Apparatus for culturing shear sensitive cells and related methods

Abstract

An apparatus for culturing cells in connection with a fluid is provided. The apparatus includes a bioreactor including a bed for culturing cells, an agitator for circulating the fluid within the bioreactor, and a controller adapted to control the agitator to operate at a first speed during a first predetermined amount of time and a second speed thereafter. A bioreactor having a fixed bed including a coating to promote cell adhesion and growth is also provided. Related methods are also disclosed.

Description

[0001] APPARATUS FOR CULTURING SHEAR SENSITIVE CELLS AND RELATED METHODS

[0002] This application claims the benefit of U.S. Provisional Patent Application Ser. Nos. 63 / 682839 and 63 / 779766, the disclosures of which are incorporated by reference.

[0003] TECHNICAL FIELD

[0004] This document relates generally to the cell culturing arts and, more particularly, to an apparatus for culturing cells and, in particular, cells sensitive to shear stress and / or grown in an adherent manner on a surface of a fixed bed, as well as related methods.

[0005] BACKGROUND

[0006] Cell culturing often involves the use of a bioreactor, which may include a fixed or packed bed designed for adherent, semi-adherent, or suspended animal cell growth using a volume of culture medium serving as an associated fluid. In typical applications, this culture medium or fluid is circulated throughout the bioreactor to provide oxygen and nutrients to the growing cells. This is typically achieved using an agitator for causing the fluid to circulate, which may take the form of a rotating impeller positioned in contact with the fluid in the bioreactor, but could also take the form of a pump or other device for circulating the fluid therein.

[0007] In situations where the cells are sensitive to shear, such as stem cells, the rotation of the impeller or otherwise causing the fluid to circulate and too high of a rate may deleteriously damage the cells when in suspension in the circulating culture medium and before adhering to the fixed bed. This is necessarily the case during an inoculation phase of operation, during which time the cells are in suspension before attachment to the fixed bed, which requires the presence of low shear conditions to promote adhesion in an efficient and effective matter.

[0008] Aside from that consideration, the damage resulting from shear can lead to a reduction in the number of viable cells (total number of cells reduction and viability), in a degradation of the cell metabolism activity. A concomitant reduction in the yield achieved from the bioprocessing operation may result. This damage may be particularly deleterious in situations where the cells need a long period to achieve adherence, such as stem cells.

[0009] Non-adherent cells may also settle in the bioreactor or the recirculation vessel, creating areas of localized cell accumulation. If stressed or rendered dead, these cells can release toxic metabolites or signaling molecules, such as cytokines or death ligands. This contamination may disrupt the metabolism of healthy cells or trigger apoptosis through activation of death receptors.

[0010] Accordingly, a need is identified for an apparatus having the ability to enhance the adhesion yields from cell culturing using a bioreactor for adherent, semi-adherent, or suspended cell growth, particularly in connection with cells sensitive to shear, such as stem cells. The apparatus would reduce or eliminate the deleterious effects of shear on the cells, yet still provide the desirable fluid circulation to transport suspended cells through, within, or otherwise in contact with the material of the bed in order to achieve adherence. Additionally or alternatively, a fixed bed associated with the bioreactor could be enhanced to improve cell adhesion and growth, thereby further improving yields.

[0011] SUMMARY

[0012] An object of this disclosure is to provide an apparatus and related method affording the ability to enhance the yields from cell culturing using a bed for adherent cell growth, particularly in connection with cells sensitive to shear. The apparatus and method involves operating an agitator at a first reduced speed for a predetermined amount of time, such as during inoculation of the bioreactor, in order to reduce or eliminate the deleterious effects of shear on the cells. The apparatus and method further involves increasing the agitator speed to operate at a second higher speed of operation to provide the desirable fluid circulation to move suspended cells through the material or otherwise within the fixed bed in order to achieve adherence. Alternating between operational speeds may also be done, including during harvesting of the cells from the fixed bed.

[0013] Another object of this disclosure is to provide an apparatus and related method affording the ability to enhance the yields from performing cell culturing by applying a coating to the fixed bed. This may be done independent of or in connection with the above -referenced apparatus and method for enhancing yields.

[0014] According to a first aspect of the disclosure, an apparatus for culturing cells in connection with a fluid, such as media including cells, is provided. The apparatus comprises a bioreactor including a bed for culturing cells, including for example during an inoculation phase, and an agitator (impeller, pump, or any other fluid circulating device) for circulating the fluid within the bioreactor. The apparatus further includes a controller adapted to control the agitator to operate at a first speed during a first predetermined amount of time and a second speed thereafter. In one possible embodiment, the first speed is lower than the second speed, and in another possible embodiment, the first speed is higher than the second speed. The controller may also be adapted to control the agitator to operate at a third speed after the second speed, which third speed may be lower than the second speed. Alternatively or additionally, the controller may be adapted to control the agitator to operate at a third higher speed before the first speed.

[0015] In these or other embodiments, the controller may be adapted to control the agitator to alternate between the first and second speeds. The controller may also be adapted to control the agitator to operate at the second higher speed during a second predetermined amount of time.

[0016] In any embodiment, the agitator may comprise a rotatable impeller located within the bioreactor, and the bed may comprise a fixed bed. The fixed bed may include a coating to promote cell adhesion and growth. The coating may be selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0017] According to another aspect of the disclosure, an apparatus for culturing cells in connection with a fluid includes a bioreactor comprising a first chamber including an agitator for circulating the fluid within the bioreactor, a second chamber connected to a first outlet of the first chamber and including a bed, and a third chamber connected to a second outlet of the second chamber for returning fluid to the first chamber. The apparatus further includes a controller adapted to control the agitator to operate at a speed sufficient for a predetermined amount of time to circulate fluid within the second chamber but without entering the third chamber for returning to the first chamber.

[0018] In some embodiments, the controller is adapted to control the agitator to operate at the second higher speed during a second predetermined amount of time. The agitator may comprise a rotatable impeller located within the bioreactor, and the bed may comprise a fixed bed, which may include a coating to promote cell adhesion and growth.

[0019] According to another aspect of the disclosure, a bioreactor includes a fixed bed with a coating to promote cell adhesion and growth. The coating may be selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0020] According to still a further aspect of this disclosure, a method for culturing cells in a bioreactor including a fixed bed and an agitator. The method includes circulating media in the bioreactor at a first rate during a first period for achieving cell homogenization, and circulating media in the bioreactor at a second rate during a second period for achieving cell adhesion to the fixed bed.

[0021] In some embodiments, the method also includes the step of circulating media in the bioreactor at a third rate during a third period for promoting cell growth. The step of circulating media in the bioreactor at a first rate may comprise operating an agitator at a first speed for a first predetermined amount of time; and the step of circulating media in the bioreactor at a second rate comprises operating the agitator at a second speed. The first speed may be lower than the second speed. Alternatively, the first speed may be higher than the second speed, followed by a third speed that is lower than the second speed.

[0022] In any case, the method may further include the step of alternating the operation of the agitator between the first and second speeds. The step of harvesting the cells during the alternating step may also be performed. The operating steps may comprise rotating an impeller as the agitator, or using a pump external to the bioreactor as the agitator.

[0023] Still further, an aspect of this disclosure relates to a method for culturing cells in connection with a fluid. The method comprises providing a bioreactor comprising a first chamber including an agitator for circulating the fluid within the bioreactor, a second chamber connected to a first outlet of the first chamber and including a bed, and a third chamber connected to a second outlet of the second chamber for returning fluid to the first chamber. The method further comprises controlling the agitator to operate at a speed sufficient to circulate fluid within the second chamber but without entering the third chamber for returning to the first chamber for a predetermined amount of time.

[0024] Yet another aspect of the disclosure relates to a method of manufacturing a bioreactor including a fixed bed comprising applying a coating to the fixed bed to promote cell growth. The method may comprise selecting the coating from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0025] Another aspect of the disclosure pertains to a method of culturing cells. The method comprises coating a fixed bed in a bioreactor with a coating to promote cell adhesion and growth, and growing cells on the coated fixed bed.

[0026] In some embodiments, the coating is selected from the group consisting of Poly-D- Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof. The coating may be applied after the bioreactor is shipped to a customer for performing the step of growing cells on the fixed bed. Alternatively, the coating may be applied before the bioreactor is shipped to a customer for performing the step of growing cells on the fixed bed.

[0027] BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0028] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of this disclosure are utilized, and the accompanying drawings of which :

[0029] FIG. 1 is a perspective view of an exemplary bioreactor for which certain aspects of this disclosure may have applicability.

[0030] FIG. 2 is a partially exploded view of further details of the bioreactor of FIG. 1.

[0031] FIG. 3 illustrates a structured fixed bed for possible use in connection with a bioreactor.

[0032] FIGS. 3A, 3B, and 3C illustrate particular details of one example of a fixed bed. FIGS. 3D, 3E and 3F illustrate alternative arrangements for forming a fixed bed. FIGS. 3G, 3H, 31, and 3J illustrate still further alternative fixed bed arrangements. FIG. 4 is a flow diagram.

[0033] FIG. 5 is another flow diagram relating to manufacturing processes.

[0034] DETAILED DESCRIPTION

[0035] Reference is now made to FIGS. 1-3, which illustrate one embodiment of a fixed bed bioreactor 100 for culturing cells. In some embodiments, the bioreactor 100 includes an external casing or housing 112 forming or including an interior compartment and a cover 114 placed on top of the housing 112 to cover or seal the interior compartment after it is populated with at least the fixed bed. In an embodiment, the cover 114 is removable. The cover 114 may include various openings or ports with removable closures or caps C for allowing for the selective introduction or removal of material, fluid, gas, probes, sensors, samplers, or the like.

[0036] Within the interior compartment of the bioreactor housing 112, several compartments or chambers may be provided for transmitting a flow of fluid, gas, or both, throughout the bioreactor 100. As indicated in FIG. 2, in some embodiments, the chambers may include a first chamber 116 at or near a base of the bioreactor 100. In some embodiments, the first chamber 116 may include an agitator for causing fluid flow or circulate within the bioreactor 100. In some embodiments, the agitator may be in the form of a drop-in, rotatable, noncontact magnetic impeller 118 (such as, for instance, a stir bar), which thus forms a centrifugal pump in the bioreactor. The agitator could also be in the form of an impeller with a mechanical coupling to the base, an external pump forming part of a fluid circulation system, or any other device for causing fluid circulation within the bioreactor, regardless of the relative location or type.

[0037] In some embodiments, as a result of the agitation provided, fluid may then flow upwardly (as indicated by arrows A in FIG. 2) into a chamber 120 along the outer or peripheral portion of the bioreactor 100, or otherwise through a fixed bed therein. FIG. 3 shows an exemplary fixed bed in the form of a structured spiral bed 122 which, in use, may contain and retain cells being grown. In some embodiments, the spiral bed 122 may be in the form of a cartridge that may be built within and as a part of or introduced into the outer chamber 120. The bed 122 can be pre-installed in the chamber during manufacture at a facility prior to shipping or installed at the point of use. Other forms of fixed beds, such as structured, monolith or packed beds, may be used instead of the version shown in the drawings.

[0038] In some embodiments, fluid exiting the chamber 120 is passed to a headspace formed in a zone between one (upper) side of the bed 122 and the cover 114, where the fluid (media) is exposed to a gas (such as oxygen). In some embodiments, fluid may then flow radially inwardly to a central chamber 126 to return to the lower portion of bed 122. In some embodiments, this central chamber 126 can be columnar in nature and may be formed by an imperforate conduit or tube 128 or rather formed by the central opening of the structured spiral bed. The flow of fluid into the central chamber 126 may be such that a waterfall is created, thereby promoting gas transfer to the fluid.

[0039] In some embodiments, the chamber 126 returns the fluid to the first chamber 116 (return arrow R) for recirculation through the bioreactor 100, such that a continuous loop results (bottom to top in this version). In some embodiments, a sensor, for example a temperature probe or sensor may also be provided for sensing the temperature of the fluid flowing or residing in the chamber 126. In some embodiments, additional sensors (such as, for example, pH, oxygen, dissolved oxygen, temperature, cell density, etc.) may also be provided at a location before the fluid enters (or re-enters) the chamber 116. However, as can be understood by the skilled artisan, this is merely an exemplary arrangement, and other forms of bioreactors exist. For example, the arrangement could be such that the fixed bed is centrally located in the bioreactor, and fluid flows to an outer chamber to the first chamber (not shown). In any case, the fluid may also flow from the top of the fixed bed to the bottom, instead of from the bottom to the top, as previously indicated. FIG. 3 shows one embodiment of a matrix material for use as a structured fixed bed in the bioreactor of the present disclosure and, in particular, one in the form of a spiral bed 122. In some embodiments, the spiral bed may comprise one or more cell immobilization layers 122a or structures. In another embodiment, the cell immobilization layers or structures may comprise one or more woven or non-woven layers or a combination of the foregoing. The layers 122a used may be laminated to each other or may comprise layers that are unattached to each other.

[0040] In one embodiment, one or more of the cell immobilization layers 122a may be provided adjacent to one or more spacer layers 122b or structures. The spacer layers 122b may be made from a mesh structure, which may be woven or nonwoven, and may have an ordered or a disordered array of openings or pores. In some embodiments, the layering may optionally be repeated several times to achieve a stacked or layered configuration. The layers 122a, 122b may be identical or different.

[0041] In some embodiments the mesh structure included in spacer layers 122b forms tortuous paths to steer the cells into the depth of the cell immobilization layers 122a (see cells L in FIG. 3A suspended or entrapped in the material of the immobilization layer 122a). As shown in FIGS. 3B and 3C, the spacer layers 122b may also form channels 122c in conjunction with the adjacent cell immobilization layers 122a for fluid and bubbles to flow therethrough (see arrows A in FIG. 3B indicative of flow between layers, and also arrows B in FIG. 3C indicative of transverse flow). Increased 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.

[0042] In some embodiments, as shown in FIGS. 3, 3A and 3B, the structured fixed bed can be subsequently spirally or concentrically rolled along an axis or core (e.g., conduit or tube 128, which may be provided in multiple component parts). 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. In some embodiments, thickness of each of the layers 122a, 122b may be between 0.1 and 5 mm, 0.1 and 10 mm, or 0.001 and 15 mm. In some embodiments, other structures can be used which form such tortuous paths. For example, FIG. 3D shows that the one or more cell immobilization layers 122a may be adapted to form a structured fixed bed 122. The one or more layers 122a provide a tortuous channel of flow (arrow B) from a linear or regular inflow (arrow A) without using additional spacer layers (but such may be used, if desired). This may be achieved, for example, by providing one or more layers of woven fibers or filaments 123, 125 that disrupt the flow.

[0043] FIG. 3E shows that such a result may be achieved using a non-woven material as the cell immobilization layer 122a. This may be achieved by forming the layer 122a as a reticulated arrangement (such as by 3-D printing) with openings 127 through which liquid may pass and return again, thus forming the tortuous channels that again promote homogeneity and also serve to further shear or divide any bubbles present in the liquid. This function may again be achieved with or without added spacer layers being present.

[0044] The orientation of the structured fixed bed 122 may be other than as shown in a bioreactor 100 as shown in FIG. 2, where the flow is arranged vertically (bottom to top, in the example provided). For example, as shown in FIG. 3F, a bioreactor 100 may include a first chamber 120 that includes a structured fixed bed 122 comprised of one or more horizontally arranged material layers. The one or more layers may comprise a woven or reticulated material, as per FIGS. 3D and 3E, or non-woven, but as illustrated in FIG. 3F, may comprise one or more cell immobilization layers 122a (three shown, but any number may be present) sandwiched by adjacent spacer layers 122b (vertical spacing exaggerated for purposes of illustration), which are optional.

[0045] The flow is thus arranged from side-to-side (left to right or right to left), with the material layer(s) (spacer or otherwise) providing for the channels for creating the tortuous flow (arrows B) from a linear or regular inflow (arrow A), but could also be vertical. The pumping action may be provided by an agitator or other pump, which may be located at the entrance end of the chamber 120, and a return path provided at the exit end, as schematically illustrated by path R. Any arrangement for providing media circulation may be used as the agitator including, for example, a pump or agitator located external to the bioreactor (such as in a separate reservoir or conduit for supplying media to the bioreactor). Additional spacer layers may also be provided between the cell immobilization layers 122a, if desired.

[0046] In another possible embodiment, and with reference to FIG. 3G, the structured fixed bed 122 comprises a three-dimensional (3D) monolith matrix 124. The matrix 124 may take the form of a scaffold or lattice formed of multiple interconnected units or objects 124 a (e.g., round or spherical beads connected by connectors), which objects have surfaces for cell adhesion. The matrix 124 may include a tortuous path for fluid and cells to flow therethrough when in use. In some embodiments, the matrix 124 may be in the form of a 3D array, lattice, scaffolding, or sponge. The matrix 124 may be single use in nature to avoid the cost and complexities involved in cleaning according to bioprocessing standards. The fixed bed 122 in any case may comprise polymeric materials compatible in cell culture applications, including, for example, Polystyrene (PS), polyethylene terephthalate (PET), polycarbonate (PC), polyvinylpyrrolidone, polybutadiene, polyvinylchloride (PVC), polyethylene (PE), polyethylene oxide, polypyrroles, polypropylene (PP), polypropylene oxide, Ethylene-vinyl acetate (EVA), Polyethylene terephthalate glycol-modified (PETG), Thermoplastic polyurethane (TPU), Polydimethylsiloxane (PDMS) , Polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (TPFE), Fluoropolymers (PFA / FEP), Polyether ether ketone (PEEK), and combinations thereof.

[0047] FIGS. 3H, 31, and 3J illustrate still other forms of fixed beds, including in combination with a bioreactor 100 in the case of FIGS. 3H and 3J. The fixed bed 122 in the depiction of FIG. 3H comprises plural spaced cell immobilization layers 122a arranged in a vertical stack between an inlet I and an outlet O (but the arrangement could be reversed or used in different orientations from the vertical one shown). The fixed bed 122 in FIG. 31 includes a single cell immobilization layer 122a of spirally wound material, which may be woven as indicated in the enlarged portion of the figure, but could be non-woven instead. The fixed bed 122 in the bioreactor 100 shown in FIG. 3J is provided with a solid core R around which a layer of material (woven or non-woven) is spirally wound without any spacers.

[0048] According to a first aspect of the disclosure, and turning back to FIG. 1, a controller 200 is provided for controlling one or more functions of the bioreactor 100, including in this example actuation of the agitator, such as impeller 118, to control a rate of fluid or media circulation within the bioreactor 100 and in particular, through or within the fixed bed 122. In the specific embodiment shown, the controller 200 may activate the impeller 118 (in the case of a magnetic coupling, by using an external driver D coupled thereto in a non-contact fashion) to rotate at a variable speed in order to control the rate of circulation of fluid within the bioreactor 100. In one example, this includes a first lower speed of rotation during an inoculation phase during which cells are introduced to the bioreactor 100 and remain in suspension, moving within the bioreactor at a particular linear speed (e.g., 0.1-0.5 cm / s) as a result of a first rate of fluid circulation, and a second higher speed of rotation during a growth phase once the cells have adhered to the particular matrix material, such as the structured fixed bed 122, as the result of a second rate of fluid circulation. The same procedure may be used in a situation where an external pump serves as the agitator, with the controller 200 serving to control the pump speed in order to vary the rate of fluid circulation.

[0049] The controller 200 as indicated in FIG. 1 may alternately actuate or drive the agitator, such as impeller 118, between predetermined periods of operation at different speeds, such as the aforementioned first speed and second speed. For example, during the inoculation phase, as shown in the diagram of FIG. 4, in the impeller 118 may be activated at the first speed (e.g., 300-800 rpm) for a first predetermined time (e.g., up to 60 minutes, and including but not limited to any time from greater than zero to 60 minutes). During this first period, the cells are added and circulated within and / or through the bed, such as the structured fixed bed 122, in order to ensure homogenous distribution.

[0050] After the first predetermined time, which may be empirically determined as the time needed for the cells to distribute within the bed (and for example could be from a few minutes to several hours), a second speed or rate of rotation, which may be lower than the first speed, is implemented (which may be done gradually over time, or in an abrupt manner). This second speed is used to circulate the fluid while reducing or eliminating the deleterious effects of shear on the cells and to facilitate achieving adhesion to the surfaces of the fixed bed when the cells make contact therewith. During this period, the cells experience little or no shear and may rest in contact with the corresponding surfaces, which thus facilitates adhesion to the material forming the fixed bed 122.

[0051] Variables that may factor into the empirically determined times may include, for example, agitation / circulation speed, cell culture media composition (e.g., the addition of additives boosting cell culture adhesion), the presence or absence of coating molecules, the presence or absence of hydrophilisation treatment of the fixed bed. Other factors may include the design of the fixed bed, the direction of the media circulation, and / or cell density at inoculation (per surface available for the cells and per volume of cell culture media liquid).

[0052] After this second predetermined time, the impeller 118 may be activated by the controller 200 to operate at a third speed. This third speed may be higher in order to circulate the fluid in the bioreactor 100 through the bed 122. This application of the third speed thus facilitates delivering nutrients to the cells once adhered to the fixed bed 122 as needed during a growth phase. In the case where the particular bioreactor 100 shown in FIG. 1 is used, the rotation of the impeller 118 at the first speed may be such that the agitation is inadequate to cause fluid to flow into the central column 126 and thus form a continuous loop. Instead, the fluid agitation simply causes fluid to flow within the chambers 116, 120, and within the structured fixed bed 122 located therein. Later, during the second higher rotational speed, the impeller 118 may cause agitation in the fluid sufficient to cause the fluid to enter the central column 126 upon exiting the upper portion of the fixed bed 122, thus forming a continuous flow loop within the bioreactor 100. However, it is possible for the flow arrangement to be reversed, such as flow moves from an inner chamber radially outwardly to a central column (the particular flow arrangement is not considered critical).

[0053] Example 1

[0054] As one example of one possible implementation of the aforementioned protocol, a test was conducted to demonstrate that cells adhere, grow, can be harvested from the scale-X hydro bioreactor and structured fixed-bed bioreactor and have a good cell behavior (viability, recovery and potency assay). For purposes of recovery / harvesting, cells could be optionally collected using the Cell Collect vibrating table, as outlined in International Patent Application PCT / EP2022 / 065264. Bioreactor cultures were done in parallel with relevant controls (flatware flasks) mimicking the bioreactor feeding strategy process (some cultures are 'sacrificed' at the end of the culture, the bioreactor could be potentially dismantled to perform the cartography of the cell density distribution inside the fixed-bed) to follow the cell density and metabolites behavior).

[0055] The parameters tested included:

[0056] Cell inoculation : the seeding efficiency and seeding kinetic are estimated by taking samples of the remaining cells in suspension after the 1-, 2-, 4- and 8-hours post inoculation. The target is or was to have most of the cells attached in less than 8 hours.

[0057] Cell growth: the cell growth is estimated by comparing the glucose and the lactate profile of the bioreactor vs. relevant TFs (tissue culture flask) / CSs (multi-stack culture flask) control performed under similar feeding conditions (ml / cells and ml / cm2). The target is to have an expected glucose consumption and lactate production.

[0058] Bioreactor control: the trends of DO / pH / temperature are monitored.

[0059] In-situ cell harvest: cell growth rate (PDL / day - population doubling level per day) and PDT (population doubling time) are estimated based on the harvested cell density. The harvest yield is estimated by measuring the remaining cells in the bioreactor after the in-situ cell lysis (based on their DNA content) but the arrangement could be in-situ cell lysis with ex-situ cell harvest. The target is to have a promising harvest yield.

[0060] Ex-situ cell harvest: harvest efficiency

[0061] Cell quality: the viability of the harvested cells is measured at the end of the run and the harvested cells are re-seeded in TFs to perform a plating / recovery efficiency. The target is to have a viability >80% with a good plating efficiency.

[0062] Without controlling the speed of the agitator per the teachings of this disclosure, the cells suffered during the inoculation step due to shear and thus did not grow well.

[0063] Process conditions and steps were then modified to reduce as much as possible the tip speed.

[0064] Inoculation step: o Bioreactor equilibration with serum-containing media. o At full bioreactor (a Scale-X Hydro bioreactor) volume (around 900ml), employ a lower impeller speed. o In intermittent cycle with agitated (e.g., 10-100 rpm) and "static" phase to prevent sedimentation and maintain cells in suspension with a very light agitation o Monitor the supernatant to evaluate floating cells and debris concentration during static phase o Media exchange at the end of the inoculation (overnight) to ensure cell growth in fresh media without dead cells and debris

[0065] During this second run, more cells attached with the inoculation at low shear and intermittent cycles. However, floating cells were still observed in the culture media during the culture. A cell activity was observed during the growth but limited in comparison of CS control.

[0066] A third run was inoculated based on the learning of the run #2 (reduced driver speed and intermittent cycles -the cycles may be a combination of a period of high speed to ensure cell distribution and a period of low speed with a rest period to allow the cells to adhere). As a result, most of the cells (85%) adhered in less than lh and few floating cells and cell debris were observed during the culture. During the cell growth, there was a very low amount of cell debris level and floating cells observed in comparison of the first and second runs. A very good metabolite activity was observed in comparison of the other runs, and in comparison of the CS control of the third run. Cells have been harvested using Accumax diluted 3X using the driver at high speed, performing on / off cycles. Harvested cells had a good viability (>80%) and were healthy (being reseed). The harvest yield was ~25% (based on the cell density harvested in CS). There were few cells remaining on the fixed bed fat the end (based on DNA) but this could be due to the preparation of the samples (bioreactor dismantling).

[0067] Example 2

[0068] As another example of one possible implementation of the aforementioned protocol, a test was completed using a scale-X hydro Bioreactor having a fixed bed, which may be any of the examples described herein and shown in the accompanying drawings or otherwise. During an inoculation phase, stem cells were introduced to the bioreactor along with the cell media. The impeller was rotated at a first speed, such as 300 rpm, for 30min to circulate the cells, and then the speed was reduced (impeller speed 100 rpm, tip speed 0.3 m / s) to a second, lower speed, to result in no circulation inside the bed. Thereafter during a growth phase, the impeller was rotated at a third speed, which was higher (330 rpm), for 30min (linear speed: 0.4cm / s - tip speed: l.lm / s)

[0069] The aforementioned speed cycling may also be performed in an alternating fashion. For instance, the impeller 118 may be operated at the first lower speed for a first predetermined time, then increased to the second higher speed for the second predetermined time. This protocol may then be repeated as necessary or desired.

[0070] The aforementioned protocol may also be used during cell harvesting. For example, cells could be harvested using trypsin-like enzymes, and the impeller rotated to cause detached cells to move at a high linear speed (l-5cm / s) and on / off cycles. Other forms of agitation, such as vibration, may also be used during cell harvesting. As an example of a harvesting protocol tested in a scale-X hydro bioreactor with a fixed-bed with stem cells:

[0071] • Bioreactor emptying

[0072] • 2 rinses PBS-EDTA (3 min 900ml - 500 rpm)

[0073] • Enzyme action Accumax diluted 3 fold (30 min 900ml)

[0074] 500rpm lOmin ( 0.8 cm / s - 1.7 m / s)

[0075] 800rpm 20min (2 cm / s - 2.5 m / s) with 3 on / off cycles

[0076] • Harvest #1 : 50 Hz (15s + 2min 200ml / min)

[0077] • Harvest #2: 60 Hz (15s + 2min 200ml / min)

[0078] • Harvest #3: 70 Hz (15s + 2min 200ml / min)

[0079] • Rinse PBS-EDTA #1 : 70 hz (15s + 2min 200ml / min)

[0080] • Rinse PBS #2: 70 hz (15s + 2min 200ml / min)

[0081] The variable speed may also be applied during the cell harvesting step.

[0082] This disclosure also proposes a method of cell culturing using the aforementioned harvesting protocol of altering the impeller speed. As illustrated in FIG. 4, the protocol involves increasing the rotational speed from one or more values during inoculation (e.g., initially higher, then lower to reduce or eliminate shear and promote cell adherence) to one or more values during growth (which may be higher than the value used to reduce shear and promote adherence).

[0083] Example 3

[0084] Cell culturing was performed using a bioreactor with a structured fixed-bed bioreactor of 5cm height. The bioreactor is operated in batch. The bioreactor is filled with 745ml, 5 cm fixed-bed height is required to reach the targeted V / S (volume / surface area). Tabie ?: v / S rafto

[0085] Fill bioreactor with 900 ml of medium to equilibrate the media overnight and apply agitation speed at 400 rpm (agitation control). Prepare a cell suspension in order to reach 15.000 (or 25.000) cells / cm2, but could range from 500 to 200,000 cells / cm2. The inoculum is prepared to 50ml. No pH (base) regulation during the inoculation because the bioreactor is not at operated at the nominal volume (pH CO2 regulation could be used during the inoculation).

[0086] While the agitation is running, (325 rpm), add the 50ml of the inoculum to reach 950 ml of final volume to the bioreactor and leave running the agitation and regulation.

[0087] Perform intermittent cycle (4 to 6 cycles according to sampling):

[0088] • Dynamic phase: 325 rpm for 30min

[0089] • Static phase: 100 rpm for 30min Take sampling of the supernatant at each static phase.

[0090] TF / CSs control are seeded at a similar cell density. When there are no or only few cells (< 10%) are found in media after the inoculation step, the agitation speed is adjusted to 375 rpm for the culture with 900 ml of media.

[0091] The cells are grown for at least 7 days

[0092] At the end of the growth phase, the cells are harvested.

[0093] The cell detachment is performed with Accumax diluted 3X in PBS-EDTA.

[0094] The duration of each cycle may be based on the classical adhesion speed of the cells. This may be based on an experimental plan but consider, for the all process (sum of all the cycles) to divide the typical adhesion time of the cell adhesion kinetic (based on the type of cell line) by 2 to 10 cycles. E.g. MSC taking 5 hours to adhere --> 5 cycles of lh = Corresponding to 5 times 30 min of dynamic phase and 30 min of static phase.

[0095] Rinse the fixed bed in-situ with PBS / EDTA twice. Stop the regulation and the agitation, empty the bioreactor, filled it with the rinse, start the agitation (without the regulation for 5 mins) and then emptied it again. Transfer PBS rinse to waste. Transfer the harvest solution (Accumax diluted 3X in PBS-EDTA) to the bioreactor. Incubate the enzyme at 500 rpm for 30 minutes.

[0096] Harvest #1: Perform some start / stop cycles of the agitation to help the cell to be detached. Remove the harvest solution from bioreactor to harvest bottle. Drain and perform a cell count.

[0097] Harvest #2: refill the bioreactor with the second enzymatic solutions.

[0098] Harvest cycle:

[0099] • Apply a vibration of 50Hz - 15s at rest

[0100] • Apply a vibration 50Hz combined with the bioreactor emptying step (at 300ml / min until, the solution reaches the bottom of the fixed-bed volume), such as by using a vibrating table.

[0101] • Stop agitation and continue to empty the bioreactor.

[0102] Harvest #3: perform a similar cycle as the harvest #2 at 60Hz Rinse: perform a similar cycle as the harvest #2 at 70hz with PBS. Reseed harvested cells in TF flask (with a centrifuge step) at standard seeding density to monitor the cell plating efficiency.

[0103] Harvest TF / CS control the same way and mimic the harvest diluted (3X) accumax during 30 min with centrifuge cycle.

[0104] An example of a typical generic process with inoculation cycles involves between 1 to 5 of complete cycles (dynamic + period of rest), as follows:

[0105]

[0106] These values are typical targeted ranges for a scale-X hydro with an impeller acting as a centrifuge pump (mixing the cell suspension and pumping the suspension inside the fixed-bed).

[0107] According to a further aspect of the disclosure, the fixed bed may be provided with a coating to facilitate cell growth, and in particular the growth of stem cells. As background, certain types of stem cells, particularly induced pluripotent stem cells (iPSCs), may benefit from the presence of specific coatings in order to facilitate their adhesion and / or growth. This is because conventional methods of hydrophilization, such as plasma or corona treatments, are not sufficient to promote cell adhesion in a short period of time.

[0108] These coatings can take the form of either simple chemical covalent links or adsorption, such as Poly-D-Lysine, DEAD groups, or collagen-derived coatings, as well as protein coatings like fibronectin, vitronectin, or collagen. Another option is the deposition of jelly-like substances such as Gelatin, Matrigel, Laminin or cryoprecipitate. The foregoing listing is considered non-exhaustive.

[0109] According to embodiments of this disclosure, a cell culture matrix in the form of a fixed bed is provided that includes a substrate lattice, which may for instance include an ordered array of fibers and pores disposed between the fibers. The ordered array of fibers is adapted to support adherent, semi-adherent, or suspended cells during cell culture. The cell culture surface further includes a positive or negative charge coating disposed on the cell culture surface, where the positive or negative charge coating promotes adhesion of cells to the cell culture surface. In various aspects of some embodiments, the positive charge coating is a polymer coating. The positive or negative charge coating can be selected from a group that includes a plasma- deposited coating, a silane-based amine coating, and a photoactive polymer coating. For the plasma-deposited coating, the plasma-deposited coating can include a diamine or a triamine. In some example, the plasma-deposited positive charge coating includes 1,3-diaminopropane. For the silane-based amine coating, the silane-based amine coating can include aminopropylsilsesquioxane (APS). For the photoactive polymer coating, the photoactive polymer coating can include at least one of an acrylamide, a methacrylamide, and an aminopropyl-methacrylamide. In some example embodiments, the photoactive polymer coating includes N-[3 (4- Benzoylbenzamido)propyl]-methacrylamide, a copolymer of acrylamide and N-[3 (4-Benzoylbenzamido)propyl]-methacrylamide, or a copolymer of N-[3 (4- Benzoylbenzamido)propyl]-methacrylamide and aminopropyl-methacrylamide. The photoactive polymer is grafted to the cell culture surface via exposure of the photoactive polymer to ultraviolet (UV) light. Other aspects of embodiments include a positive charge coating that includes a polycationic polymer, including at least one of polyethyeneimine and polyallylamine. Any type of molecule that can enhance cell adhesion may be employed.

[0110] The coatings may be applied by way of covalent links or chemical adsorptions. The coatings used should be stable in time and compatible with sterilizable processes (autoclavable or plasma treatment), and should be selected so as to not negatively impact porosity. The coatings may be applied to any portion or all of the structure of the fixed bed, including for example, a woven or non-woven structure, which for example comprise a polyethylene (PET) sheet and a polypropylene (PP) mesh. Any polymeric materials compatible in cell culture applications may be used for any purpose described herein, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. The coatings may also potentially be combined with a change of the composition of the substrate or its hydrophilization treatment.

[0111] Various alternatives to manners of forming a bioreactor including a coated fixed bed are illustrated in FIG. 5. Specifically, the uppermost flow diagram illustrates applying the coating as a step in the bioprocessing, the middle flow diagram illustrates applying the coating to the fixed bed before manufacturing the bioreactor, and the lowermost diagram illustrates applying the coating post-manufacturing, but before bioprocessing occurs. In terms of providing the coatings, one option is to combine the modification of the surface treatment and structure of the cell surface (porosity) to improve the cell adhesion. An alternative is to use reversible coatings to support cell attachment and recovery (such as the Nipam - a temperature-responsive polymer used in cell culture). The coatings may be applied during the process of manufacturing the fixed bed, or afterwards, such as for example using dip coating, spray coating, electrophoresis, spin coating, roll-to-roll or brush coating, self-assembled monolayers, interfacial polymerization, polymer grafting, layer-by-layer assembly, enzymatic coating, cell-mediated deposition, or other forms of providing coatings. All embodiments disclosed may comprise the coated or modified fixed bed.

[0112] Summarizing, this disclosure relates to the foregoing items alone or in any combination :

[0113] 1. An apparatus for culturing cells in connection with a fluid, comprising: a bioreactor including a bed for culturing cells; an agitator for circulating the fluid within the bioreactor; a controller adapted to control the agitator to operate at a first speed during a first predetermined amount of time and a second speed thereafter.

[0114] 2. The apparatus of item 1, wherein the first speed is lower than the second speed.

[0115] 3. The apparatus of item 1, wherein the first speed is higher than the second speed.

[0116] 4. The apparatus of any of items 1-3, wherein the controller is adapted to control the agitator to operate at a third speed after the second speed.

[0117] 5. The apparatus of item 4, wherein the third speed is lower than the second speed.

[0118] 6. The apparatus of any of items 1-3, wherein the controller is adapted to control the agitator to operate at a third higher speed before the first speed.

[0119] 7. The apparatus of any of items 1-6, wherein the controller is adapted to control the agitator to alternate between the first and second speeds.

[0120] 8. The apparatus of any of items 1-7, wherein the controller is adapted to control the agitator to operate at the second higher speed during a second predetermined amount of time.

[0121] 9. The apparatus of any of items 1-8, wherein the agitator comprises a rotatable impeller located within the bioreactor.

[0122] 10. The apparatus of any of items 1-9, wherein the bed comprises a fixed bed.

[0123] 11. The apparatus of item 10, wherein the fixed bed includes a coating adapted to promote cell adhesion and growth. 12. The apparatus of item 11, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0124] 13. An apparatus for culturing cells in connection with a fluid, comprising: a bioreactor comprising a first chamber including an agitator for circulating the fluid within the bioreactor, a second chamber connected to a first outlet of the first chamber and including a bed, and a third chamber connected to a second outlet of the second chamber for returning fluid to the first chamber; and a controller adapted to control the agitator to operate at a speed sufficient for a predetermined amount of time to circulate fluid within the second chamber but without entering the third chamber for returning to the first chamber.

[0125] 14. The apparatus of item 13, wherein the controller is adapted to control the agitator to operate at the second higher speed during a second predetermined amount of time.

[0126] 15. The apparatus of item 13 or item 14, wherein the agitator comprises a rotatable impeller located within the bioreactor.

[0127] 16. A bioreactor having a fixed bed including a coating to promote cell growth.

[0128] 17. The bioreactor of item 16, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0129] 18. A method for culturing cells in a bioreactor including a fixed bed and an agitator, comprising: circulating media in the bioreactor at a first rate during a first period for achieving cell homogenization; and circulating media in the bioreactor at a second rate during a second period for achieving cell adhesion to the fixed bed.

[0130] 19. The method of item 18, further including the step of circulating media in the bioreactor at a third rate during a third period for promoting cell growth.

[0131] 20. The method of item 18 or item 19, wherein the step circulating media in the bioreactor at a first rate comprises operating an agitator at a first speed for a first predetermined amount of time; and the step of circulating media in the bioreactor at a second rate comprises operating the agitator at a second speed. 21. The method of any of items 18-20, wherein the first speed is lower than the second speed.

[0132] 22. The method of any of items 18-20, wherein the first speed is higher than the second speed, followed by a third speed that is lower than the second speed.

[0133] 23. The method of any of items 18-20, further including the step of alternating the operation of the agitator between the first and second speeds.

[0134] 24. The method of item 23, further including the step of harvesting the cells during the alternating step.

[0135] 25. The method of any of items 20-24, wherein the operating steps comprise rotating an impeller as the agitator, or using a pump external to the bioreactor as the agitator.

[0136] 26. A method for culturing cells in connection with a fluid, comprising: providing a bioreactor comprising a first chamber including an agitator for circulating the fluid within the bioreactor, a second chamber connected to a first outlet of the first chamber and including a bed, and a third chamber connected to a second outlet of the second chamber for returning fluid to the first chamber; and controlling the agitator to operate at a speed sufficient to circulate fluid within the second chamber but without entering the third chamber for returning to the first chamber for a predetermined amount of time.

[0137] 27. A method of manufacturing a bioreactor including a fixed bed comprising applying a coating to the fixed bed to promote cell growth.

[0138] 28. The method of item 27, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0139] 29. A method of culturing cells, comprising: coating a fixed bed in a bioreactor with a coating to promote cell growth; and growing cells on the fixed bed.

[0140] 30. The method of item 29, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

[0141] 31. The method of item 29, wherein the coating is applied after the bioreactor is shipped to a customer for performing the step of growing cells on the fixed bed. 32. The method of item 29, wherein the coating is applied before the bioreactor is shipped to a customer for performing the step of growing cells on the fixed bed.

[0142] As used herein, the following terms have the following meanings:

[0143] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0144] "About," "substantially," or "approximately," as used herein referring to a measurable value, such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0145] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0146] The disclosures of the following documents are incorporated herein by reference, as are any other patent applications or publications referenced herein : International Patent Application PCT / EP2020 / 084317; U.S. Provisional Patent Application Ser. No. 63 / 153,082, filed Feb. 24, 2021; International Patent Application Ser. No. PCT / EP2020 / 084317, filed Dec. 2, 2020; U.S. Provisional Patent Application Ser. No. 62 / 942,345, filed Dec. 2, 2019; U.S. Provisional Patent Application Ser. No. 63 / 004,706, filed Apr. 3, 2020; U.S. Provisional Patent Application Ser. Nos. 62 / 758,152, 62 / 733,375, and 62 / 608,261; U.S. Patent Application Publication No. 2018 / 0282678; International Patent Application PCT / EP2018 / 076354; U.S. Provisional Patent Application 62 / 711,070; and U.S. Provisional Patent Application 62 / 725,545.

[0147] While embodiments of the present apparatus have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the protection under the applicable law and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

25CLAIMS1. An apparatus for culturing cells in connection with a fluid, comprising: a bioreactor including a bed for culturing cells; an agitator for circulating the fluid within the bioreactor; a controller adapted to control the agitator to operate at a first speed during a first predetermined amount of time and a second speed thereafter.

2. The apparatus of claim 1, wherein the first speed is lower than the second speed.

3. The apparatus of claim 1, wherein the first speed is higher than the second speed.

4. The apparatus of any of claims 1-3, wherein the controller is adapted to control the agitator to operate at a third speed after the second speed.

5. The apparatus of claim 4, wherein the third speed is lower than the second speed.

6. The apparatus of any of claims 1-3, wherein the controller is adapted to control the agitator to operate at a third higher speed before the first speed.

7. The apparatus of any of claims 1-6, wherein the controller is adapted to control the agitator to alternate between the first and second speeds.

8. The apparatus of any of claims 1-7, wherein the controller is adapted to control the agitator to operate at the second higher speed during a second predetermined amount of time.

9. The apparatus of any of claims 1-8, wherein the agitator comprises a rotatable impeller located within the bioreactor.

10. The apparatus of any of claims 1-9, wherein the bed comprises a fixed bed.

11. The apparatus of claim 10, wherein the fixed bed includes a coating to promote cell adhesion and growth.

12. The apparatus of claim 11, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

13. An apparatus for culturing cells in connection with a fluid, comprising: a bioreactor comprising a first chamber including an agitator for circulating the fluid within the bioreactor, a second chamber connected to a first outlet of the first chamber and including a bed, and a third chamber connected to a second outlet of the second chamber for returning fluid to the first chamber; and a controller adapted to control the agitator to operate at a speed sufficient for a predetermined amount of time to circulate fluid within the second chamber but without entering the third chamber for returning to the first chamber.

14. The apparatus of claim 13, wherein the controller is adapted to control the agitator to operate at the second higher speed during a second predetermined amount of time.

15. The apparatus of claim 13 or claim 14, wherein the agitator comprises a rotatable impeller located within the bioreactor.

16. A bioreactor having a fixed bed including a coating to promote cell adhesion and growth.

17. The bioreactor of claim 16, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations thereof.

18. A method for culturing cells in a bioreactor including a fixed bed and associated with an agitator, comprising: circulating media in the bioreactor at a first rate during a first period for achieving cell homogenization; andcirculating media in the bioreactor at a second rate during a second period for achieving cell adhesion to the fixed bed.

19. The method of claim 18, further including the step of circulating media in the bioreactor at a third rate during a third period for promoting cell growth.

20. The method of claim 18, wherein the step circulating media in the bioreactor at a first rate comprises operating an agitator at a first speed for a first predetermined amount of time; and the step of circulating media in the bioreactor at a second rate comprises operating the agitator at a second speed.

21. The method of claim 20, wherein the first speed is lower than the second speed.

22. The method of claim 20, wherein the first speed is higher than the second speed, followed by a third speed that is lower than the second speed.

23. The method of claim 20, further including the step of alternating the operation of the agitator between the first and second speeds.

24. The method of claim 20, further including the step of harvesting the cells during the alternating step.

25. The method of any of claims 20-24, wherein the operating steps comprise rotating an impeller as the agitator.

26. A method for culturing cells in connection with a fluid, comprising: providing a bioreactor comprising a first chamber including an agitator for circulating the fluid within the bioreactor, a second chamber connected to a first outlet of the first chamber and including a bed, and a third chamber connected to a second outlet of the second chamber for returning fluid to the first chamber; and controlling the agitator to operate at a speed sufficient to circulate fluid within the second chamber but without entering the third chamber for returning to the first chamber for a predetermined amount of time.

27. A method of manufacturing a bioreactor including a fixed bed comprising applying a coating to the fixed bed to promote cell growth.2828. The method of claim 27, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

29. A method of culturing cells, comprising: coating a fixed bed in a bioreactor with a coating to promote cell growth; and growing cells on the fixed bed.

30. The method of claim 29, wherein the coating is selected from the group consisting of Poly-D-Lysine, DEAD groups, collagen-derived coatings, protein coatings such as fibronectin, vitronectin, or collagen, jelly-like substances, such as Gelatin, Matrigel, Laminin, or cryoprecipitate, a reversible coating, such as Nipam, or combinations or derivatives thereof.

31. The method of any of claims 29-30, wherein the coating is applied after the bioreactor is shipped to a customer for performing the step of growing cells on the fixed bed.

32. The method of any of claims 29-30, wherein the coating is applied before the bioreactor is shipped to a customer for performing the step of growing cells on the fixed bed.

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