents of the culture chamber, pulsating the fluid passing through the culture chamber, and changing the direction of the fluid flow through the culture chamber.

[0016] Additional aspects of the present disclosure are, in part, set forth in the detailed description, the drawings, and any of the following claims, and in part, derived from the detailed description or learned by practice of the present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure as claimed.

[0017] A more complete understanding of the present disclosure may be obtained by reference to the following detailed description in conjunction with the accompanying drawings. **Brief Description of the Drawings**

[0018] [Figure 1A] A photograph showing the accumulation of biomaterials extending between layers of a fixed-bed substrate according to an embodiment of the present disclosure. [Figure 1B] A photograph showing a woven mesh substrate stained with crystal violet that holds a DNA biofilm throughout the mesh and blocks the pores of the mesh according to an embodiment of the present disclosure. [Figure 2A] A schematic illustration of the step of applying a pre-recovery solution to a fixed-bed bioreactor according to an embodiment of the present disclosure. [Figure 2B] A schematic illustration of the step of applying a recovery solution to a fixed-bed bioreactor according to an embodiment of the present disclosure. [Figure 2C] A schematic illustration of the step of applying a post-recovery solution to a fixed-bed bioreactor according to an embodiment of the present disclosure. [Figure 3] A graph showing cell recovery yields when DNase is not used and when DNase is used in a post-recovery wash solution according to an embodiment of the present disclosure. [Figure 4] A graph showing cell recovery yields when DNase is not used and when various DNase products are used in a post-recovery wash solution according to an embodiment of the present disclosure. [Figure 5A] A flowchart showing a method for recovering cells according to an embodiment of the present disclosure. [Figure 5B]A flowchart showing a method for recovering cells according to an embodiment of the present disclosure.

Mode for Carrying Out the Invention

[0019] Various embodiments of the present disclosure are considered with reference to the drawings, which illustrate various aspects of a packed bed bioreactor system and related methods using the bioreactor system according to non-limiting embodiments of the present disclosure. The following description is intended to provide an effective description of the bioreactor system, and various aspects of the bioreactor system and method are specifically considered in detail throughout the present disclosure with reference to non-limiting embodiments, and these embodiments are interchangeable with each other within the context of the present disclosure.

[0020] According to an embodiment of the present disclosure, a system and method for improved cell recovery efficiency when recovering adherent cells from a fixed bed bioreactor are provided. To recover adherent cells, according to one aspect of the embodiment, cells growing on the surface of a substrate can be recovered using a cell dissociation reagent (CDR). CDR is used to separate cells from a culture substrate in adherent cell culture. The components of CDR are enzymes having proteolytic activity. The enzyme digests surface adhesion proteins and separates the cells from the substrate. Trypsin is a commonly used CDR in cell passage. However, excessive trypsinization can lead to irreversible cell damage. There are several trypsin alternative products developed for gentler separation. Accutase (registered trademark) (manufactured by Innovative Cell Technologies, Inc.) is a natural enzyme mixture having proteolytic and collagenolytic enzyme activities. This is a non-mammalian, non-bacterial reagent. TrypLE (registered trademark) (manufactured by Life Technologies Corporation) is a product free from animal origin derived from recombinant bacteria, and a cGMP-compliant version is also available on the market. Both Accutase (registered trademark) and TrypLE (registered trademark) are very gentle on the recovered cells.

[0021] However, as discussed above, cell retrieval from fixed-bed bioreactors is more difficult compared to two-dimensional culture environments. While bioreactors capable of extracting viable cells from FBR substrates have been demonstrated, retrieval efficiency may be more consistent, especially in high-density cell cultures. Experimental results have shown that cell retrieval efficiency from several fixed-bed reactors (FBRs) is inconsistent using current retrieval processes, ranging from 50% to 90%. A possible underlying cause of low retrieval efficiency may be the accumulation of DNA on the FBR substrate. DNA can originate from dead or low-viability cells introduced by the inoculant, or from cells damaged during the dynamic culture process (e.g., by shear forces from the fluid flow). This DNA, circulating within the system, can then adhere to the substrate, as shown in Figure 1A, and in the most severe cases, is present on those substrates near the inlet of the FBR. As shown in Figure 1B, this accumulation of DNA on the FBR substrate can cause cells to form large aggregates, which may not only fail to pass through the substrate's pores during the recovery process, but may also form biofilms that block pores within the substrate. The accumulation of aggregates and the blocking of openings within the substrate can impair the uniformity of cell and fluid flow, potentially negatively impacting not only recovery but also the health and uniformity of the actual cell culture. As a result, recovery efficiency was significantly reduced. This DNA problem is more serious in high-density cell cultures.

[0022] As used herein, “recovery efficiency” refers to the percentage of cells in a cell culture operation that can be recovered from the bioreactor by releasing and removing cells from the reactor in situ, compared to the total number of cells present in the cell culture. The total number of cells present in the cell culture can be determined by adding the number of recovered cells to the number of cells in the bioreactor detected when the bioreactor is manually dismantled after the recovery process is complete and the substrate is stained with crystal violet to determine the number of remaining cells on the substrate that were not successfully recovered.

[0023] According to one embodiment, a method for recovering cells from a bioreactor is provided. As shown in Figure 2A, a bioreactor 110 is provided that houses a cell culture substrate 112 for culturing adherent cells. The bioreactor 110 includes an inlet and an outlet at both ends of the bioreactor 110 and the substrate 112. Tubes connected to the inlet and outlet are used to perfuse the cell culture medium through the culture space within the bioreactor 110 that houses the substrate 112. In addition, one or more washing solutions (pre-recovery washing solution 116, recovery washing solution 118, and post-recovery washing solution 120) can be provided and connected to tubes to supply the washing solutions to the bioreactor 110. These washing solutions can flow through the bioreactor 110 and be collected in containers 117, 119, 121 when they exit the bioreactor 110, or (for example, in Figure 2B during the incubation period using the recovery solution) can be perfused in a loop. One or more pumps 114 and valves may be used to control the flow path and speed. The process begins with step 100, which involves removing the cell culture medium by passing a pre-recovery solution 116, such as phosphate-buffered saline (PBS) solution, through a bioreactor 110. Following the pre-recovery solution wash, a cell recovery solution 118, such as Accutase® or TrypLE® (these are provided as examples only, and it is intended that any suitable CDR may be used). The cell recovery solution is pumped into the bioreactor 110 and incubated for a certain period to separate the cells from the substrate 112. At the end of the predetermined incubation period, the separated cells in the bioreactor are recovered by flushing them into a collection container 119 with the help of pressurized air pumped through the inlet or outlet of the bioreactor 110 to the opposite end of the bioreactor 110. A post-recovery solution 120, such as PBS solution, then flows through the bioreactor 110 to remove any remaining separated cells.

[0024] As one aspect of the embodiment, the recovery efficiency can be further improved by adding an endonuclease to one or more of the recovery solutions. For example, a deoxyribonuclease such as deoxyribonuclease I (DNase I) can be added to either the pre-recovery solution, the recovery solution, or the post-recovery solution. In one or more specific embodiments, the endonuclease is added to the post-recovery solution. DNase I is an endonuclease that hydrolyzes phosphodiester bonds and cleaves or nicks either double-stranded or single-stranded DNA into oligonucleotides. DNase has been used in tissue digestion and single cell suspension preparation. The purpose of adding DNase to one of the recovery solutions in the embodiments of the present disclosure is to digest the DNA accumulated on the bioreactor substrate in order to improve the recovery efficiency. As mentioned, in certain embodiments, DNase I is added to the post-recovery solution; however, DNase I can be added to the pre-recovery solution and / or the cell recovery solution. Mg +2 , Ca +2 or Mn +2 such divalent metal ions can also be added to ensure full DNase I activity.

[0025] As shown in Figures 3 and 4, the use of DNase I in the post-recovery washing solution significantly improves cell recovery efficiency when using either Accutase® or TrypLE as the CDR. Figure 3 compares two experiments in which HEK293T cells were cultured for 3 days each in the same Corning® Ascent® FBR. Cells were then recovered with and without DNase added to the post-recovery solution. The DNase product used is sold as Bensonase® (MilliporeSigma). The experiment was repeated three times for each recovery condition, and the average cell recovery yield percentage is shown by the column in Figure 3, with variability shown by the range bar for each column. As shown in Figure 4, DNase I from different vendors all yielded similar improved results. In each case, HEK293T cells were cultured at 22,000 cells / cm³ in the Corning® Ascent® FBR. 2 Cells were seeded at the following density. After 3 days of culture, cells were harvested at 1×TrypLE in the absence or presence of 25 U / mL of DNase I from three different vendors in the post-harvest solution. The harvested cell yield was ×1000 cells / cm². 2 As shown in [figure], therefore, embodiments of the present disclosure provide an innovative cell recovery process that can remove DNA accumulation on a bioreactor substrate and thus significantly improve cell recovery efficiency. DNase I can be added to the recovery process at different stages of the pre-recovery washing solution, the cell recovery solution, and / or the post-recovery washing solution.

[0026] As shown in Figure 5A, embodiments of the present disclosure include a method 400 for recovering adherent cells from a fixed-bed reactor, the method 400 comprising: step S1 providing a bioreactor containing cells for recovery; step S2 providing a washing solution containing a cell dissociation reagent to a culture chamber; and step S3 removing and collecting cells separated from the culture chamber via at least one inlet and / or outlet after providing the washing solution. Step S2 may include additional steps such as step S4 providing a pre-recovery solution into the culture chamber; step S5 providing a recovery solution into the culture chamber for a predetermined incubation time; flushing the recovery solution out of the culture chamber following the predetermined incubation time; and step S6 providing a post-recovery solution into the culture chamber following the flushing of the recovery solution.

[0027] According to embodiments of the present disclosure, a method for recovering cells from a bioreactor may include providing a bioreactor containing cells for recovery. The bioreactor itself has at least one inlet and / or outlet, as well as a culture chamber to which cells are attached to a substrate. In one embodiment, the bioreactor has an inlet and an outlet on both sides of the bioreactor, with the substrate positioned between the inlet and the outlet so that a fluid (e.g., cell culture medium, nutrients, cells, and cell byproducts) can flow substantially linearly from the inlet, through the substrate, and to the outlet. This simple linear flow path is thought to improve the uniformity of the fluid flow and therefore improve the uniformity and recovery of the cell culture. However, embodiments are not intended to be limited to this arrangement and may include other flow designs.

[0028] The method also includes providing a washing solution containing a cell dissociation reagent (CDR) into the culture chamber. The washing solution is stored in a reservoir or container outside the bioreactor and can be supplied to the culture chamber at a desired time. After the washing solution has been supplied, the separated cells are removed from and collected from the culture chamber via at least one inlet and / or outlet. The bioreactor system may include a recovery container fluidly connected to at least one inlet and / or outlet for collecting the recovered cells. In one embodiment, the reactor has an inlet through which cell culture medium is perfused in a pathway to a substrate, and the cell culture medium exits the bioreactor through an outlet. In a further embodiment, the direction of flow through the bioreactor is reversed when supplying the washing solution. That is, the washing solution is stored in a container fluidly connected to the outlet of the bioreactor, flows into the culture chamber through the outlet, and then flows into and / or through the substrate. The washing solution can then also exit the culture chamber through the inlet. Alternatively, the flow may be reversed again for a certain incubation period so that the washing solution flows out through the outlet. According to a further embodiment, the washing solution may be perfused into a recirculation loop through the cell culture chamber so that the washing solution exits the bioreactor and then, optionally, re-enters the bioreactor after undergoing some type of readjustment to maintain the optimal properties of the washing solution. Alternatively, the washing solution may flow through the bioreactor only once and be collected in a container after exiting the bioreactor.

[0029] According to an additional embodiment of the embodiment, the cell dissociation reagent comprises a proteolytic enzyme such as Accutase® or TrypLE®. The washing solution may further comprise an endonuclease such as deoxyribonuclease. The deoxyribonuclease may include deoxyribonuclease I (DNase I). The washing solution may comprise, for example, Mg +2 Ca +2 , or Mn +2It can also contain divalent metal ions such as those mentioned above.

[0030] In some embodiments, the method may further include providing a pre-recovery solution into the culture chamber. The pre-recovery solution may include a phosphate-buffered saline (PBS) solution. The pre-recovery solution is introduced into the bioreactor after cell culture is complete to remove any cell culture medium from the fixed bed substrate and prepare the fixed bed for recovery. After providing the pre-recovery solution, the method may further include providing a recovery solution into the culture chamber for a predetermined incubation time. During this incubation time, the CDR in the recovery solution helps separate the cells from the substrate so that they can be removed from the fixed bed. Following the predetermined incubation time, the recovery solution is flushed out of the culture chamber. Flushing of the recovery solution acts to flush out most or all of the cells from the bioreactor. Following flushing of the recovery solution, a post-recovery solution is provided into the culture chamber. The post-recovery solution can be used for additional washing of the substrate and to recover any additional cells that were not flushed with the recovery solution. This method may further include removing the post-recovery solution from the culture chamber and collecting the residual cells removed by the post-recovery solution. In the embodiments, at least one of the pre-recovery solution, recovery solution, and post-recovery solution includes the washing solution mentioned above.

[0031] According to embodiments of the present disclosure, a method for recovering cells from a bioreactor may include providing a bioreactor having cells for recovery. The bioreactor includes an inlet, an outlet, and a culture chamber to which cells are attached to a substrate. The method also includes providing a pre-recovery solution into the culture chamber; providing a recovery solution into the culture chamber for a predetermined incubation time; flushing the recovery solution out of the culture chamber following the predetermined incubation time; and providing a post-recovery solution into the culture chamber following the flushing of the recovery solution. The separated cells are recovered during the flushing of the recovery solution and, optionally, during the flushing of the post-recovery solution.

[0032] In some embodiments, providing a pre-recovery solution into the culture chamber removes the cell culture medium from the culture chamber. The pre-recovery solution includes, for example, a washing solution such as phosphate-buffered saline (PBS). Other acceptable cell washing solutions may also be used. Following the provision of the pre-recovery solution, the pre-recovery solution may be discharged from the bioreactor before the recovery solution is filled into the bioreactor, or the recovery solution may be supplied simultaneously with the removal of the pre-recovery solution. The recovery solution includes a cell dissociation reagent (CDR). The cell dissociation reagent includes at least one of a calcium chelating agent and a proteolytic enzyme. According to some embodiments, flushing the recovery solution from the culture chamber may include replacing the recovery solution with a pressurized gas flowing into the culture chamber and pushing it out through one of the bioreactor's inlet and outlet. Flushing the recovery solution also includes removing cells separated from the substrate. The separated and flushed cells are then collected in a recovery collection container.

[0033] In some embodiments, providing a post-recovery solution into a culture chamber includes introducing the post-recovery solution into the culture chamber. If any cells remain in the fixed bed following flushing with the recovery solution, these residual cells can be removed from the culture chamber by the post-recovery solution and subsequently collected. The post-recovery solution may include a washing solution such as PBS or another acceptable cell washing solution. In one embodiment, at least one of the pre-recovery solution, recovery solution, and post-recovery solution comprises an endonuclease. The endonuclease may include, for example, a deoxyribonuclease such as deoxyribonuclease I (DNase I). In further embodiments, at least one of the pre-recovery solution, recovery solution, and post-recovery solution comprising an endonuclease may include, for example, Mg +2 Ca +2 , or Mn +2 It further contains divalent metal ions such as [list of ions]. The presence of divalent metal ions can increase the effectiveness of endonucleases.

[0034] In further embodiments of the embodiment, the method may include mechanically agitating the culture chamber. Mechanical agitation of the culture chamber can occur in at least one of the pre-recovery solution, recovery solution, and post-recovery solution contained within the culture chamber. Mechanical agitation may include at least one of stirring the contents of the culture chamber, shaking or vibrating the contents of the culture chamber, pulsating the fluid passing through the culture chamber, and changing the direction of the fluid flow through the culture chamber.

[0035] Referring to Figures 2A to 2C, the fixed-bed bioreactor 110 enables not only the culture of adherent cells and the recovery of cell byproducts (e.g., transfected viruses) but also the recovery of the cells themselves. The cell culture substrate can take various forms, but is preferably a porous material provided in a monolithic structure (such as a foam substrate or a single woven mesh sheet) or in a packed bed configuration of multiple substrate material pieces or layers. In one embodiment, for example, the multiple substrate material pieces are sheets or discs of a porous polymer made from one or more polymer fibers. For example, the porous polymer material can be a woven mesh substrate material provided as a laminate of sheets or discs in a container.

[0036] According to one embodiment, the bioreactor system disclosed herein can pressurize the bioreactor and generate a safe flow rate that does not damage cells or recovered products. The pressurization for recovery can be automated and fully integrated into the bioreactor controller system. Current commercially available products, which tend to use non-uniform substrates, capture cells non-uniformly within the substrate during attachment. This leads to non-uniform cell growth and inefficient transfection of subsequent DNA plasmids into the cells. This negatively impacts the entire virus produced by the cells. In addition, because many cells are captured, it is not possible to efficiently extract the virus from within the cells for use in subsequent gene therapy. Currently, as a workaround, detergents are used to lyse the cells and release the virus in situ; however, the addition of detergents adds cost and complexity to downstream virus purification steps. Since the pressure delivered in combination with various washing solutions can facilitate cell release from bioreactor substrates, embodiments of this disclosure eliminate the need for cell lysis. In embodiments of this disclosure, preferred cell culture substrates enhance the uniformity of the cell flow and release for recovery. An example of such substrate materials is disclosed in U.S. Patent No. 11,434,460, the contents of which are incorporated herein by reference in their entirety. The cell culture substrate is porous to allow cells, culture medium, nutrients, and cell byproducts to perfuse through the substrate, and to allow used culture medium containing cell secretion materials (e.g., recombinant proteins, antibodies, viral particles, DNA, RNA, sugars, lipids, biodiesel, inorganic particles, butanol, metabolic byproducts) to pass through and be recovered. Further details of the cell culture substrate according to the embodiment are provided below.

[0037] Embodiments of the present disclosure include a bioreactor and a cell culture substrate used therein, the cell culture substrate comprising a substrate that is a cell growth matrix and / or packed bed system for adhesion-dependent cells, enabling easy and effective scale-up to any practical production scale for cells or cell-derived products (e.g., proteins, antibodies, viral particles). In one embodiment, the substrate has good mechanical strength and provides a structurally defined surface area for adherent cells to adhere and proliferate, forming a highly uniform, multi-degree interconnected fluid network when assembled in a packed bed or other bioreactor. In certain embodiments, a mechanically stable, non-degradable woven mesh can be used to assist in the generation of adherent cells. Uniform cell seeding of such a substrate, as well as efficient recovery of cells or other products of the bioreactor, is achievable. In addition, embodiments of the present disclosure can assist in cell culture to achieve a confluent monolayer or multilayer of adherent cells on the disclosed substrate and avoid the formation of 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. The structurally defined substrates of one or more embodiments enable complete cell removal from the bioreactor packed bed and consistent cell recovery. Another embodiment of the present disclosure also provides a method of cell culture using a bioreactor having a substrate for bioprocessing the production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0038] In one or more embodiments, the cell culture substrate facilitates the adhesion and proliferation of colonization-dependent cells in a high volume-density format. The substrate can be assembled and used within a bioreactor system, such as a perfused fixed-bed bioreactor as described herein, and can provide a uniform cell distribution during the inoculation step while preventing the formation of large and / or uncontrollable cell aggregates within the fixed bed. Thus, the substrate eliminates diffusion limitations during bioreactor operation. In addition, the substrate enables easy and efficient cell recovery from the bioreactor.

[0039] The substrate can be formed from a substrate material in the form of a thin sheet-like structure having first and second sides separated by a relatively small thickness. In other words, the thickness of the sheet-like substrate is small relative to the width and / or length of the first and second sides of the substrate. In addition, multiple holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometric shape that allows cells to adhere to the surface of the substrate material as if it were a two-dimensional (2D) surface, while also allowing appropriate 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 penetrating in the thickness direction, a number of filaments fused into a mesh-like layer, or a number of filaments woven into a mesh layer. The physical structure of the substrate has a high surface-to-volume ratio for culturing colonization-dependent cells. According to various embodiments, the substrate can be placed or filled in a bioreactor in a particular manner to obtain uniform cell seeding, uniform medium perfusion, and efficient cell harvesting.

[0040] The cell culture substrate can be a woven mesh layer made from a first set of fibers extending in a first direction and a second set of fibers extending in a second direction. The woven fibers of the substrate form multiple openings. The size and shape of the openings can vary depending on the type of weaving (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments, etc.). The openings can be defined by a certain width or diameter. The woven mesh can be considered as a two-dimensional sheet or layer on a macroscale. However, a close examination of the woven mesh reveals a three-dimensional structure resulting from the rising and falling of the intersecting fibers of the mesh. Therefore, the thickness of the woven mesh can be greater than the thickness of a single fiber.

[0041] The woven mesh can consist of monofilament or multifilament polymer fibers. In one or more embodiments, the monofilament fibers may have a diameter in the range of about 10 μm to about 1000 μm. At the microscale level, due to the scale of the fibers compared to cells (e.g., the fiber diameter being larger than that of cells), the surface of the monofilament fibers is presented as a normal 2D surface for adherent cells to attach and proliferate. Such fibers are woven into a mesh having a defined pattern and a certain amount of structural stiffness. The fibers can be woven into a mesh having openings in the range of about 25 μm × 25 μm to about 1000 μm × 1000 μm. These ranges of filament diameter and opening diameter are examples of some embodiments but are not intended to limit the possible feature sizes of the mesh by all embodiments.

[0042] The base mesh can be made from monofilament or multifilament fibers of polymer materials suitable for cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh base material may have different structural patterns or weaves, including, for example, knitting, warp knitting, or weaving (plain weave, twill weave, Dutch weave, five-needle weave).

[0043] The surface chemical properties of mesh filaments may need to be modified to provide desired cell adhesion properties. Such modifications can be made through chemical treatment of the mesh's polymer material or by grafting cell adhesion molecules onto the filament surface. Alternatively, the mesh can be coated with a thin layer of a biocompatible hydrogel exhibiting cell adhesion properties, such as collagen or Matrigel®. Alternatively, the surface of the mesh filament fibers can be made to have cell adhesion properties through treatment processes with various types of plasma, process gases, and / or chemicals known in the industry.

[0044] The woven mesh substrate may be provided in a plurality of disks having a central hole configured to surround the central column of the bioreactor described herein. Multiple such disks can be stacked in the outer region of the bioreactor to form a packing bed.

[0045] According to some embodiments, the cell culture substrate is a soluble foamed scaffold comprising an ionotropically crosslinked polygalacturonic acid compound selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof, and at least one first water-soluble polymer having surface activity.

[0046] Embodiments disclosed herein enable not only the attachment and growth of cells to a cell culture substrate, but also the viable recovery of cultured cells. The inability to recover viable cells is a significant drawback in current platforms and leads to difficulties in constructing and maintaining a sufficient number of cells for proliferative capacity. According to one aspect of the embodiments of this disclosure, it is possible to recover viable cells from a cell culture substrate, including 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the recovered cells, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. Cells can be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase®.

[0047] It will be understood that various disclosed embodiments may involve specific features, elements, or steps described in relation to that particular embodiment. It will also be understood that while certain features, elements, or steps are described in relation to one particular embodiment, they may be substituted or combined with alternative embodiments in various not-executed combinations or sequences.

[0048] It should also be understood that, as used herein, the terms “the,” “a,” or “an” mean “at least one,” and should not be limited to “only one” unless the opposite is explicitly indicated. Therefore, for example, a reference to “opening” includes examples having two or more such “openings” unless the context otherwise explicitly indicates.

[0049] In this specification, a range can be expressed as "approximately" from one particular value and / or "approximately" to another particular value. Where such a range is expressed, examples include that particular value and / or other particular value. Similarly, where a value is expressed as an approximation using the antecedent "approximately," it will be understood that the particular value forms another aspect. It will further be understood that each endpoint of a range is important, whether related to or independent of the other endpoints.

[0050] All numerical values ​​expressed herein, whether stated or not, should be interpreted as including "approximately" unless otherwise explicitly indicated. However, it should be further understood that each numerical value cited is intended to be equally precise, whether expressed as "approximately" or not. Therefore, both "dimensions less than 10 mm" and "dimensions less than approximately 10 mm" include embodiments of "dimensions less than approximately 10 mm" and "dimensions less than 10 mm."

[0051] Unless otherwise expressly stated, no method described herein is intended to be construed as requiring its steps to be carried out in a specific order. Therefore, if a method claim does not actually list the order in which its steps are followed, or if it is not specifically stated in the claims or specification that the steps should be limited to a particular order, no particular order is ever intended to be inferred.

[0052] It should be understood that while various features, elements, or steps of a particular embodiment may be disclosed using the transitional phrase "including," alternative embodiments are implied, including those that may be described using the transitional phrase "consisting of" or "essentially derived from." Therefore, for example, alternative embodiments implied by a method comprising A+B+C include embodiments in which the method consists of A+B+C, and embodiments in which the method is essentially derived from A+B+C.

[0053] While several embodiments of this disclosure are illustrated in the accompanying drawings and described in the preceding detailed description, it should be understood that this disclosure is not limited to the disclosed embodiments and that numerous rearrangements, modifications, and substitutions are possible without departing from the disclosure as described and defined by the following claims.

Claims

1. A method for recovering cells from a bioreactor, wherein the method is To provide a bioreactor containing cells for harvesting, wherein the bioreactor comprises at least one inlet and / or outlet, and a culture chamber to which the cells are attached to a substrate, To provide a washing solution containing a cell dissociation reagent (CDR) in the culture chamber, A method comprising, after providing the washing solution, removing and collecting cells separated from the culture chamber through at least one inlet and / or outlet.

2. The method according to claim 1, wherein the cell dissociation reagent comprises an endonuclease.

3. The method according to claim 2, wherein the endonuclease comprises deoxyribonuclease.

4. The method according to claim 3, wherein the deoxyribonuclease comprises deoxyribonuclease I (DNase I).

5. The method according to any one of claims 2 to 4, wherein the washing solution further comprises divalent metal ions.

6. The aforementioned divalent metal ion is Mg ＋２ Ca ＋２ , or Mn ＋２ The method according to claim 5, including the method described in claim 5.

7. To provide the pre-recovery solution in the culture chamber, After providing the pre-recovery solution, the recovery solution is provided in the culture chamber for a predetermined incubation period. Following the predetermined incubation time, the recovered solution is flushed out of the culture chamber. The method according to any one of the prior claims, further comprising providing the recovered solution into the culture chamber following the flushing of the recovered solution.

8. The method according to claim 7, wherein at least one of the pre-recovery solution, the recovery solution, and the post-recovery solution includes the washing solution.

9. The method according to claim 7 or 8, wherein the separated cells are recovered during the flushing of the recovery solution.

10. The method according to any one of claims 7 to 9, further comprising removing the post-recovery solution from the culture chamber and collecting the residual cells removed by the post-recovery solution.

11. A method for recovering cells from a bioreactor, wherein the method is To provide a bioreactor containing cells for recovery, wherein the bioreactor comprises an inlet, an outlet, and a culture chamber to which the cells are attached to a substrate, To provide the pre-recovery solution in the culture chamber, The recovery solution is provided in the culture chamber for a predetermined incubation period, Following the predetermined incubation time, the recovered solution is flushed out of the culture chamber. The process includes providing the recovered solution into the culture chamber following the flushing of the recovered solution, A method in which separated cells are recovered during the flushing of the recovery solution.

12. The method according to claim 11, wherein providing the pre-recovery solution into the culture chamber includes removing the cell culture medium from the culture chamber.

13. The method according to claim 11 or 12, wherein the pre-recovery solution includes a washing solution.

14. The method according to claim 13, wherein the washing solution comprises phosphate-buffered saline (PBS).

15. The method according to any one of claims 11 to 14, wherein the recovered solution comprises a cell dissociation reagent (CDR).

16. The method according to claim 15, wherein the cell dissociation reagent comprises at least one of a calcium chelating agent and a proteolytic enzyme.

17. The method according to any one of claims 11 to 16, wherein the flushing of the recovered solution from the culture chamber includes replacing the recovered solution using pressurized gas flowing into the culture chamber.

18. The method according to any one of claims 11 to 17, wherein the flushing includes removing cells separated from the substrate.

19. The method according to claim 18, further comprising collecting the cells separated from the culture chamber and flushed in a collection container.

20. The method according to any one of claims 11 to 19, wherein providing the recovered solution into the culture chamber includes flowing the recovered solution into the culture chamber.

21. The method according to any one of claims 11 to 20, further comprising collecting residual cells removed from the culture chamber with the recovery solution.

22. The method according to any one of claims 11 to 21, wherein the recovered solution contains PBS.

23. The method according to any one of claims 11 to 22, wherein at least one of the pre-recovery solution, the recovery solution, and the post-recovery solution contains an endonuclease.

24. The method according to claim 23, wherein the endonuclease comprises deoxyribonuclease.

25. The method according to claim 24, wherein the deoxyribonuclease comprises deoxyribonuclease I (DNase I).

26. The method according to any one of claims 23 to 25, wherein at least one of the pre-recovery solution containing the endonuclease, the recovery solution, and the post-recovery solution further contains a divalent metal ion.

27. The aforementioned divalent metal ion is Mg ＋２ Ca ＋２ , or Mn ＋２ The method according to claim 26, including the method described in claim 26.

28. The method according to any one of claims 11 to 27, further comprising mechanically shaking the culture chamber.

29. The method according to claim 28, wherein the mechanical agitation of the culture chamber occurs in at least one of the pre-recovery solution, the recovered solution, and the post-recovery solution located within the culture chamber.

30. The method according to claim 28 or 29, wherein the mechanical agitation includes at least one of stirring the contents of the culture chamber, shaking or vibrating the contents of the culture chamber, pulsating the fluid passing through the culture chamber, and changing the direction of the fluid flow through the culture chamber.

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