Systems and methods for harvesting cells from fixed-bed bioreactors

Abstract

A bioreactor for culturing and harvesting cells is provided. The bioreactor includes a vessel with an interior cavity and one or more media ports for passing fluid between the interior cavity and an exterior of the vessel, and a substrate disposed in the interior cavity and having a surface for adhering cells thereto. The bioreactor also includes an agitator arranged to transmit mechanical energy to at least one of the vessel, the substrate, and a fluid in the interior cavity. The agitator transmits mechanical energy sufficient to contribute to either detaching cells adhered to the substrate or dislodging cells entrapped in the substrate.

Description

Attomey Docket No. SP24-269SYSTEMS AND METHODS FOR HARVESTING CELLS FROM FIXED-BED BIOREACTORSCROSS 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 / 722,211 filed on November 19, 2024, the content of which is relied upon and incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to the cell culture bioreactors and, in particular, to systems and methods to harvest cells and / or cell by-products from bioreactors for culturing adherent cells.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 therapies (CGT) are innovative technologies in modem medicine to treat diseases caused by genetic disorders. 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. With more than 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 / hl-2021-report / ), there is an unmet need for a robust manufacturing platform to meet the rapidly increasing clinical demands.

[0004] 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-Atorney Docket No. SP24-269 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.

[0005] Another example of a high-density culture system for anchorage dependent cells is a packed bed bioreactor system. 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, media 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.

[0006] 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. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during cells harvesting step. It is based on loosening the packed bed matrix and agitation or stirring of packed bed particles to allow porous matrices to collide and thus detach the cells. However, this approach is laborious and may cause significant cells damage, thus reducing overall cell viability. Contributing to the inefficiencies in harvesting are the often complex flow designs in packed bed bioreactors, where complicated flow paths make it difficult to efficiently removes cells from within the reactor without manually disassembling and harvesting the cells. The channeling and cell-trapping effects described above also decrease the potential for efficient cell harvesting.

[0007] In other current solutions available on the market, cells cannot be easily harvested from perfusion bioreactors using standard enzymatic methods. Instead, virus recovery via cellAttorney Docket No. SP24-269 lysis with detergent is used, which adds time and complexity to down-stream purification of the virus.

[0008] The ability to harvest viable cells aseptically from the fixed-bed substrate is valuable in the industry. This ability to harvest viable cells enables bioreactors to be used as a seed train for manufacturing scale and production platform for cell therapy and regenerative medicine purposes. However, compared to cells cultured in a two-dimensional environment, cell harvesting from a fixed-bed bioreactor is challenging. As discussed herein, the type of substrate used for adherent cell culturing can contribute to this challenge by entrapping cells. In addition, many bioreactors use complicated or circuitous fluid flow pathways through the bioreactor, which make efficient harvesting difficult. For example, bioreactors having embedded or integrated media conditioning adds to the complexity of the bioreactor design and flow path, which can negatively impact the ability to harvest cells, the harvest efficiency in fixed bed reactors systems generally can be improved, including in terms of consistency and especially for high density cell cultures, or bioreactors having integrated media conditioning spaces. Lower cell harvest efficiency will result in the higher cost for both manufacturing and patients.

[0009] Thus, there remains a need for systems and method that enable consistent and efficient harvesting of adherent cells in fixed bed bioreactors.SUMMARY

[0010] According to embodiments of this disclosure, a bioreactor for culturing and harvesting cells according to a first Aspect is provided. In embodiments, the bioreactor includes a vessel having an interior cavity and one or more media ports for passing fluid between the interior cavity and an exterior of the vessel. The bioreactor also can include a substrate disposed in the interior cavity and having a surface for adhering cells thereto. The bioreactor also includes an agitator arranged to transmit mechanical energy to at least one of the vessel, the substrate, and a fluid in the interior cavity. The agitator is able to transmit mechanical energy sufficient to contribute to either detaching cells adhered to the substrate or dislodging cells entrapped in the substrate.Atorney Docket No. SP24-269

[0011] In a second Aspect, the bioreactor according to the first Aspect is provided, wherein the vessel can further include an integrated media reservoir to contain cell culture media and not contain the substrate.

[0012] In a third Aspect, the bioreactor according to the second Aspect is provided, wherein the bioreactor further includes one or more media conditioning ports to deliver fluid to the integrated media reservoir for conditioning cell culture media in the integrated media reservoir.

[0013] In a fourth Aspect, the bioreactor according to the second or third Aspects is provided, wherein the integrated media reservoir is disposed in a center of the vessel and the substrate surrounds the integrated media reservoir.

[0014] In a fifth Aspect, the bioreactor according to any one of the first through fourth Aspects is provided, wherein the vessel further includes a housing forming an interior compartment. The interior compartment includes a first chamber containing the substrate. The first chamber has a fluid input end and a fluid output end. The interior compartment also includes a second chamber within the first chamber, the second chamber defined by an imperforate wall between the first chamber and the second chamber for returning fluid having passed through the substrate from the fluid input end to the fluid output end back to the fluid input end of the first chamber.

[0015] In a sixth Aspect, the bioreactor according to the fifth Aspect is provided, wherein the interior compartment further includes a third chamber for recirculating fluid through the substrate.

[0016] In a seventh Aspect, the bioreactor according to the sixth Aspect is provided, wherein the third chamber is disposed below or above the first chamber.

[0017] In an eighth Aspect, the bioreactor according to the sixth or seventh Aspect is provided, wherein the third chamber includes an impeller.

[0018] In a ninth Aspect, the bioreactor according to any one of the first through eighth Aspects, wherein the agitator can vibrate at least one of the vessel, the substrate, and the fluid in the interior cavity.

[0019] In a tenth Aspect, the bioreactor according to any one of the first through ninth Aspects is provided, wherein the vessel is disposed on top of the agitator.

[0020] In an eleventh Aspect, the bioreactor according to the tenth Aspect is provided, wherein the agitator is a vibration table having a platform sized and shaped to support theAttorney Docket No. SP24-269 vessel thereon, and the platform can move in such a way as to cause vibrations to travel through at least one of the vessel, the substrate, and the fluid in the interior cavity.

[0021] In a twelfth Aspect, the bioreactor according to the tenth or eleventh Aspect is provided, wherein the agitator has one or more anchor points to be coupled to the vessel.

[0022] In a thirteenth Aspect, the bioreactor according to the twelfth Aspect is provided, wherein the one or more anchor points can couple to the vessel such that the platform and the vessel move in conjunction with one another.

[0023] In a fourteenth Aspect, the bioreactor according to any one of the tenth through thirteenth Aspects is provided, wherein the agitator is attached to the bottom of the vessel.

[0024] In a fifteenth Aspect, the bioreactor according to any one of the first through ninth Aspects if provided, wherein the agitator includes a membrane to oscillate at a predetermined frequency.

[0025] In a sixteenth Aspect, the bioreactor according to the fifteenth Aspect is provided, wherein the membrane is disposed around the substrate between the substrate and an inner wall of the vessel.

[0026] In a seventeenth Aspect, the bioreactor according to the fifteenth Aspect is provided, wherein the membrane is disposed in the integrated media reservoir.

[0027] In an eighteenth Aspect, the bioreactor according to the fifteenth Aspect is provided, wherein the membrane is disposed on at least one of a top wall and a bottom wall of the vessel.

[0028] In a nineteenth Aspect, the bioreactor according to the fifteenth Aspect is provided, wherein the substrate surrounds the membrane.

[0029] In a twentieth Aspect, the bioreactor according to any one of the preceding Aspects is provided, further including a controller to control the agitator.

[0030] In a twenty-first Aspect, the bioreactor according to the twentieth Aspect is provided, wherein the controller can control at least one of a frequency of motion and a time of operation of the agitator.

[0031] In a twenty-second Aspect, the bioreactor according to the twenty-first Aspect is provided, wherein the frequency of motion is from about 50 Hz to about 31 kHz.

[0032] In a twenty-third Aspect, the bioreactor according to any one of the first through twenty-first Aspects is provided, wherein the agitator can generate ultrasonic waves.Attorney Docket No. SP24-269

[0033] In a twenty-fourth Aspect, the bioreactor according to any one of the precedingAspects is provided, further including a vessel rotator able to rotate the vessel about a longitudinal axis of the vessel sufficient to generate a centrifugal force to contribute to either detaching cells adhered to the substrate or dislodging cells entrapped in the substrate.

[0034] In a twenty-fifth Aspect, a bioreactor for culturing and harvesting cells is provided. The bioreactor can include a vessel having an interior cavity and one or more media ports for passing fluid between the interior cavity and an exterior of the vessel; and a substrate disposed in the interior cavity and having a surface for adhering cells thereto. The interior cavity can include a first chamber housing the substrate, the first chamber having a fluid input end and a fluid output end. The interior cavity can also include a second chamber within the first chamber, the second chamber defined by an imperforate wall between the first chamber and the second chamber for returning fluid having passed through the substrate from the fluid input end to the fluid output end back to the fluid input end of the first chamber.

[0035] In a twenty-sixth Aspect, the bioreactor according to the twenty-fifth Aspect is provided, wherein the second chamber includes an integrated media reservoir to contain cell culture media and not contain the substrate.

[0036] In a twenty-seven Aspect, the bioreactor according to the twenty-sixth Aspect is provided, wherein the bioreactor further includes one or more media conditioning ports to deliver fluid to the integrated media reservoir for conditioning cell culture media in the integrated media reservoir.

[0037] In a twenty-eighth Aspect, the bioreactor according to any one of the twenty-fifth through twenty-seventh Aspects is provided, the interior compartment further including a third chamber for recirculating fluid through the substrate.

[0038] In a twenty-ninth Aspect, the bioreactor according to the twenty-eighth Aspect is provided, wherein the third chamber is disposed below or above the first chamber.

[0039] In a thirtieth Aspect, the bioreactor according to the twenty-eighth or twenty-ninth Aspect is provided, the third chamber including an impeller.

[0040] In a thirty-first Aspect, the bioreactor according to any one of the twenty-fifth through thirtieth Aspects is provided, further including a harvest collection vessel fluidly connected to the interior cavity and able to receive contents harvested from the substrate.

[0041] In a thirty-second Aspect, the bioreactor according to any one of the twenty -fifth through thirty-first Aspects can further include a harvest solution container fluidly connectedAttorney Docket No. SP24-269 to the interior cavity and able to hold a harvest solution. The harvest solution is a solution able to assisting in detaching cells adhered to the substrate when the harvest solution is transferred from the harvest solution container to the interior cavity.

[0042] In a thirty-third Aspect, the bio reactor according to any one of the twenty-fifth to thirty-second Aspects can further include a pressurized fluid delivery mechanism configured to deliver fluid under pressure to the interior cavity at a pressure sufficient to detach cells from the substrate.

[0043] In a thirty-fourth Aspect, the bioreactor according to the thirty-third Aspect is provided, wherein the pressurized fluid delivery mechanism includes at least one of a pump, a syringe, a piston, a bladder, and a pressurized gas.

[0044] In a thirty-fifth Aspect, the bioreactor according to the thirty-third or thirty-fourth Aspect is provided, wherein the pressurized fluid delivery mechanism is configured to deliver the harvest solution to the interior cavity.

[0045] In a thirty-sixth Aspect, the bioreactor according to any one of the thirty-first to thirty-fifth Aspects can further include a one-way valve within the interior cavity, the oneway valve being configured to allow fluid to flow in a direction toward the harvest collection vessel during a cell harvesting operation.

[0046] In a thirty-seventh Aspect, the bioreactor according to any one of the thirty-second to thirty-sixth Aspects is provided, wherein the harvest solution is a cell disassociation reagent.

[0047] In a thirty-eighth Aspect, the bioreactor according to any one of the first to thirtyseventh Aspects can further include a valve to redirect a flow of fluid within the vessel during cell harvesting.

[0048] In a thirty-ninth Aspect, the bioreactor according to the thirty-eighth Aspect is provided, wherein the valve can block a recirculation path of fluid within the interior cavity during cell harvesting.

[0049] In a fortieth Aspect, the bioreactor according to the thirty-ninth Aspect is provided, wherein, during cell harvesting, the fluid is redirected to an exterior of the interior cavity via an outlet fluidly connected to a harvest collection vessel.

[0050] In a forty-first Aspect, a method of culturing and harvesting cells from a bioreactor is provided, the method including: providing a bioreactor of any one of Aspects twenty -five to forty; seeding cells onto the substrate, the cells being adherent cells that adhere to or areAttorney Docket No. SP24-269 entrapped by the substrate; recirculating cell culture media through the vessel along a recirculation path, the recirculation path passing through the first chamber and the second chamber during the culturing of the cells; conditioning the cell culture media within the second chamber; and harvesting cells from the substrate by detaching or dislodging the cells from the substrate and removing the cells from the interior cavity.

[0051] In a forty-second Aspect, the method according to the forty-first Aspect is provided, wherein the harvesting comprises generating vibrations that pass through at least one of the vessel, the cell culture media, and the substrate, thereby assisting in the detaching or dislodging of the cells.

[0052] In a forty-third Aspect, the method according to the forty-second Aspect can further include, prior to generating the vibrations, placing the vessel on a vibration table configured to transmit the vibrations to the vessel.

[0053] In a forty-fourth Aspect, the method according to the forty-second Aspect is provided, wherein generating the vibrations comprises oscillating a membrane in the interior cavity.

[0054] In a forty-fifth Aspect, the method according to any one of the forty-first to the forty-fourth Aspects is provided, further comprising redirecting a flow of cell culture media in the interior cavity during the harvesting.

[0055] In a forty-sixth Aspect, the method according to any one the forty-first to forty-fifth Aspects can further include reversing a flow direction of cell culture media in the interior cavity during the harvesting.

[0056] 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 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 disclosed.BRIEF DESCRIPTION OF THE DRAWINGS

[0057] 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 wherein:Attorney Docket No. SP24-269

[0058] Figure 1 is a perspective view of a bioreactor with a vibration device for cell harvesting, according to embodiments.

[0059] Figure 2 is a perspective view of a bioreactor with a vibration device for cell harvesting, according to embodiments.

[0060] Figure 3 is a perspective view of a bioreactor with a vibration device for cell harvesting, according to embodiments.

[0061] Figure 4 is a perspective view of a bioreactor with a vibration device for cell harvesting, according to embodiments.

[0062] Figure 5 is a perspective view of a bioreactor with a vibration device for cell harvesting, according to embodiments.

[0063] Figure 6 is a perspective view of a bioreactor with a pressurized fluid flow and / or flow redirection device for cell harvesting, according to embodiments.

[0064] Figure 7 is a perspective view of a bioreactor with a pressurized fluid flow and / or flow redirection device for cell harvesting, according to embodiments.

[0065] Figure 8 is a perspective view of a bioreactor with a pressurized fluid flow and / or flow redirection device for cell harvesting, according to embodiments.DETAILED DESCRIPTION

[0066] Various embodiments of the present disclosure will be discussed with reference to the figures, which illustrate various aspects of packed-bed bioreactor systems and related methods of using the bioreactor systems according to non-limiting embodiments of the present disclosure. The following description is intended to provide an enabling description of the bioreactor systems and the various aspects of the bioreactor systems and methods will be specifically discussed in detail throughout the disclosure with reference to the non-limiting embodiments, these embodiments are interchangeable with one another within the context of the disclosure.

[0067] Current commercial products, which tend to use non-uniform substrates, trap cells nonuniformly in the substrate during attachment. This leads to a nonuniform cell growth and subsequent inefficient transfection of the DNA plasmids into the cells. This negatively impacts the total virus produced by the cells. In addition, because many of the cells become trapped, they cannot efficiently recover the virus inside the cells for later use in gene therapy.Attorney Docket No. SP24-269Currently as a work-around, detergents are used to lyse cells and release virus in situ; however, the addition of detergent adds cost and complexity to downstream virus purification steps. The embodiments of this disclosure reduce or eliminate the need for cell lysis using mechanical harvesting techniques, sometimes in combination with the various harvest solutions, to facilitate cell release from the bioreactor substrate.

[0068] In embodiments, the bioreactor can be a perfusion bioreactor, wave bioreactor, cylindrical bioreactor, bag bioreactor, moving bed bioreactor, packed-bed bioreactor, fixed- bed bioreactor, fibrous bioreactor, membrane bioreactor, batch bioreactor, or continuous bioreactor. The bioreactor can be made from or comprise a suitable material or materials, including, for example, stainless steel, glass aluminum, or plastic. In embodiments, the bioreactor can allow for downstream processing, such as analysis of products, filtration, concentration, inactivation, and / or purification.

[0069] In embodiments, a bioreactor disclosed herein can comprise a process controller. The process controller can, in embodiments, be configured to control operations of a bioreactor and can include or be connected to a plurality of sensors, a computer, a server, or a network. In embodiments, the bioreactor can include one or more sensors, including, for example, a temperature sensor (e.g., thermocouple), flow rate sensor, gas sensor, or any other sensor. In embodiments, the process controller can be operable to control aspects of a product manufacturing process, and can be coupled to sensors disposed in the bioreactor or in a perfusion pathway connected to the bioreactor to, for example, control temperature, volume flow rate or gas flow rate into, out of, or within the bioreactor. The process controller can include a display, including, for example, a computer monitor, a mobile device app, or an analog display, that can be accessed by a user to determine the state of the system.

[0070] In embodiments, the bioreactor disclosed herein can include and / or contain sensors for monitoring different parameters. In aspects of embodiments, a sensor disclosed herein can be located in any compartment of a bioreactor disclosed herein. In some embodiments, sensors described herein can be a gas sensor (e.g., oxygen, nitrogen, or carbon dioxide), pH sensor, temperature sensor, cell density sensor, or dissolved oxygen sensor. In embodiments, the sensors disclosed herein can measure, amongst other things, biomass or cell density, the dissolved oxygen partial pressure, oxygen content, the pTl value, the temperature, certain concentrations of nutrients, such as lactate, ammonium, carbonates, glucose or any metabolic product or product to be metabolized which could for example reflect the cell density. InAttorney Docket No. SP24-269 embodiments, cell density (biomass density) can be determined by electrical impedance analysis or electrical impedance spectroscopy using an arrangement of measuring electrodes. In aspects of embodiments, a bioreactor according to this disclosure can comprise sensors for measuring culture parameters. In embodiments, a sensor disclosed herein can be in contact with cell culture media in the bioreactor.

[0071] In embodiments, a bioreactor disclosed herein can use regulation loops according to disclosed parameters. The regulation loop can, for example, modulate the quantity of oxygen injected according to the value of the dissolved oxygen partial pressure present or the quantity of dissolved oxygen consumed by the cells; the speed of circulation of the culture medium; inject CO2 according to the pH value obtained by the sensors or any other type of regulation used in this type of culture.

[0072] In embodiments, a bioreactor disclosed herein may include heating and / or cooling devices, designed to heat and / or cool the cell culture media. The heating device can include an electrical element, an electrical coil or any other heating means generally used in the field of cell culture, such as for example, a thermostatically controlled double jacket. The cooling device may be in suitable cooling device such as a Peltier element. In embodiments, the bioreactor includes at least one inlet for the introduction of gas and / or culture medium and at least one outlet for the collection of the culture medium contained in the bioreactor. In some embodiments, a mix of gas or gaseous mixture and culture medium can be supplied through the same supply line.

[0073] In embodiments, the cell culture media can be circulated through the bioreactor via a circulator such as a pump or impeller, such as a rotatable, non-contact magnetic impeller, a blade or screw system, or an external circulation system. The circulator can include a disk blade turbine, a curved blade turbine, a turbine impeller with pitched blades, or a three-blade propeller.

[0074] In embodiments, the bioreactor described herein includes cell culture substrate formed as a fixed bed. The fixed bed, in embodiments, is a structured fixed bed (which means that it is formed of an easily replicated, generally homogenous, substantially fixed structure, and thus is not randomly oriented or unstructured, and can take a variety of shapes or sizes while meeting this qualification). The structured fixed bed described herein can provide for a large cell growth surface within a small volume while still allowing circulation of media and cells. The structure fixed bed may include a tortuous path for cells and cell culture media. InAtorney Docket No. SP24-269 embodiments, a spacer layer facilitates the tortuous path. The structure fixed bed can include one or more cell immobilization layers having a surface that allows cells to adhere and grow upon and forming a cell immobilization section. Adjacent to the cell immobilization layers are one or more spacer layers. The spacer layer can include a structure which forms a spacer section. In embodiments, adjacent to the cell immobilization layers are one or more spacer layers. The spacer layer can include a structure which forms a spacer section. The spacer section, in embodiments, allows passage of cells and media through an open but tortuous path. The structure or nature of the space layers can be chosen such that the spacer layers create a tortuous, open path for cells and culture media to travel in parallel to the surface of the spacer and cell immobilization layers. In embodiments, the tortuous path or channel formed by the spacer sections creates turbulence which facilitates cell and cell media incursion into the immobilization lyaers.

[0075] In embodiments, the spacer layer can be a mesh or comprise a mesh structure. The mesh structure or mesh can include a network or web-like patern of fibers, filaments, wire, or thread. In embodiments, the network can define pores, opening or perforations formed of a three-dimensional weave (e.g., woven fibers). In embodiments, the spacer layer and / or the cell immobilization layers of a spacer section and an immobilization section can be made of a biocompatible polymer, including, for example, polyester, polyethylene, polypropylene, polyamide, plasma treated polyethylene, plasma treated polyester, plasma treated polypropylene, or plasma treated polyamide. In embodiments, the spacer layer or cell immobilization layer can include silica, polystyrene, agarose, styrene divinylbenzene, polyacrylonitrile or latex. In embodiments, the layers can be hydrophilic or hydrophobic. In embodiments, the cell immobilization layer can be woven or nonwoven. In embodiments, a cell immobilization section and a spacer section can be alternately positioned. In embodiments, alternately positioned sections can alternate in a vertical position or in a horizontal position. In embodiments, one or more layers of cell immobilization layers can be superimposed on one or more spacer layers (or vice versa). In embodiments, a structured fixed bed disclosed herein can be tightly or loosely rolled to a structure such as a spiral structure or varying shape or cylindrical roll.

[0076] Reference is now made to Figure 1, which illustrates one example of a bioreactor 100, according to embodiments of this disclosure, for culturing cells. In embodiments, the bioreactor 100 includes an external casing or housing 112 forming an interior compartmentAttorney Docket No. SP24-269 and a (optionally) removable cover 114 for covering the interior compartment, which may include one or more openings or ports P, optionally with removable covers or caps C for allowing the selective introduction or removal of fluid, gas (including by way of a sparger), probes, sensors, samples, or the like. In embodiments, the bioreactor 100 may be used in connection with an external reservoir and conduits (e.g., inputs and outputs), which may form a continuous loop for circulating fluid to the bioreactor 100.

[0077] Within the interior compartment formed by the bioreactor housing 112, several compartments or chambers may be provided for holding, transmitting, and / or directing a flow of fluid (liquid and / or gas) throughout the bio reactor 100. In embodiments, the chambers may include a first chamber 116 enclosed by the outer wall(s) of the bioreactor 100 comprising or designed to hold a substrate 118. In embodiments, the chambers may include a second chamber 120. The second chamber 120 can be an integrated media conditioning chamber, where media to be circulated through the substrate 118 is conditioned. Media conditioning, as used herein, refers to adjusting or maintaining desired properties of the media circulated through the bioreactor 100. For example, the media can be cell culture media used to nourish the cells during cell culture, and the media conditioning chamber can be used to adjust the temperature, content (e.g., gas contents, dissolved oxygen, cell analytes and nutrients, etc.), pH, and other properties. The media can also be conditioned, in a case of harvesting cells, to have the desired properties for assisting in harvesting the cells from the first chamber 116, including, for example, the presence of one or more cell harvesting solutions. The second chamber 120 may also include one or more sensors, used to monitor a state of the media in the second chamber. The sensors can be inserted through the one or more ports P. In embodiments, the integrated media chamber can also be located in a headspace chamber 121 above the substrate. In embodiments, the chambers may also include a third chamber 122 at or near the base of the bioreactor 100. The third chamber 122 can include a circulator 124, such as an impeller. As a result of the provided circulator, fluid may flow upward through the first chamber 116 and thus through the substrate 118 contained therein. In embodiments, the fluid can then be recirculated down through the second chamber 120 and back to the circulator 124, where the cycle can be repeated. In this way, conditioned media can be circulated through the bioreactor during various cell culture processes.

[0078] The bioreactor 100 of Figure 1 can present challenges for harvesting cells. Due to the circuitous path of media flow and integrated media conditioning, harvesting cells is notAttorney Docket No. SP24-269 straight forward. For example, it is difficult to deliver a concentrated harvest solution, or reverse flow and / or deliver high volume flowrates to efficiently harvest cells using high shear force to remove the cells from the substrate. Accordingly, embodiments of this disclosure enable improved harvesting through the use of physical agitation (e.g., to loosen or detach cells from the substrate) and changing the flow direction (e.g., to loosen or detach cells and to efficiently extract cells). The ability to remove cells efficiently enables use of the bioreactor, for example, in a seed train (i.e., a series of processes to proliferate cells) and use the cells in therapy applications where cell removal from the bioreactor is a necessary step.

[0079] According to embodiments, the entire bioreactor vessel or a sub-component that includes the fixed bed or substrate can be mechanically vibrated to remove the adherent cells from the substrate surfaces. In embodiments, the vibration is designed to use high-frequency motion to eject or loosen cells that are either adhered or entrapped in the substrate. The high- frequency vibration can also cause cavitation in the fluid, resulting in shear and removal of cells from the substrate. However, excessive vibration or cavitation can result in cell damage or death, so an optimum frequency and time are required depending on the design of the bioreactor, the fixed bed and substrate, cell type, and energy delivery method. Thus, embodiments of this disclosure include systems and method where the frequency is adjustable for the specific need.

[0080] In embodiments, the bioreactor 100 can include an agitator designed to generate the vibrations used for harvesting. In embodiments, the agitator can include a vibration unit 130 that can be attached to the bioreactor 100 prior to or after cell growth and proliferation is complete. In embodiments, the bioreactor 100 could be relocated to the vibration unit 130 to deliver the mechanical energy, such that multiple bioreactors can be used with a single vibration unit, or the vibration unit 130 can be attached to or permanently integrated with the bioreactor 100. In aspects of embodiments, the vibration unit 130 is a vibration table, shake table, piezoelectric transducer, capacitive transducer, ultrasonic transducer, or other device capable of producing vibrations are a frequency useful for harvesting cells from the bioreactor. The possible vibration frequency ranges from single or tens of megahertz to single megahertz. For example, the frequency can range from about 10 Hz to 1 MHz, 20 Hz to 50 kHz, or 30 Hz to 35 kHz, although embodiments are limited to these ranges.

[0081] As shown in Figure 1, the bioreactor 100 can be placed on top of the vibration unit 130. However, embodiments are not limited to this arrangement, and a vibration unit could beAtorney Docket No. SP24-269 placed on the sides or top of the bioreactor, as well. In embodiments, a subcomponent of the bioreactor containing the fixed bed substrate (and cells) can be connected directly to a vibration source placed within the outer vessel of the bioreactor, the subcomponent containing the fixed bed can be removed from the outer vessel of the bioreactor and placed in a separate vibration unit and / or harvesting device. The vibration can be the main driver or a secondary driver for enhancing cell harvest / removal from fixed bed. For example, in addition to the vibration, one or more cell harvesting solutions can be deployed within the fixed bed to facilitates cell detachment, or pressurized flow can be used to assist in cell detachment via shear force from the pressurized fluid. Additional forces, such as centrifugal force, can also be used to assist in cell detachment and separation from the fixed bed. For example, the bioreactor 100 or a subcomponent containing the fixed bed can be rotated such that centrifugal force aids in removal of the cells.

[0082] As discussed above, the source of vibration (i.e., agitator) can be integrated into the bioreactor. Embodiments of an integrated agitator are shown in Figures 2-5. These embodiments can include, for example, a thin wall or membrane in the bioreactor vessel in targeted locations that can vibrate when atached or coupled by a transducer. The vibration transfers to the fluid to aid in cell harvest / removal from mesh. In Figure 2, the vibration membrane 132 is placed at the botom of the bioreactor 100 or botom of the third chamber 122. In Figure 3, the vibration membrane 134 is placed within the second chamber 120 or the integrated media conditioning chamber. In Figure 4, the vibration membrane 136 is placed at the top wall of the bioreactor 100. In Figure 5, the vibration membrane 138 surrounds the fixed bed 118. Alternatively, the vibration membrane 138 could be positioned on the outside of the second chamber 120 or inside the first chamber 116.

[0083] According to embodiments, cell harvesting can also be achieved or assisted by reversing or altering the direction of the flow of fluid through the bioreactor. For example, the integrated media reservoir bioreactor could be modified to include alternative flow paths to facilitate flow of high velocity media through the fixed bed and collection in a separate vessel (or, in some embodiments, in the reservoir). The bioreactor 100 shown in Figures 1-5 continuously recirculates media from the reservoir through the fixed bed. A removal process to remove cells from the substrate could be ineffective if the cells that are removed from the substrate continue to be recirculated back into the fixed bed, potentially re-entrapping them. Several methods for seting up an isolated flow path are conceived in Figures 6-8. ValvingAttorney Docket No. SP24-269 and flow design changes can be incorporated to disable circulation of the fluid between reservoir and fixed bed when harvesting is in process. If the recirculation loop is interrupted, additional components can then be used to drain and collect fluid and directly flush reactor with high pressure media. In some embodiments, a cell harvesting solution can be used to assist in cell detachment. For example, the harvest solution may contain an enzyme to assist is cell detachment. Cells can be collected into a harvest collection container. Embodiments include several designs and methods to interrupt the recirculation loop, including, for example, using a valve (i.e., butterfly valve) to restrict or redirect flow. Embodiments include a movable device placed into the flow path to block off recirculation through the bed and / or direct it out to a collection vessel. Embodiments also include an inflatable structure or bag within the bioreactor that, when needed, inflates to block the recirculation loops and / or redirect flow to the harvesting pipeline. In some embodiments, a movable plate 200 could function similar to a syringe pump or piston head to pulse and / or drive fluid through bed at high flow rate, redirecting cells through a harvesting line 202 to a harvesting collection vessel 204, as shown in Figure 6.

[0084] As shown in Figure 7, a harvest solution or other fluid 210 can be driven through the bioreactor having a one-way valve 212 at high pressure, sufficient to shear cells from the fixed bed substrate. Then, a collection vessel 214 can collect the harvest cells. The injection of the harvest solution or other fluid can be made downstream of the one-way valve 212, and hydrostatic pressure above the one-way valve can present continued recirculation of cells through the fixed bed. Figure 8 shows a similar arrangement to that in Figure 7, but uses a temporary or moveable fluid flow block 220 to block recirculation of fluid.

[0085] According to these embodiments, it is possible to feed media with or without a concentrated harvest-enabling enzyme at high flow rates through the mesh and into a collection vessel.

[0086] In some embodiments, fluid exiting the first chamber 116 is passed to a chamber on one (upper) side of the fixed bed, where the fluid is exposed to a gas (such as oxygen or nitrogen). In some embodiments, fluid may then flow radially inwardly to a central return chamber (e.g., second chamber 120). In some embodiments, the central return chamber can be columnar in nature and may be formed by an imperforate conduit or tube or rather formed by the central opening of the structured spiral fixed bed. In some embodiments, the chamber returns the fluid to the third chamber for recirculation through the bio reactor 100, such that aAtorney Docket No. SP24-269 continuous loop results (“botom to top” in this version). In some embodiments, a sensor, for example a temperature probe or sensor T may also be provided for sensing the temperature of the fluid in the second chamber 120. In some embodiments, additional sensors (such as, for example, pH, oxygen, dissolved oxygen, temperature) may also be provided at a location before the fluid enters (or re-enters) the chamber.

[0087] Embodiments of a matrix material for use as a structured fixed bed in the bioreactor of the present disclosure include a spiral bed. In some embodiments, one or more cell immobilization layers are provided adjacent to one or more spacer layers made from a mesh structure. In some embodiments, the layering may optionally be repeated several times to achieve a stacked or layered configuration. In some embodiments, the mesh structure included in spacer layers forms a tortuous path for cells and fluid to flow when layered between two immobilization layers. Homogeneity of the cells is maintained within the structured fixed bed as a result of this type of arrangement. In some embodiments, other spacer structures can be used which form such tortuous paths. In some embodiments, the structured fixed bed can be subsequently spirally or concentrically rolled along an axis or core. In some embodiments, the layers of the structured fixed bed are firmly wound. In some embodiments, the diameter of the core, the length and / or amount of the layers will ultimately define the size of the assembly or matrix.

[0088] According to embodiments of this disclosure, systems and methods for improved cell harvesting efficiencies when harvesting adherent cells from fixed bed bioreactors are provided. According to embodiments, harvesting the cells growing on the surface of the substrate can be assisted by a cell dissociation reagent (CDR. CDR is used in adherent cell culture to detach cells from culture substrate. A component of a CDR is an enzyme with proteolytic activity. The enzyme digests the surface adhesion proteins and causes the separation of cells from the substrate. Trypsin is a commonly used CDR in cell passaging; however, excessive trypsinization can lead to irreversible cell damage. There are several trypsin alternative products developed for gentler detachment. Accutase® (by Innovative Cell Technologies, Inc.) is a natural enzyme mixture with proteolytic and collagenolytic enzyme activity. It is non-mammalian, non-bacterial reagent. TrypLE® (by Life Technologies Corporation) is a recombinant bacterial-derived animal origin-free product, and cGMP- compliant version is also available in the market. Both Accutase® and TrypLE® are very gentle to harvested cells.Attorney Docket No. SP24-269

[0089] However, as discussed above, cell harvesting from a fixed-bed bioreactor is more challenging compared to a two-dimensional culture environment. Although bioreactors capable of recovering viable cells from FBR substrates have been demonstrated, the harvest efficiency could be more consistent, especially for high density cell culture.

[0090] As used herein, “harvest efficiency” refers to a percentage of cells in a cell culture run that can be harvested from the bioreactor by release and removal of the 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 cells harvested with the number of cells detected in the bioreactor as determined by manually disassembling the bioreactor after the harvesting process is completed and crystal violet staining the substrate to determine the number of remaining cells on the substrate that were not successfully harvested.

[0091] As an aspect of embodiments, harvesting efficiency can be further improved by adding an endonuclease to one or more harvest solutions used in a harvesting process. For example, a deoxyribonuclease, such as deoxyribonuclease I (DNase I), can be added to any harvest solution. DNase I is an endonuclease that hydrolyzes phosphodiester bonds and breaks or cleaves either double or single stranded DNA into oligonucleotides. DNase has been used in tissue digestion and single-cell suspension preparation. The purpose of addition of DNase to one of the harvest solutions in embodiments of this disclosure is to digest DNA accumulated on bioreactor substrate to improve harvest efficiency. As mentioned, in a particular embodiment, DNase I is added to the post-harvest solution; however, DNase I can be added to pre-harvest solution and / or cell harvest solution. Divalent metal ions, such as Mg+2, Ca+2or Mn+2, can also added to ensure full DNase I activity.

[0092] According to embodiments of this disclosure, a method for harvesting cells from a bioreactor can include providing the bioreactor comprising cells for harvesting. The bioreactor itself has at least one inlet and / or outlet, and a culture chamber in which the cells are adhered to a substrate. As an aspect of embodiments, the bioreactor has an inlet and an outlet on opposite sides of the bioreactor, with the substrate in between the inlet and outlet, so that fluid (e.g., cell culture media, nutrients, cells, and cell byproducts) can flow in a substantially linear direction from the inlet, through the substrate, and to the outlet. This simple linear flow path is believed to improve fluid flow uniformity, and thus cell cultureAttorney Docket No. SP24-269 uniformity and harvesting. However, embodiments are not intended to be limited to this arrangement and can include other flow designs.

[0093] According to additional aspects of embodiments, the cell dissociation reagent comprises a proteolytic enzyme, such as Accutase® or TrypLE®. The wash solution may further include an endonuclease, such as a deoxyribonuclease, for example. The deoxyribonuclease can include deoxyribonuclease I (DNase I). The wash solution can further include a divalent metal ion, such as Mg+2, Ca+2or Mn+2, for example.

[0094] In aspects of embodiments, the method can further include providing a pre-harvest solution in the culture chamber. The pre-harvest solution can include a phosphate buffered saline (PBS) solution. The pre-harvest solution is flowed into the bioreactor after the cell culture is completed to remove any cell culture media from the fixed bed substrate and to prepare the fixed bed for harvesting. The method can further include, after providing the preharvest solution, providing a harvest solution in the culture chamber for a predetermined incubation time. During this incubation time, a CDR in the harvest solution helps the cells detach from the substate so that they can be removed from the fixed bed. Following the predetermined incubation time, the harvest solution is flushed from the culture chamber. The flushing of the harvest solution acts to also flush the majority or entirety of the cells from the bioreactor. Following the flushing of the harvest solution, a post-harvest solution is provided in the culture chamber. The post-harvest solution can be used for an additional wash of the substrate and to harvest any additional cells that were not flushed with the harvest solution. The method can further include removing the post-harvest solution from the culture chamber and capturing residual cells removed by the post-harvest solution. At least one of the preharvest solution, the harvest solution, and the post-harvest solution includes the wash solution mentioned above, according to embodiments.

[0095] According to embodiments of this disclosure, a method for harvesting cells from a bioreactor can include providing the bioreactor having cells for harvesting. The bioreactor includes an inlet, an outlet, and a culture chamber in which the cells are adhered to a substrate. The method also includes providing a pre-harvest solution in the culture chamber; providing a harvest solution in the culture chamber for a predetermined incubation time; following the predetermined incubation time, flushing the harvest solution from the culture chamber; and following the flushing of the harvest solution, providing a post-harvest solutionAttorney Docket No. SP24-269 in the culture chamber. The detached cells are harvested during the flushing of the harvest solution and, optionally, during a flushing of the post-harvest solution.

[0096] In aspects of embodiments, the providing of the pre-harvest solution into the culture chamber removes cell culture media from the culture chamber. The pre-harvest solution comprises a wash solution, such as phosphate-buffered saline (PBS), for example. Other accepted cell washing solutions may also be used, as well. Following providing the preharvest solution, the pre-harvest solution may be drained from the bioreactor prior to fdling the bioreactor with the harvest solution, or the harvest solution may be supplied concurrently with removal of the pre-harvest solution. The harvest solution includes a cell dissociation reagent (CDR). The cell dissociation reagent comprises at least one of a calcium chelator and a proteolytic enzyme. According to aspects of embodiments, the flushing of the harvest solution from the culture chamber can include using pressurized gas flowed into the culture chamber to displace the harvest solution and force it out through one of the inlet and the outlet of the bioreactor. The flushing of the harvest solution also includes removing cells that have been detached from the substrate. Cells that have been detached and flushed are then captured in a harvest collection container.

[0097] In aspects of embodiments, the providing of the post-harvest solution in the culture chamber includes flowing the post-harvest solution into the culture chamber. If any cells remain in the fixed bed following flushing of the harvest solution, these residual cells can be removed from the culture chamber by the post-harvest solution and captured then. The postharvest solution can comprise a wash solution, such as PBS, or another accepted cell wash solution. As an aspect of embodiments, at least one of the pre-harvest solution, the harvest solution, and the post-harvest solution includes an endonuclease. The endonuclease can include a deoxyribonuclease, such as deoxyribonuclease I (DNase I), for example. In a further aspect, the at least one of the pre-harvest solution, the harvest solution, and the postharvest solution that includes the endonuclease further includes a divalent metal ion, such as Mg+2, Ca+2or Mn+2, for example. The presence of the divalent metal ion can increase the effectiveness of the endonuclease.

[0098] In further aspects of embodiments, the method can include mechanically agitating the culture chamber. The mechanical agitating of the culture chamber can occur during at least one of the pre-harvest solution, the harvest solution, and the post-harvest solution being in the culture chamber. The mechanical agitation can include at least one of stirring the contents inAttorney Docket No. SP24-269 the culture chamber, shaking or vibrating contents of the culture chamber, pulsing of fluid through the culture chamber, and altering a flow direction of fluid through the culture chamber.

[0099] The substrate used in the fixed bed or packed bed can take a variety of forms, but is preferably a porous material provided in a monolithic structure (such as a foam substrate or single woven mesh sheet) or in a packed-bed configuration of a plurality of pieces of substrate material or layers. In one embodiment, for example, the plurality of pieces of substrate material 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 stack of sheets or discs within the vessel.

[0100] According to an aspect of embodiments, the bioreactor systems disclosed herein can pressurize the bioreactor and generate a safe flow rate that will not damage the cells or harvested product. The action of the pressurization for harvesting can be automated and fully integrated into a bioreactor controller system.As an aspect of embodiments of this disclosure, a suitable cell culture substrate will enhance the flow uniformity and release of cells for harvesting. Examples of such substrate materials are disclosed in U.S. Patent No. 11,434,460, the content of which is incorporated herein by reference in its entirety. The cell culture substrate is porous to allow perfusion of cells, media, nutrients, and cell by-products through the substrate and to allow spent media with cell secreted material (e.g., recombinant protein, antibody, virus particles, DNA, RNA, sugars, lipids, biodiesel, inorganic particles, butanol, metabolic byproducts) to pass through the substrate and be harvested. Further details of the cell culture substrate according to embodiments are provided below.

[0101] Embodiments of the present disclosure include bioreactors and cell culture substrates used therein, including substrates that are cell growth matrices and / or packed-bed systems for anchorage dependent cells that enable 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, a substrate is provided with a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, mechanically stable, non-degradable woven meshes can be used to support adherent cell production. Uniform cell seeding of such aAttorney Docket No. SP24-269 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 achieve confluent monolayer or multilayer of adherent cells on the disclosed substrate, and can avoid formation of 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. The structurally defined substrate of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of bioreactor. In another embodiment of the present disclosure, a method of cell culturing is provided using bioreactors with the substrate for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0102] In one or more embodiments, the cell culture substrate supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. The substrate can be assembled and used in a bioreactor system, such as a perfused fixed bed bioreactor as described herein, and provide uniform cell distribution during the inoculation step, while preventing formation of large and / or uncontrollable cell aggregates inside the fixed bed. Thus, the substrate eliminates diffusional limitations during operation of the bioreactor. In addition, the substrate enables easy and efficient cell harvest from the bioreactor.

[0103] The substrate can be formed with a substrate material that of a thin, sheet-like construction 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, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were 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 meshlike layer; 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 to obtain uniform cell seeding, uniform media perfusion, and efficient cell harvest.Attorney Docket No. SP24-269

[0104] 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. 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.). An opening can be defined by a certain width or diameter. A woven mesh may be considered, 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.

[0105] The woven mesh can be comprised of monofilament or multifilament polymer fibers. In one or more embodiments, a monofilament fiber may have a diameter in a range of about 10 pm to about 1000 pm. On a microscale level, due to the scale of the fiber compared to the cells (e.g., the fiber diameters being larger than the cells), the surface of monofilament fiber is presented as regular 2D surface for adherent cells to attach and proliferate. Such fibers are woven into a mesh that has a defined pattern and a certain amount of structural rigidity. Fibers can be woven into a mesh with openings ranging from about 25 pm x 25 pm to about 1000 pm x 1000 pm. These ranges of the filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments.

[0106] 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 structure patterns or weaves, including, for example knitted, warp-knitted, or woven (plain weave, twilled weave, dutch weave, five needle weave).

[0107] 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 mesh or 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 adhesiveAttorney Docket No. SP24-269 properties through the treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry.

[0108] The woven mesh substrate may be provided in a number of discs with a center hole configured to surround the center column of the bioreactor described herein. A plurality of such discs can be stacked in the outer region of the bioreactor to form the packed bed.

[0109] According to some embodiments, the cell culture substrate is a dissolvable foam 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.

[0110] The embodiments disclosed herein enable not only cell attachment and growth to a cell culture substrate, but also the viable harvest of cultured cells. The inability to harvest viable cells is a significant drawback in current platforms, and it leads to difficulty in building and sustaining a sufficient number of cells for production capacity. According to an aspect of embodiments of this disclosure, it is possible to harvest viable cells from the cell culture substrate, including between 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the cells that are harvested, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase®.

[0111] It will be appreciated that the various disclosed embodiments may involve particular features, elements or steps that are described in connection with that particular embodiment. It will also be appreciated that a particular feature, element or step, although described in relation to one particular embodiment, may be interchanged or combined with alternate embodiments in various non-illustrated combinations or permutations.

[0112] It is also to 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 explicitly indicated to the contrary. Thus, for example, reference to “an opening” includes examples having two or more such “openings” unless the context clearly indicates otherwise.

[0113] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, examples include from theAttorney Docket No. SP24-269 one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0114] All numerical values expressed herein are to be interpreted as including “about,” whether or not so stated, unless expressly indicated otherwise. It is further understood, however, that each numerical value recited is precisely contemplated as well, regardless of whether it is expressed as “about” that value. Thus, “a dimension less than 10 mm” and “a dimension less than about 10 mm” both include embodiments of “a dimension less than about 10 mm” as well as “a dimension less than 10 mm.”

[0115] 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 no way intended that any particular order be inferred.

[0116] While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase “comprising,” it is to be understood that alternative embodiments, including those that may be described using the transitional phrases “consisting” or “consisting essentially of,” are implied. Thus, for example, implied alternative embodiments to a method comprising A+B+C include embodiments where a method consists of A+B+C, and embodiments where a method consists essentially of A+B+C.

[0117] Although multiple embodiments of the present disclosure have been illustrated in the accompanying Drawings and described in the foregoing Detailed Description, it should be understood that the disclosure is not limited to the disclosed embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the disclosure as set forth and defined by the following claims.

Claims

Attorney Docket No. SP24-269CLAIMS:

1. A bioreactor for culturing and harvesting cells, the bioreactor comprising: a vessel comprising an interior cavity and one or more media ports configured for passing fluid between the interior cavity and an exterior of the vessel; a substrate disposed in the interior cavity and comprising a surface configured for adhering cells thereto; and an agitator arranged to transmit mechanical energy to at least one of the vessel, the substrate, and a fluid in the interior cavity, wherein the agitator is configured to transmit mechanical energy sufficient to contribute to either detaching cells adhered to the substrate or dislodging cells entrapped in the substrate.

2. The bioreactor of claim 1, wherein the vessel further comprises an integrated media reservoir configured to contain cell culture media and not contain the substrate.

3. The bioreactor of claim 2, wherein the bioreactor further comprises one or more media conditioning ports configured to deliver fluid to the integrated media reservoir for conditioning cell culture media in the integrated media reservoir.

4. The bioreactor of claim 2 or claim 3, wherein the integrated media reservoir is disposed in a center of the vessel and the substrate surrounds the integrated media reservoir.

5. The bioreactor of any one of claims 1-4, wherein the vessel further comprises: a housing forming an interior compartment, the interior compartment comprising: a first chamber comprising the substrate, the first chamber having a fluid input end and a fluid output end; and a second chamber within the first chamber, the second chamber defined by an imperforate wall between the first chamber and the second chamber for returning fluid having passed through the substrate from the fluid input end to the fluid output end back to the fluid input end of the first chamber.Attorney Docket No. SP24-2696. The bioreactor of claim 5, the interior compartment further comprising a third chamber for recirculating fluid through the substrate.

7. The bioreactor of claim 6, wherein the third chamber is disposed below or above the first chamber.

8. The bioreactor of claim 6 or claim 7, the third chamber comprising an impeller.

9. The bioreactor of any one of claims 1-8, wherein the agitator is configured to vibrate at least one of the vessel, the substrate, and the fluid in the interior cavity.

10. The bioreactor of any one of claims 1-9, wherein the vessel is disposed on top of the agitator.

11. The bioreactor of claim 10, wherein the agitator is a vibration table comprising a platform sized and shaped to support the vessel thereon, and the platform is configured to move in such a way as to cause vibrations to travel through at least one of the vessel, the substrate, and the fluid in the interior cavity.

12. The bioreactor of claim 10 or claim 11, wherein the agitator comprises one or more anchor points configured to be coupled to the vessel.

13. The bioreactor of claim 12, wherein the one or more anchor points are configured to couple to the vessel such that the platform and the vessel move in conjunction with one another.

14. The bioreactor of any one of claims 10-13, wherein the agitator is attached to the bottom of the vessel.

15. The bioreactor of any one of claims 1 -9, wherein the agitator comprises a membrane configured to oscillate at a predetermined frequency.Attorney Docket No. SP24-26916. The bioreactor of claim 15, wherein the membrane is disposed around the substrate between the substrate and an inner wall of the vessel.

17. The bioreactor of claim 15, wherein the membrane is disposed in the integrated media reservoir.

18. The bioreactor of claim 15, wherein the membrane is disposed on at least one of a top wall and a bottom wall of the vessel.

19. The bioreactor of claim 15, wherein the substrate surrounds the membrane.

20. The bioreactor of any one of the preceding claims, further comprising a controller configured to control the agitator.

21. The bioreactor of claim 20, wherein the controller is configured to control at least one of a frequency of motion and a time of operation of the agitator.

22. The bioreactor of claim 21, wherein the frequency of motion is from about 50 Hz to about 31 kHz.

23. The bioreactor of any one of claims 1-21, wherein the agitator is configured to generate ultrasonic waves.

24. The bioreactor of any of the preceding claims, further comprising a vessel rotator configured to rotate the vessel about a longitudinal axis of the vessel sufficient to generate a centrifugal force to contribute to either detaching cells adhered to the substrate or dislodging cells entrapped in the substrate.

25. A bioreactor for culturing and harvesting cells, the bioreactor comprising: a vessel comprising an interior cavity and one or more media ports configured for passing fluid between the interior cavity and an exterior of the vessel; andAttorney Docket No. SP24-269 a substrate disposed in the interior cavity and comprising a surface configured for adhering cells thereto, wherein the interior cavity comprises: a first chamber comprising the substrate, the first chamber having a fluid input end and a fluid output end; and a second chamber within the first chamber, the second chamber defined by an imperforate wall between the first chamber and the second chamber for returning fluid having passed through the substrate from the fluid input end to the fluid output end back to the fluid input end of the first chamber.

26. The bioreactor of claim 25, wherein the second chamber comprises an integrated media reservoir configured to contain cell culture media and not contain the substrate.

27. The bioreactor of claim 26, wherein the bioreactor further comprises one or more media conditioning ports configured to deliver fluid to the integrated media reservoir for conditioning cell culture media in the integrated media reservoir.

28. The bioreactor of any one of claims 25-27, the interior compartment further comprising a third chamber for recirculating fluid through the substrate.

29. The bioreactor of claim 28, wherein the third chamber is disposed below or above the first chamber.

30. The bioreactor of claim 28 or claim 29, the third chamber comprising an impeller.

31. The bioreactor of any one of claims 25-30, further comprising a harvest collection vessel fluidly connected to the interior cavity and configured to receive contents harvested from the substrate.

32. The bioreactor of any one of claims 25-31, further comprising a harvest solution container fluidly connected to the interior cavity and configured to hold a harvest solution,Atorney Docket No. SP24-269 the harvest solution being a solution configured to detach cells adhered to the substrate when the harvest solution is transferred from the harvest solution container to the interior cavity.

33. The bioreactor of any one of claims 25-32, further comprising a pressurized fluid delivery mechanism configured to deliver fluid under pressure to the interior cavity at a pressure sufficient to detach cells from the substrate.

34. The bioreactor of claim 33, wherein the pressurized fluid delivery mechanism comprises at least one of a pump, a syringe, a piston, a bladder, and a pressurized gas.

35. The bioreactor of claim 33 or claim 34, wherein the pressurized fluid delivery mechanism is configured to deliver the harvest solution to the interior cavity.

36. The bioreactor of any one of claims 31-35, further comprising a one-way valve within the interior cavity, the one-way valve being configured to allow fluid to flow in a direction toward the harvest collection vessel during a cell harvesting operation.

37. The bioreactor of any one of claims 32-36, wherein the harvest solution is a cell disassociation reagent.

38. The bioreactor of any one of claims 1-37, further comprising a valve configured to redirect a flow of fluid within the vessel during cell harvesting.

39. The bioreactor of claim 38, wherein the valve is configured to block a recirculation path of fluid within the interior cavity during cell harvesting.

40. The bioreactor of claim 39, wherein, during cell harvesting, the fluid is redirected to an exterior of the interior cavity via an outlet fluidly connected to a harvest collection vessel.

41. A method of culturing and harvesting cells from a bioreactor, the method comprising: providing a bioreactor of any one of claims 25-40;Attorney Docket No. SP24-269 seeding cells onto the substrate, the cells being adherent cells that adhere to or are entrapped by the substrate; recirculating cell culture media through the vessel along a recirculation path, the recirculation path passing through the first chamber and the second chamber during the culturing of the cells; conditioning the cell culture media within the second chamber; and harvesting cells from the substrate by detaching or dislodging the cells from the substrate and removing the cells from the interior cavity.

42. The method of claim 41, wherein the harvesting comprises generating vibrations that pass through at least one of the vessel, the cell culture media, and the substrate, thereby assisting in the detaching or dislodging of the cells.

43. The method of claim 42, further comprising, prior to generating the vibrations, placing the vessel on a vibration table configured to transmit the vibrations to the vessel.

44. The method of claim 42, wherein generating the vibrations comprises oscillating a membrane in the interior cavity.

45. The method of any one of claims 41-44, further comprising redirecting a flow of cell culture media in the interior cavity during the harvesting.

46. The method of any one of claims 41-45, further comprising reversing a flow direction of cell culture media in the interior cavity during the harvesting.

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