Cell harvesting device

Abstract

Provided is a cell harvesting device. The cell harvesting apparatus comprises: an agitator adapted to receive a bioreactor; and a connector adapted to couple the agitator to the bioreactor.

Description

[0001] This application is a divisional application of the Chinese application with the application date of June 6, 2022, application number 202221397679.9, and invention name “System for harvesting cells”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 196337 filed on June 3, 2021, U.S. Provisional Patent Application Serial No. 63 / 295673 filed on December 31, 2021, U.S. Provisional Patent Application Serial No. 63 / 310753 filed on February 16, 2022, U.S. Provisional Patent Application Serial No. 63 / 323308 filed on March 24, 2022, and U.S. Provisional Patent Application Serial No. 63 / 330981 filed on April 14, 2022, the disclosures of which are incorporated herein by reference. Technical Field

[0004] This document relates generally to bioreactor systems with enhanced cell harvesting capabilities, and more particularly to systems for harvesting cells, and cell harvesting devices. Background Art

[0005] Certain high-cell-density bioreactors used for biomanufacturing include structures to promote cell retention / adhesion and growth, such as fixed beds (either structured or packed). The arrangement of the fixed-bed material affects local fluid, heat, and mass transfer. In many cases, maximizing cell culture in a given space is very intensive.

[0006] For some biomanufacturing applications, the cells grown in the fixed bed are themselves harvested from the bioreactor after the growth phase. This may be when the cell harvest is used as a seed culture to amplify cells to inoculate another (e.g., production) bioreactor, or when the cell itself is the product of interest (e.g., to produce a cell bank or for cell therapy applications). In order to harvest living cells from the fixed bed, chemical reagents such as trypsin can be used. However, this generally results in a limited amount of cell separation individually, typically due to the densely packed nature of the fixed bed material in a typical bioreactor, which makes it more difficult for chemical reagents to circulate throughout the bed and increase the yield of the cells harvested.

[0007] Cells also attach to the fixation bed via small, cable-like proteins called integrins. Furthermore, cells produce other proteins, like collagen and glycosaminoglycans, that form a mesh-like extracellular matrix. Even when using a detachment solution such as trypsin to achieve cell separation from the structure, these proteins can cause harvested cells to clump together. This can be undesirable for recovery purposes and can result in lower-than-expected cell yields.

[0008] Therefore, there exists a demonstrated need for a method of increasing the yield of cells harvested from a bioreactor. Summary of the Invention

[0009] According to one aspect of the present disclosure, a method for harvesting cells is provided. The method includes providing a bioreactor including a fixed bed structure capable of cell entrapment or adhesion and cell growth, adding cells to the bioreactor via a culture medium, and allowing the cells to become entrapped and / or adhere to the fixed bed structure and grow within the bioreactor. The method further includes agitating the bioreactor and moving the liquid level relative to the fixed bed structure, and introducing a cell separation solution including an enzyme mixture into the bioreactor, wherein a portion of the cells is separated from the fixed bed structure without clumps or aggregates in the portion of the cells.

[0010] In one embodiment, the agitating step and the moving step are performed simultaneously. The moving step can include at least partially draining the bioreactor of fluid, such as by moving the liquid level from near the top of the fixed bed structure to near the bottom of the fixed bed. The moving step can include adding fluid to the bioreactor, such as, for example, by adding additional cell separation solution to the bioreactor. The moving step can include moving the structure for cell retention / adhesion and growth relative to the bioreactor to shift the position of the liquid level.

[0011] Prior to the moving step, the liquid level can be above the fixed bed structure, and the moving step can involve raising and lowering the liquid level multiple times (but can also involve only one time) (e.g., from the top of the fixed bed structure to the bottom of the fixed bed structure). The agitating step can include vibrating the bioreactor. The introducing step can include an enzyme that cleaves integrins and a different enzyme that cleaves the extracellular matrix as an enzyme mixture.

[0012] According to another aspect of the present disclosure, a system for harvesting cells is provided. The system includes a bioreactor comprising a structure for cell retention / adhesion and growth; a cell harvesting mechanism adapted to agitate the bioreactor and to shift a fluid level relative to the structure; and a container comprising a cell separation solution in fluid communication with the bioreactor. The cell separation solution comprises an enzyme mixture adapted to separate a portion of cells from the structure for cell retention / adhesion and growth, and upon separation, the portion of cells does not form clumps or aggregates.

[0013] In one embodiment, the structure for cell retention / adhesion and growth includes a fixed bed, such as a 3D printed fixed bed. The structure for cell retention / adhesion and growth includes a fixed bed having multiple cell immobilization layers, which are arranged, for example, in a stacked or spiral configuration and are in direct contact with adjacent layers or have intervals between these adjacent layers. The cell harvesting mechanism includes an apparatus and / or a pump for vibrating or shaking the bioreactor (particularly the structure for cell retention / adhesion and growth). The cell harvesting mechanism can form part of a docking station for a bioreactor, which can include a harvesting container for harvesting cells to introduce into another bioreactor.

[0014] In these or other embodiments, the bioreactor can be tilted relative to the horizontal plane to facilitate draining of fluid from the structure for cell entrapment / adhesion and growth. A compactor can be provided for compacting the structure for cell entrapment / adhesion and growth, either internally or externally to the structure. The enzyme mixture includes an enzyme that cleaves integrins and a different enzyme that cleaves the extracellular matrix.

[0015] The cell harvesting apparatus includes an actuator for moving a structure for cell retention / adhesion and growth relative to a bioreactor to shift a liquid level. A controller for controlling the cell harvesting mechanism may be provided to agitate the bioreactor and shift the liquid level relative to the structure for cell retention / adhesion and growth. The controller is adapted to control delivery of an enzyme mixture to the bioreactor.

[0016] Another aspect of the present disclosure relates to a system for harvesting cells, comprising: a bioreactor including a structure for cell retention / adhesion and growth; an agitator adapted to agitate the bioreactor; an actuator adapted to move a liquid level relative to the structure for cell retention / adhesion and growth; and a container comprising a cell separation solution in fluid communication with the bioreactor. The cell separation solution comprises an enzyme mixture for separating cells from the structure for cell retention / adhesion and growth without generating clumps or aggregates.

[0017] In one embodiment, the agitator comprises a vibrator. The actuator may comprise a linear actuator and / or a pump. A controller may also be provided to control the actuator and / or the agitator.

[0018] Yet another aspect of the present disclosure relates to a system for harvesting cells. The system includes a bioreactor comprising a structure for cell retention / adhesion and growth. The cell harvesting mechanism is adapted to agitate the bioreactor while filling and flushing the bioreactor with a fluid. A container includes a cell separation solution in fluid communication with the bioreactor.

[0019] In one embodiment, the cell separation solution includes an enzyme mixture for separating cells without producing clumps or aggregates. The cell harvesting mechanism may include a device for applying vibrational energy to the bioreactor, and specifically a device for applying vibrational energy to a structure for cell retention / adhesion and growth, such as by vibrating or shaking the bioreactor and / or the structure, and / or a device for partially or completely filling, emptying, and flushing the bioreactor. The filling, emptying, and flushing equipment may include one or more pumps. The cell harvesting mechanism may form part of a docking station for the bioreactor.

[0020] Still another aspect of the present disclosure relates to a method for separating cells from a fixed bed bioreactor, comprising: adding an enzyme mixture for separating cells without producing clumps or aggregates to the fixed bed bioreactor; and adjusting the position of the liquid level in the fixed bed bioreactor while vibrating the bioreactor. The adjusting step may include filling and flushing the bioreactor with a fluid, including by repeatedly filling and flushing the bioreactor with the fluid. The method may further include the step of delivering the separated cells from the bioreactor to another bioreactor. Still further, the method may include tilting the bioreactor and / or compacting the fixed bed in the bioreactor. The adjusting step may include moving the fixed bed relative to the bioreactor.

[0021] Another disclosed aspect is a method for separating cells in a bioreactor. The method includes vibrating the bioreactor and tilting and emptying the bioreactor. The vibrating step, tilting step, and emptying step can be performed simultaneously.

[0022] The present disclosure also relates to a system for harvesting cells. The system includes a bioreactor comprising a fixed bed for growing adherent cells; and a compactor for compacting the fixed bed to facilitate cell removal. A vibrator may be provided to vibrate the bioreactor or the fixed bed. The compactor may be located inside or outside the fixed bed.

[0023] Still further, the present disclosure relates to a system for harvesting cells. The system includes a preculture container comprising a structure for adherent cell growth; and a vibrator adapted to vibrate a bioreactor to separate the cells from the structure. A bioreactor downstream of the preculture container is configured to receive the separated cells. A pump may also be provided for pumping fluid into or out of the preculture container to move the fluid level relative to the structure, and a controller may be provided for controlling the pump and, if applicable, the vibrator. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A bioreactor system according to one aspect of the present disclosure is schematically illustrated.

[0025] Figure 2 A more detailed example of a bioreactor system according to another aspect of the present disclosure is shown.

[0026] Figure 2A Shown are examples of connectors used to connect a bioreactor to an agitator such as a shaker.

[0027] Figure 2B is a flow chart illustrating an exemplary use of a bioreactor system according to the present disclosure.

[0028] Figure 3 A bioreactor is depicted and shows how fluid moves or pulses within an associated bed during application of vibration to enhance cell harvest.

[0029] Figure 3A 、 Figure 3B 、 Figure 3C 、 Figure 3D 、 Figure 3E and Figure 3F Various other forms of bioreactor arrangements are presented that may benefit from the various inventive aspects disclosed herein.

[0030] Figure 4 A further embodiment of a bioreactor system according to another aspect of the present disclosure is schematically illustrated.

[0031] Figure 4A 、 Figure 4B and Figure 4C A bioreactor integrated into a docking station including a shaking table is shown.

[0032] Figure 5 Different ways of applying mechanical energy to empty a fixed bed are demonstrated.

[0033] Figure 6 and Figure 7 Tilting of the bioreactor during emptying to aid fluid recovery is demonstrated.

[0034] Figure 8 、 Figure 9 and Figure 10 Various embodiments of compactors for compacting fixed beds are presented.

[0035] Figures 11 to 16 A bioreactor comprising a dynamic fixed bed is demonstrated.

[0036] Figures 17 to 19 Examples of various aspects of control for bioprocessing operations are presented.

[0037] Figures 20 to 24 Details of exemplary experiments performed according to various aspects of the present disclosure are presented, wherein: Figure 22 (Continued) Yes Figure 22 The continuation of . DETAILED DESCRIPTION

[0038] In one aspect, the present disclosure relates to a bioreactor with enhanced cell harvesting capabilities. Figure 1 This can be achieved by a system 10 including a bioreactor 12, such as a bioreactor including a structure for adherent or suspended cell growth, such as a fixed bed 14. The fixed bed 14 can include, for example, a structured fixed bed, a 3D printed matrix, a bed composed of one or more woven or non-woven materials, such as, for example, one or more sheets of such materials in direct contact with an inserted spacer, bead, hollow fiber, or having such inserted spacers, beads, hollow fibers, or any other suitable cell culture structure for promoting adherent cell growth or cell growth via entrapment (e.g., see below). Figures 3A to 3F ). The bed 14 can be designed in any desired shape, orientation, or form, including, for example, a 3D porous monolith, stacked layers (see, for example, U.S. Pat. No. 11,111,470, the disclosure of which is incorporated herein by reference), vertically arranged parallel layers, layers arranged in a spiral or wound configuration, or a packed bed (see, for example, U.S. Pat. No. 8,137,959, the disclosure of which is incorporated herein by reference).

[0039] The system 10 further includes a cell harvesting mechanism 16 for increasing the yield of cells harvested from the bioreactor 12. The cell harvesting mechanism 16 can be integrated with the bioreactor 12 and / or can be engaged or used only when cell harvesting is desired, such as by being connected to or interacting with the bioreactor. As further outlined in the following description, the harvesting mechanism 16 can also be built into a docking station for the bioreactor 12 or disposed externally to the integrated system.

[0040] In one example, if Figure 2As shown, the cell harvesting apparatus 16 can include a first device 18 for agitating the bioreactor 12 and a second device 20 for moving or changing the level of fluid within the bioreactor 12, such as, in one example, by filling the bioreactor 12 with a fluid chemical mixture (see below) so that the level is at or above the top portion of the fixed bed, and draining the bioreactor 12 to move the level of fluid from the fill level to or below the bottom of the fixed bed 14. Alternatively or additionally, the second device 20 can generate a reciprocating or "back and forth" movement of a portion of the fluid between the interior and exterior of the fixed bed bioreactor 12. This back and forth movement of the fluid can be generated by an actuator, such as one or more pumps, to create a pulse action, in which the fluid is partially drained and then partially introduced into the bioreactor, or it can involve complete draining and refilling of the bioreactor 12. To this end, the bioreactor 12 can be associated with an inlet 24, an outlet, or a drain 26, each of which can be associated with a suitable pump 22a, 22b and a vent 28. The bioreactor 12 may comprise a rigid container, or may comprise a disposable or single-use container or bag.

[0041] The first device 18 can be any device that applies agitation energy to the bioreactor 12 or to the fixed bed 14, for example. The portion of the bioreactor 12 that applies energy can include any portion thereof that causes vibration of the fixed bed 14 in a manner sufficient to cause cell separation. The device 18 can include, for example, a shaker, a vortex device, an agitator in the form of an oscillator, or another device for applying mechanical energy to the bioreactor and / or a structure for adherent cell growth / entrapment, such as the fixed bed 14. The device 18 can be inside or outside the bioreactor 12. The vibratory motion can be oscillatory, reciprocating or periodic, harmonic or random. The frequency can be 20 Hz to 100 Hz, or more specifically, 50 Hz to 80 Hz. The amplitude can be low, such as 0.5 mm to 5.0 mm, or more specifically, 2 mm to 3 mm.

[0042] The second apparatus 20 may include, for example, one or more fluid transfer devices, such as a bidirectional pump 22a or a reversible pump 22b, or other means to transfer fluid to and from the bioreactor 12. The second apparatus 20 may recirculate fluid emptied during an emptying mode into the bioreactor 12 during a filling mode, or may introduce fresh fluid into the bioreactor 12 during such a filling mode. The second apparatus 20 may also perform only an emptying, purging, or filling cycle, and may do so using only one such cycle (e.g., one emptying or filling) or multiple cycles.

[0043] The second device 20 can be integrated with the first device 18 so that the devices work in tandem or in parallel. Alternatively, the devices 18, 20 can be part of a single device. In either case, a controller (e.g., a computer or processor) can be provided that manages the algorithm or process for combining agitation and fluid movement to and from the bioreactor 12, either automatically or as a result of operator commands.

[0044] The system 10 can further include a harvest container 30, a waste container 32, and a supply container 34 containing a cell separation solution, such as a cell separation solution including an enzyme (possibly including an enzyme cocktail, as further outlined in the description below), each in fluid communication with the bioreactor 12. Optional containers 36, 38, and 40 for supplying a flush solution and an inactivation solution can also be in fluid communication with the bioreactor 12. For example, container 34 can provide an enzymatic and / or chemical cell separation solution (e.g., trypsin / PBS / EDTA, which can be heated to 37 degrees Celsius), container 36 can provide an optional flush solution (e.g., PBS / EDTA), container 38 can include another form of an optional flush buffer solution (e.g., PBS), and container 40 can provide an optional inactivation solution (e.g., STI serum). Any or all of these containers (and the solutions therein) may optionally be agitated and may be part of a recirculation loop 42 to allow recirculation with the bioreactor 12 (because the amount of enzyme (e.g., trypsin) required to separate the cells may be higher than the nominal volume of the bioreactor), and possibly with the reservoir 44. In particular, the harvest vessel 30 may be agitated and optionally temperature controlled to prevent sedimentation of the harvested cells, such as before use to inoculate another bioreactor that may be located upstream (e.g., see Figure 4 The system 10 may also be adapted to preheat the separation solution and / or maintain the temperature of the cell separation solution (typically 37° C.).

[0045] Using the above system, the agitation applied to the bioreactor 12 is combined with the movement of the internal fluid of the bioreactor 12 to enhance cell harvest. For example, the system 10 can vibrate, pulse or shake the bioreactor container while circulating the separation solution via external pumping using pulsation or back and forth fluid movement. Alternatively, the separation solution can be moved internally by using internal circulation within the bioreactor (such as via an agitator) or by using external recirculation-circulation or perfusion. For example, the vibration can be at a selected frequency (e.g., 20 Hz to 300 Hz, including, for example, 60 Hz to 80 Hz), and the pulses of the fluid are applied for multiple cycles (e.g., between 1 and 10, and at a flow rate between 0.1 L / min and 5 L / min).

[0046] Such agitation produces the greatest energy transfer at the level of the gas phase adjacent to the bioreactor 12. By dynamically adjusting (e.g., raising and / or lowering) the level of the gas phase during the vibration / shaking / agitation action, note that Figure 3 The liquid level in the bioreactor 12 and along the fixed bed 14 (note Figure 3 By removing the second device 20 (e.g., a pump) from the bottom portion of the fixed bed 14 near the bioreactor 12 (line L), the cells are more efficiently separated from the material of the fixed bed. Thus, the yield or harvest of cells from the bioreactor 12 is increased in an easy and relatively inexpensive manner without significantly increasing cost or complexity.

[0047] To maintain the integrity of the bioreactor 12 during agitation, the bioreactor 12 can be attached to the system 10, and specifically to the first device 18, using a connector 46. The connector 46 can include a mechanical structure for coupling the device 18 to the bioreactor 12 and should be sufficiently rigid to transfer mechanical energy to the bioreactor 12. To prevent mechanical damage, the connector 46 should be properly fitted to the bioreactor 12 and should maintain and protect any fragile parts of the bioreactor (e.g., pH and DO probes P) from damage.

[0048] exist Figure 2A In the illustrated example shown, the connector 46 includes a ring-shaped part 46a for engaging the lid or cover of the bioreactor 12 with overhangs 46b. These overhangs 46b are releasably connected (e.g., by clamps 46c) to supports 46d. The supports 46d are directly attached to the device 18 in a manner that allows mechanical energy transmission while maintaining the safety of the bioreactor 12 during agitation.

[0049] As an example, system 10 may operate as follows:

[0050] In batch mode using concentrated enzymes such as trypsin (the amount of enzyme is equivalent to the nominal volume of the bioreactor 12);

[0051] In perfusion mode for enzymes (inlet and outlet); or

[0052] • Recirculation (eg using an external loop to recycle the enzyme).

[0053] As another aspect and reference Figure 2B Flowchart, Figure 2 One exemplary use of the illustrated system 10 may involve performing the following steps after the seeding and cell growth phase:

[0054] 1. After the growth phase of the bioreactor is complete and the cells are trapped and growing inside the bed, empty the bioreactor 12 using a drain (e.g., bottom line), such as to a waste container (e.g., Figure 2 Container 32).

[0055] 2. Optionally, flush the bioreactor 12 by adding flush buffer, mixing, and emptying the flush buffer (this step can be performed several times, for example between 1 and 5 times, and can also be done in perfusion by continuously filling and emptying the bioreactor).

[0056] 3. Add the separation solution to the bioreactor so that its level reaches or exceeds the height (or length) of a fixed bed, such as one comprising trypsin, which may be preheated and diluted in an appropriate buffer (eg, which may contain a chelating agent).

[0057] 4. Optionally, wait for a period of time (eg, 1 minute to 60 minutes), potentially maintaining the temperature of the fluid in the recirculation loop at 8°C to 37°C.

[0058] 5. Move the liquid level, such as by draining or otherwise pumping the solution out of the bioreactor (optionally performing a cycle of the separation solution back and forth to the bioreactor 12 to fill and empty the fixed bed 14 (e.g., several cycles—1 to 10 times at a flow rate between 0.1 L / min and 5 L / min) or optionally circulating the separation solution (e.g., through a recirculation loop)). During this step, mechanical energy (e.g., from vibration or other agitation) is applied to the bioreactor 12 and is combined with the movement of the liquid level relative to the bed 14. The draining / emptying of the solution can occur once, or more fills / empties can be performed to more effectively separate the cells.

[0059] 6. Harvest cells from bioreactor 12 (eg, empty using the drain line).

[0060] 7. Optionally, flush the bioreactor 12 and combine the flushing with the harvest.

[0061] 8. Optionally, add enzyme inhibitors to the harvest (use serum, soybean trypsin inhibitor, etc.).

[0062] As mentioned above, the volume of the isolated enzyme can be higher than the nominal volume of the bioreactor 12. For example, in a bioreactor 12 having a volume of 30 m 2 Bed and 0.023ml / cm 2 (150ml / 6600cm 2, recommended for CS / CF10), the amount of trypsin added to the bioreactor should be about 7 L. Therefore, trypsinization should be done in recirculation, in perfusion, or in several steps (steps 3 to 6 - see above). For a nominal volume of about 3 L, trypsinization can be performed in two steps, benefiting from a flushing step.

[0063] According to any aspect of the present disclosure or otherwise, yet another aspect of the present disclosure relates to the concept of using a cell separation solution in the form of an enzyme cocktail associated with harvesting cells from a bed bioreactor. The proposal is to introduce an enzyme cocktail into the fixed bed during cell harvesting, the enzyme cocktail comprising a mix of different enzymes that cleave integrins and extracellular matrix, allowing the cells to be separated with high yield without the aggregation problems faced by using a single enzyme such as trypsin. For example, the enzyme cocktail may include: (1) a serine protease such as trypsin (preferential cleavage: Arg-|-Xaa, Lys-|-Xaa); and / or (2) one or more of the following: (a) chymotrypsin (preferential cleavage: Leu-|-Xaa, Tyr-|-Xaa, Phe-|-Xaa, Met-|-Xaa, Trp-|-Xaa, Gln-|-Xaa, Asn-|-Xaa); (b) elastase (preferential cleavage: hydrolysis of proteins including elastin, collagen types III and IV, fibronectin and immunoglobulins (white A, typically with a large hydrophobic group at P1); (c) collagenase I (preferential cleavage: cleavage of the collagen triple helix at 775-Gly-|-Ile-776 in the α-1 (I) chain, approximately three-quarters of the length of the molecule from the N-terminus); and / or (d) cysteine ​​proteases (e.g., papaverine, etc.). Examples of commercially available enzyme mixtures (containing different enzyme mixtures with the following activities: trypsin, chymotrypsin, elastase, type I collagenase) are Accutase and Accumax (distributed by Innovative Cell Technologies). DNA enzymes can also be used as cleavage / anti-aggregation agents.

[0064] The use of such enzyme mixtures prevents the formation of clumps, aggregates, or prevents a single enzyme solution from being too aggressive to the cells (e.g., an enzyme solution that reduces cell viability, etc.). The enzyme mixture can be used in a single solution or added sequentially to the bioreactor, including mixing of enzymes in batch mode (the amount of enzyme is equivalent to the nominal volume of the bioreactor), perfusion (inlet and outlet) mode, or recirculation mode.

[0065] As an example of a protocol that can be used during cell harvesting, the following steps are involved after the seeding and cell growth phase:

[0066] 1. Empty the bioreactor 12 using the drain (eg, bottom line) to a waste container.

[0067] 2. Optionally, rinse by adding rinse buffer, applying mixing, and emptying the rinse buffer (this step can be done multiple times and can also be done in perfusion mode).

[0068] 3. Add the separation solution including the enzyme mixture (enzyme mix) to the bioreactor so that its level is at or above the height (or length) of the fixed bed which can be preheated.

[0069] 4. Optionally, wait for a period of time (eg, 2 to 30 minutes) while maintaining the temperature at 22°C to 37°C (this step can be done in both batch and recirculation modes).

[0070] 5. Shifting the level of the solution so that it travels through the fixed bed and until it is below or above the other end of the fixed bed, or shifting the level, such as by full recirculation to fill and empty the fixed bed (from 1 to 10 cycles), optionally circulating the enzyme mix (such as via a recirculation loop) and applying low amplitude vibration (shaking / agitation) of 10 Hz to 200 Hz to the bioreactor 12, combined with back and forth pulsing motion inside the fixed bed 14 to detach the cells.

[0071] 6. Harvest cells from the bioreactor (eg, using the drain line).

[0072] 7. Flush the bioreactor 12 and pool the flush product with the harvest.

[0073] 8. Optionally, add enzyme inhibitors to the harvest (use serum, soybean trypsin inhibitor, chelating agents, diluents, etc...).

[0074] Another aspect of the present disclosure also includes using an enzyme or a mixture of enzymes in combination with a mechanical energy application (e.g., vibration) device, preferably before and during the above-mentioned liquid level movement step, to recover cells from a fixed-bed bioreactor. After recovery or harvesting, such cells can be lysed using reagents or mechanical action (e.g., microfluidizer, homogenizer) outside the bioreactor to release intracellular viruses or cell-associated viruses. This also includes using an enzyme or a mixture of enzymes in combination with a vibration device to recover cells from a fixed-bed bioreactor for transfection via electroporation in a second reactor.

[0075] Any or all aspects of the present disclosure may be applied or combined to other forms of fixed beds. Figures 3A to 3B, the fixed bed 14 may include a structured fixed bed 122 for cells (adherent or otherwise) comprising one or more cell immobilization layers 122a which may be wound into a spiral as shown. The one or more layers 122a provide a tortuous flow channel (arrow B) from a linear or regular flow (arrow A) without the use of additional spacer layers (although such spacer layers may be used if desired). For example, Figure 3C As shown, this can be achieved by providing a layer of woven fibers or filaments 123, 125 that disrupt the flow.

[0076] Figure 3D It is shown that such results can be achieved using a nonwoven material as the cell immobilization layer 122a. This can be achieved by forming the layer 122a into a mesh arrangement (e.g., by 3D printing) with openings 127 through which the fluid can pass and return, thereby forming tortuous channels that again promote uniformity and also serve to further shear or separate any bubbles present in the fluid. This function can be achieved with or without an added spacer layer.

[0077] The orientation of the structured fixed bed 122 may differ from that shown in the bioreactor 12, in which the fluid is arranged vertically (in Figure 3 From bottom to top in the example provided). For example, Figure 3E As shown, the horizontally arranged bioreactor 100 may include a first chamber 120 comprising a structured fixed bed 122 comprised of one or more horizontally arranged layers of material. Figure 3C and Figure 3D , these one or more layers may comprise woven or mesh materials, but as Figure 3E As shown, one or more cell immobilization layers 122a (three are shown, but any number may be present) may be sandwiched between adjacent spacer layers 122b (vertical spacing is exaggerated for illustrative purposes). Thus, the flow is arranged from side to side (left to right or right to left), with one or more material layers (spacers or otherwise) providing pathways for generating a tortuous flow (arrow B) from a linear or regular flow (arrow A), and thereby further separating any bubbles present in the fluid. Pumping action may be provided by an agitator or other pump located at the inlet end of the chamber 120, with a return path provided at the outlet end, as schematically shown by path R. Additional spacer layers may be provided between the cell immobilization layers 122a, if desired.

[0078] In another possible embodiment and with reference to Figure 3FIn one embodiment, the structured fixed bed 122 comprises a three-dimensional (3D) integral matrix 124 of a support or lattice form formed by a plurality of interconnected units or objects 124a, which has a surface for cell adhesion. Matrix 124 may include a tortuous path for fluid and cells to flow through when in use. In certain embodiments, matrix may be in the form of a 3D array, lattice, scaffold or sponge. Matrix 124 is preferably disposable in nature to avoid cleaning the involved cost and complexity according to bioprocessing standards.

[0079] According to another aspect of the present disclosure and with reference to Figure 4 , the system 100 includes a bioreactor used as a pre-culture vessel 112 (e.g., a seed culture) for producing cells that are used to inoculate another vessel, such as a production bioreactor 128. The pre-culture vessel 112 may include a structured fixed bed 122, such as a fixed bed including one or more spirally wound layers, such as Figure 3A Alternatively, the fixed bed 122 can be designed in a horizontal stacking form, such as Figure 3E as shown (where flow is horizontal, rather than vertical), or any other known form, including others disclosed herein.

[0080] like Figure 4A 、 Figure 4B and Figure 4C As indicated, a bioreactor serving as a pre-culture vessel 112 (or any other bioreactor described herein) can be associated with a docking station 150. This station 150 can include an integrated shaker 152 on which the vessel 112 can rest. An integrated pump 154 ​​and transfer line 156 are provided for fluid delivery as part of a cell culture system, including upstream and downstream processing with one or more additional bioreactors as needed, or possibly for delivering a cell separation solution to the pre-culture vessel.

[0081] According to another aspect of the present disclosure and with reference to Figure 5 , a system 200 can be provided that includes a bioreactor 212 that includes a fixed bed 222 and an agitator. The agitator can include either or both of an external vibrator, such as a table 240 on which the bioreactor 212 is placed, or alternatively, an internal vibrator 250 placed within the bioreactor 212 to transmit vibrations to the fixed bed 222. Because the fixed bed 222 is typically formed of a hydrophilic material, it tends to retain fluid during cell harvesting. During cell harvesting, applying vibrations before, during, or after draining the bioreactor 212 can cause any fluid within the fixed bed 222 to be released, thereby potentially further improving the recovery of any cells remaining in the trapped fluid.

[0082] refer to Figure 6 and Figure 7 Yet another aspect of the present disclosure relates to the concept of tilting or skewing the bioreactor 310, and specifically the fixed bed 322 of the bioreactor, in order to improve fluid recovery during harvesting. By tilting or skewing the fixed bed 322, the force of gravity is in the vertical direction and the magnitude of the drag force (F_Drag) is reduced. Since the magnitude of the gravity force applied to the volume of fluid is the same, but the residual F_Drag is reduced, the magnitude of the gravity force is higher than the drag force F_Drag. This volume of fluid can then escape from the cell immobilization layer 322a (e.g., nonwoven) and enter the interior of the spacer layer 322b (e.g., mesh - see arrow E). Within the mesh spacer layer 322b, the applied drag force is minimized (ultimately to zero) and the volume of fluid can flow all the way along the spacer layer. As Figure 7 As shown, the tilting can be at an angle α of, for example, 30 to 45 degrees relative to the horizontal plane H, and can be achieved by tilting the entire bioreactor 312 including the fixed bed 322, or by tilting only the fixed bed 322 if the fixed bed can be tilted within the bioreactor 312.

[0083] Another aspect of the present disclosure for improving cell harvest includes compressing or compacting the fixed bed. Figure 8 This can be achieved by associating a fixed bed 422 of a bioreactor (not shown) with an internal compactor for compacting the fixed bed. In one example, the compactor can include a cylindrical wall 450 having interdigitated members 452, 454 that are movable relative to each other in a radial direction. In the illustrated version, the wall 450 is internal to the fixed bed 422, but it could also be external thereto.

[0084] The compactor further includes an actuator for causing movement relative to the fixed bed in order to provide compaction force thereto. The actuator may include a connecting rod 460 connected to a power device 462 (which may include a motor or a hand crank) for engaging members 452, 454 and causing radial movement. When the position of the bed 422 is fixed, this movement compacts or compresses the bed 422 and thus forces the release of any retained fluid, including separated cells (due to vibration, introduction of a separation solution, or both). The squeezing action provided can be repeated as needed to maximize the release of fluid.

[0085] Figure 9Another possible version of an arrangement for compressing a fixed bed 522 is shown. This arrangement can include opposing members 552, 554 connected to a telescoping member 556, which can include a linear actuator. Member 556 can pass through an inner wall 558 of the bioreactor 512. Thus, due to the outer wall 562 of the bioreactor 512, actuation causes the members 552, 554 to be pushed outward against the bed 522, compressing the bed.

[0086] Figure 10 Yet another version is shown. In this arrangement, opposing members 652, 654 can be mounted within a fixed bed 622 connected to an internal actuator, such as a rotatable member 656 (which can be linear or curved). Member 656 is actuated to rotate, forcing members 652, 654 to move apart and thereby compress the fixed bed 622 to release the fluid therein.

[0087] According to further aspects of the present disclosure, the bioreactor 700 may include a container 712 and a fixed bed 714 for culturing or growing cells in conjunction with a fluid culture medium. The fixed bed 714 may be moved relative to the container 712 to change the liquid level. For example, Figure 11 and Figure 12 As shown, the container 712 can include a main portion 712a for receiving a certain volume of fluid and a fixed bed 714 in one position, and an auxiliary portion 712b for receiving a portion of the volume of fluid (fluid culture medium M) and the fixed bed in a second position (the terms "main" and "auxiliary" have nothing to do with the shape or size of the corresponding parts of the container, although in the embodiment shown, the auxiliary portion is shown as being smaller and cylindrical, while the main portion is larger and cubic).

[0088] The auxiliary portion 712b of the container 712 is adapted to receive the fixed bed 714 and may also be adapted to move relative to the main portion 712a of the container 712. Figure 13 As shown, the auxiliary portion 712b, including the fixed bed 714, can be lowered into the main portion 712a of the vessel 712, thereby causing the fluid to pass through the fixed bed 714 (at a height H2) and enter the volume of the auxiliary portion 712b above the fixed bed 714 (formed by the height H1) (thereby changing the total volume of the vessel 712). The reverse movement then causes the fluid to flow back through the fixed bed 714 and into the main portion 712a of the vessel 712. The raising and lowering can be accomplished using an actuator 716, such as a linear actuator.

[0089] Using this arrangement, the fixed bed 714 remains immersed at all times, and the resulting flow action causes the fluid (culture medium) to pass back and forth through the fixed bed 714 to promote cell viability and growth. The speed of relative (e.g., vertical) movement can be controlled to produce a desired flow rate through the fixed bed 714, which will depend in part on the porosity or density of the arrangement. The variability of the flow rate can also be controlled according to the nature of the bioprocess (e.g., high flow rate to ensure uniform cell circulation or during cell harvesting, or low flow rate to protect shear sensitive cells). In any case, it should be understood that the desired flow can be generated without using circulation, such as by an internal agitator, to move the fluid throughout the container, instead of relying on the movement of the fixed bed 714 relative to the container 712.

[0090] Go to Figure 14 , schematically illustrates an arrangement in which the position of the auxiliary portion 712b of the vessel 712 remains fixed relative to the main portion 712a. In this version, the fixed bed 714 is moved between positions within the auxiliary portion 712b (note that Figure 14 A first or lower position 714' on the left, and a second or raised position 714" on the right, which can represent one or more of the same or different auxiliary portions 712b). A lid or cover 718 can also be provided, along with an actuator (not shown) for raising and lowering the fixed bed 714, which can be inside or outside the container 712.

[0091] In any case, from the first position, the fixed bed 714 can be raised within the auxiliary portion 712b, which will cause the fluid to pass through it in its path. However, because the flow rate may be relatively slow, it may cause some of the fluid to flow into the previously emptied space in the auxiliary portion 712b, as shown in FIG. Figure 15 712a, 712b, the fluid eventually reaches equilibrium, passing through the fixed bed 712 in the process. The movement of the fixed bed 714 can then be reversed, as indicated by arrow A, to move within the auxiliary portion 712b, again causing the fluid in front of the fixed bed to pass through it during this movement. This movement can be repeated, and the speed controlled, to provide the desired amount of fluid (fluid culture medium M) flow to promote cell growth and viability.

[0092] The auxiliary portion 712b described above, whether movable or non-movable, may be formed of a generally rigid material, may be cylindrical in nature, and may be hollow for receiving a fixed bed. Figure 15 and Figure 16 It is also possible to form the auxiliary portion 712b of the container from a foldable or flexible material. Figure 16As shown, the auxiliary portion 712b can be telescopic and include multiple telescopic parts (such as sliding tubes nested together) to fold and move the fixed bed 714 within the fluid in the main portion 712a of the container (note the positions 712b to 712b') and also receive fluid extracted from the main portion through movement. Figure 16 An arrangement is shown in which the auxiliary portion 712b is made flexible, like a bellows or an accordion, and can therefore be folded when the fixed bed 714 moves together with the auxiliary portion 712b.

[0093] Figure 17 A docking station 800 is shown for a bioreactor 802. The docking station 800 can include a controller 804 having a display 806 for displaying various parameters associated with the ongoing bioprocessing operation and also allowing input to control various aspects of the bioprocessing operation. For example, the docking station 800 can include various auxiliary containers 808 associated with pumps 810 connected by conduits. The controller 804 can be used to control these pumps to control the flow of fluids into or out of the bioreactor 802, as well as to control the mixing of the fluids in the bioreactor, such as by controlling an agitator (not shown) therein, the drive of which can form part of the docking station 800.

[0094] like Figure 18 As shown, docking station 800 can be associated with harvesting module 801, including an external agitator, which is shown in the form of a shaker 812. As shown, bioreactor 802 can be moved from docking station 800 to shaker 812 to facilitate harvesting cells according to the teachings of the present disclosure. Shaker 812 can be controlled independently or by controller 804, and can also be integrally formed with docking station 800.

[0095] Go to Figure 19 , a controller 804 associated with the docking station 800 can be used to control the cell harvesting operation. For example, once the desired cell density is reached, the cell harvesting manifold 813 can be connected to the bioreactor 802. The bioreactor 802 can be emptied and flushed with a buffer from a container 814, and then an enzyme mixture can be introduced, such as from a corresponding container 816. Pumping fluid to / from the bioreactor 802 can be accomplished using a pump 810 associated with the docking station 800, which can again be controlled by the controller 804.

[0096] The bioreactor 802 can then be transferred to an agitator or shaker table 812. The shaking can be accomplished along with harvesting into a suitable container 818. Multiple flushes can be accomplished, such as by using the controller 804 to control the pumping of fluids into and out of the bioreactor 802 using a pump 810 associated with the docking station 800 and connected to a manifold 813. In this way, the entire cell growth and harvesting process (including as disclosed herein) can be controlled by the controller 804.

[0097] Examples

[0098] Experiments were performed to evaluate the feasibility of the cell harvesting technique disclosed herein. Adherent HEK293 cells from a cryopreserved cell bank (18H003, ECACC) were used for all experiments. Plasticware culture and bioreactor culture were performed in DMEM (4.5 g / L glucose) supplemented with 5% fetal bovine serum. Inoculation was performed at 20,000 cells / cm under all conditions. 2 Up to 25,000 cells / cm 2 Before cell culture in bioreactors, cells are pre-cultured in T-flasks and multilayer plastic containers to achieve the desired inoculum. Passaging is performed every 3 to 4 days (at the mid-exponential phase).

[0099] The cells were seeded in scale-X hydro (2.4m 2 ), carbo 10m 2 and 30m 2 bioreactor and using a recirculation loop (0.17 mL / cm 2 ) were maintained in batch mode for 4 hours before starting culture growth. The bioreactor culture conditions are detailed in Table 1:

[0100] Table 1. Bioreactor culture conditions

[0101]

[0102] Daily samples of the culture medium and the fixed bed (via sampling carriers) were taken and cell growth was assessed by glucose and lactate curves, respectively, and by direct cell counts on the sampling carriers.

[0103] Harvesting was performed from the bioreactor after 4 to 6 days of expansion. Before being harvested, the bioreactor was emptied and rinsed with a DPBS solution containing 5mM EDTA (DPBS-EDTA), preheated to 37°C. Subsequently, the separation solution preheated to 37°C was added to the bioreactor and then incubated for 20 to 25 minutes under agitation (0.5cm / s) and temperature control (37°C). The bioreactor was then moved to the harvesting module and subjected to vibration while the container was emptying. Various combinations of vibration frequency and duration were explored and are detailed in Figure 22 and Figure 22 (Continued).

[0104] In some cases, multiple rinses with enzyme solution were performed after the first harvest. In all cases, the bioreactor was rinsed with DPBS-EDTA after harvest. In some cases, vibration was applied during some or all rinse steps. Samples of the fixed-bed support and supernatant were taken at different steps during the harvest process to determine harvest efficiency and cell viability. For this study, two isolating enzyme solutions were tested: TrypLE TM Select, 1X or 5X (Gibco) and Accumax (Innovative Cell Technologies), 1X or diluted 1:3 in DPBS-EDTA.

[0105] An overview of the cell harvest protocol is given in Figure 20 In brief, cells are expanded in bioreactor 802 using culture medium from associated container 803 in a recirculating arrangement for 4 to 6 days (1). On the day of harvest, bioreactor 802 is rinsed with buffer from container 814 and associated waste container 815 (2), then filled with enzyme solution from container 816 and incubated for 20 to 25 minutes (3). Subsequently, bioreactor 802 is emptied into harvest container 818 with synchronous vibration (4). Finally, a rinse is performed using buffer 814 to facilitate cell recovery (5). It will be appreciated that steps 1 to 3 are performed using bioreactor 802 mounted on controller 800. Steps 4 and 5 are performed using bioreactor 802 mounted on harvest module 801.

[0106] Use hemocytometer to measure cell density and the viability in harvested cells by trypan blue dye exclusion method (Trypan Blue dye exclusion).By cell lysis (scale-X cell counting kit, Univ. Walsells Technology Co., Ltd. (Univercells Technologies)), the biomass estimation on the sampling carrier is performed, then crystal violet staining is carried out and nucleus counting is carried out with hemocytometer.Nucleus counting on the fixed bed fiber is used to estimate the total cell number inside bioreactor.After cell harvesting, dismantle bioreactor, and take out the part for fixed bed from representative position, to assess the cell density remaining on fixed bed.

[0107] Cells harvested from scale-X hydro (n=5) bioreactors, carbo 10 (n=1) bioreactors, and carbo 30 (n=1) bioreactors were seeded into T-flasks to assess repopulation efficiency, cell morphology, and cell recovery at the first passage. Population doubling time (PDT) and cell morphology were assessed in comparison to control cells collected from plastic vessels. In one case, cells harvested from a scale-X hydro bioreactor were used to seed a second hydro, providing proof of concept for a "fixed bed to fixed bed" seed culture. Inoculation conditions using hydro-harvested cells were identical to those previously described.

[0108] In the first phase, tests were performed using TrypLE 1X and 5X as isolation solutions. However, the conditions tested tended to release cells with a large proportion of aggregates, thereby affecting the total cell recovery rate (data not shown). In contrast, the use of Accumax was found to produce single-cell suspensions under the conditions tested. Therefore, all experiments described in this document were performed using Accumax, either at 1X or at a 1:3 dilution.

[0109] Five cell harvesting experiments were performed using the standard version of the scale-X hydro. In all experiments, the Accumax incubation was followed by a harvesting step, followed by several rinse steps, as described in the Materials and Methods sections. An evaluation of different harvesting methods showed that using the Accumax for one harvest cycle followed by two rinse cycles, all accompanied by vibrations between 50 Hz and 70 Hz, was the most effective in recovering cells ( Figure 22 and Figure 22 (Continued)). An additional rinse step using DPBS-EDTA enabled moderate additional cell release. Applying vibration during the rinse further increased the amount of cells recovered.

[0110] In all experiments, analysis of sample carriers before and after harvest indicated that >90% of the cells had detached from the fixed bed material ( Figures 21 to 22 (continued) In summary, 2.7 to 5.1 x 10 cells can be harvested as a single cell suspension from the scale-X hydro bioreactor. 9 After incubation with Accumax, perform one harvest cycle followed by two rinse cycles with Accumax, all accompanied by vibrations between 50Hz and 70Hz to obtain the highest single cell yield. 3 cells / cm 2 ) and close to the confluence point (400-500x10 3 cells / cm 2 ), suggesting that optimizing cell number at harvest may be crucial to achieving the highest harvest yield. Of note, cell harvesting in plasticware is typically performed at 200x10 3 cells / cm 2 This highlights the advantage of fixed-bed bioreactors in being able to harvest cells at higher densities.

[0111] Following proof-of-concept and optimization of the hydrodynamic scale, additional cell harvest experiments were performed to investigate the scalability of the carbo 10 (n=1) and 30 bioreactors (n=1). In both harvests, >98% of the cells were detached from the fixed bed material. Up to 51x10 cells were collected as single cell suspensions from the scale-X carbo 30. 9 cells, thus providing sufficient cells for 8,500 cells / cm 2 Inoculated with scale-X nitro 600. As shown in the hydro experiment, it is possible to efficiently harvest up to 450x10 3 cells / cm 2 This translates to a cell density of 10,000 cells / cm in carbo 30. 2 Ability to inoculate with nitro 600. Of note, in both experiments in carbo, a manual inversion step was added to facilitate bioreactor emptying and comparison with scale-X hydro data. It is estimated that a complete harvest without this procedure would require two additional flushing steps (for a total of five) beyond those listed in this study.

[0112] In all experiments performed for this study (hydro, carbo 10 and 30), the harvested cells showed excellent viability (between 84% and 96%) ( Figures 21 to 22 (continued)In the runs using diluted Accumax, no aggregates were observed in the collected suspension.

[0113] Repopulation experiments were performed with cells harvested at all scales and showed excellent repopulation in T-flasks with PDT of 29 to 37 hours (compared to 29 to 39 hours for control cells from T-flasks; Figure 23 In one case, cells harvested from a scale-X hydro were used to inoculate a second hydro, where appropriate growth was observed ( Figure 24 The cells can then be harvested from the second hydro, providing proof-of-concept for fixed-bed to fixed-bed seed culture.

[0114] The above results show that from scale-X carbo 30m 2 Harvested cells should be sufficient to produce 10,000 HEK293 cells / cm 2 Inoculation scale-X nitro 600m 2 Importantly, the scale-X carbo 30 is equivalent to twelve 40-layer plasticware containers, typically handled with specialized equipment such as large incubators, automated manipulators, and shakers. A simple evaluation comparing a seed culture process using plasticware with the scale-X carbo showed a dramatic reduction (95%) in equipment footprint.

[0115] Under the conditions tested for this study, multiple washes were required with Accumax to achieve adequate cell recovery. Despite the need for multiple washes, it is estimated that total enzyme usage remained lower compared to conventional plasticware processes (for scale-X carbo 30m 2 9.0 L for 12xCF40 and 9.6 L for 12xCF40). In addition, it was shown that it is feasible to reduce the amount of enzyme per surface through some optimization.

[0116] Considering the harvested volume, scale-X 30m 2 Resulting in high inoculum cell density and lower seed volume: 15 L more than conventional vessels (3 L for 3 harvests and 2 3 L for rinsing): 28.8 L for 12 CF-40s (considering the following recommended volumes: 20 ml enzyme + 25 ml medium and 15 ml rinse per layer)

[0117] The scale-X bioreactor can be harvested in a closed system by a single operator within 2 h, whereas harvesting cells from multiple CF-40s requires more operators and sterile connections under the LAF.

[0118] While these preliminary estimates clearly point to significant savings in seed culture generation costs using scale-X carbo, more complete cost modeling may reveal even larger differences due to labor, total operational footprint, sterility risks, media usage, etc.

[0119] In general, the present disclosure may be considered to relate to any or all of the following in any combination or arrangement:

[0120] 1. A method for harvesting cells, comprising:

[0121] Providing a bioreactor including a fixed bed structure capable of cell retention or attachment as well as cell growth;

[0122] adding cells to the bioreactor via culture medium;

[0123] allowing cells to become entrapped and / or adhere to the fixed bed structure and grow within the bioreactor;

[0124] introducing a cell separation solution including an enzyme mixture into the bioreactor;

[0125] agitating a portion of the bioreactor; and

[0126] The level of the cell separation solution is moved relative to the fixed bed structure; wherein a majority of the cells are separated from the fixed bed structure without forming clumps or aggregates among the majority of the cells.

[0127] 2. The method according to item 1, wherein the stirring step and the moving step are performed simultaneously.

[0128] 3. The method according to item 1 or item 2, wherein the moving step comprises at least partially emptying the bioreactor of the cell separation solution.

[0129] 4. The method of any one of items 1 to 3, wherein the moving step comprises moving the liquid level from above or near the top of the fixed bed structure to below or near the bottom of the fixed bed.

[0130] 5. The method of any one of items 1 to 4, wherein the moving step comprises adding a fluid to the bioreactor.

[0131] 6. The method according to any one of items 1 to 5, wherein the adding step comprises adding additional cell separation solution to the bioreactor.

[0132] 7. The method according to any one of items 1 to 6, wherein the liquid level is above the fixed bed structure before the moving step.

[0133] 8. The method according to any one of items 1 to 7, wherein the moving step comprises raising and lowering the liquid level a plurality of times.

[0134] 9. The method of any one of items 1 to 8, wherein the agitating step comprises vibrating the fixed bed directly or indirectly, such as at a frequency between about 20 Hz and about 300 Hz and at an amplitude between about 0.5 mm and about 5 mm.

[0135] 10. The method according to any one of items 1 to 9, wherein the introducing step comprises introducing an enzyme that cleaves integrins and a different enzyme that cleaves the extracellular matrix as an enzyme mixture.

[0136] 11. A system for harvesting cells, comprising:

[0137] bioreactors, including structures for cell retention / adhesion and growth;

[0138] a cell harvesting mechanism adapted to agitate the bioreactor and shift the fluid level relative to the structure; and

[0139] A container includes a cell detachment solution in fluid communication with the bioreactor, the cell detachment solution including an enzyme mixture for detaching cells from the structure for cell entrapment / adhesion and growth without generating clumps or aggregates.

[0140] 12. The system of claim 11, wherein the structure for cell retention / adhesion and growth comprises a fixed bed, such as a 3D printed fixed bed.

[0141] 13. The system of claim 11 or 12, wherein the structure for cell retention / adhesion and growth comprises a fixed bed having a plurality of cell immobilization layers, the plurality of cell immobilization layers being arranged, for example, in a stacked or spiral configuration and either in direct contact with adjacent layers or with spaces between adjacent layers.

[0142] 14. The system according to any one of items 11 to 13, wherein the cell harvesting mechanism comprises a device for vibrating or shaking the bioreactor.

[0143] 15. The system of any one of items 11 to 14, wherein the cell harvesting mechanism comprises a pump for moving the liquid level.

[0144] 16. The system according to any one of items 11 to 15, wherein the cell harvesting mechanism comprises a device for applying vibrational energy to the bioreactor, and in particular a device for applying vibrational energy to a structure for cell entrapment / adhesion and growth.

[0145] 17. The system according to any one of items 11 to 16, wherein the cell harvesting mechanism forms part of a docking station for a bioreactor.

[0146] 18. The system according to any one of items 11 to 17, wherein the bioreactor comprises a harvesting vessel for harvesting cells for introduction into another bioreactor.

[0147] 19. The system of any one of items 11 to 18, wherein the bioreactor is tilted relative to a horizontal plane to facilitate fluid evacuation from the structure for cell entrapment / adhesion and growth.

[0148] 20. The system according to any one of items 11 to 19, further comprising a compactor for compacting the structure for cell entrapment / adhesion and growth, the compactor being inside or outside the structure.

[0149] 21. The system according to any one of items 11 to 20, wherein the enzyme mixture comprises an enzyme that cleaves integrins and a different enzyme that cleaves the extracellular matrix.

[0150] 22. The system according to any one of items 11 to 21, wherein the cell harvesting device comprises an actuator for moving the structure for cell retention / adhesion and growth relative to the bioreactor to move the position of the liquid level.

[0151] 23. The system of any one of items 11 to 22, further comprising a controller for controlling the cell harvesting mechanism to agitate the bioreactor and move the liquid level relative to the structure for cell retention / adhesion and growth.

[0152] 24. The system according to any one of items 11 to 23, wherein the controller is adapted to control the delivery of the enzyme mixture to the bioreactor.

[0153] 25. A system for harvesting cells, comprising:

[0154] bioreactors, including structures for cell retention / adhesion and growth;

[0155] an agitator, suitable for agitating the bioreactor;

[0156] an actuator for moving the fluid level relative to the structure for cell entrapment / adhesion and growth; and

[0157] A container includes a cell detachment solution in fluid communication with the bioreactor, the cell detachment solution including an enzyme mixture for detaching cells from the structure for cell entrapment / adhesion and growth without generating clumps or aggregates.

[0158] 26. The system of item 25, wherein the agitator comprises a vibrator.

[0159] 27. The system of item 25 or item 26, wherein the actuator comprises a linear actuator.

[0160] 28. The system of any one of items 25 to 27, wherein the actuator comprises a pump.

[0161] 29. The system of any one of items 25 to 28, further comprising a controller for controlling the actuator.

[0162] 30. A system for harvesting cells, comprising:

[0163] bioreactors, including structures for cell retention / adhesion and growth;

[0164] a cell harvesting mechanism adapted to agitate the bioreactor while filling and flushing the bioreactor with fluid;

[0165] A container comprising a cell separation solution is in fluid communication with the bioreactor.

[0166] 31. The system according to item 30, wherein the cell separation solution comprises an enzyme mixture for separating cells without generating clumps or aggregates.

[0167] 32. The system of item 30 or item 31, wherein the cell harvesting mechanism comprises a device for vibrating or shaking the bioreactor.

[0168] 33. The system of any one of items 30 to 32, wherein the cell harvesting mechanism comprises equipment for partially or completely filling, emptying and flushing the bioreactor.

[0169] 34. The system of item 33, wherein the filling, emptying and flushing means comprises one or more pumps.

[0170] 35. The system of any one of items 30 to 34, wherein the cell harvesting mechanism comprises a device for applying vibrational energy to the bioreactor, and in particular a device for applying vibrational energy to a structure for cell entrapment / adhesion and growth.

[0171] 36. The system of any one of items 30 to 35, wherein the cell harvesting mechanism forms part of a docking station for a bioreactor.

[0172] 37. A method for separating cells from a fixed-bed bioreactor, comprising:

[0173] adding an enzyme mixture for separating cells without producing clumps or aggregates to a fixed bed bioreactor; and

[0174] The position of the liquid level in the fixed bed bioreactor is adjusted while vibrating the bioreactor.

[0175] 38. The method of item 37, wherein the conditioning step comprises filling and flushing the bioreactor with a fluid.

[0176] 39. The method of item 36 or item 37, wherein the conditioning step comprises repeatedly filling and flushing the bioreactor with fluid.

[0177] 40. The method according to any one of items 37 to 39, further comprising the step of transferring the separated cells from the bioreactor to another bioreactor.

[0178] 41. The method according to any one of items 37 to 40, further comprising the step of tilting the bioreactor.

[0179] 42. The method according to any one of items 37 to 41, further comprising the step of compacting the fixed bed in the bioreactor.

[0180] 43. The method of any one of items 37 to 42, wherein the adjusting step comprises moving the fixed bed relative to the bioreactor.

[0181] 44. A method for separating cells in a bioreactor, comprising:

[0182] Vibrating bioreactors; and

[0183] Tilt and drain the bioreactor.

[0184] 45. The method according to item 44, wherein the vibrating step, the tilting step and the emptying step are performed simultaneously.

[0185] 46. ​​A system for harvesting cells, comprising:

[0186] bioreactors, including fixed beds for adherent cell growth; and

[0187] Compactors, used to compact the fixed bed.

[0188] 47. The system according to item 46, further comprising a vibrator for vibrating the bioreactor or the fixed bed.

[0189] 48. The system of item 46 or item 47, wherein the compactor is located inside or outside the fixed bed.

[0190] 49. A system for harvesting cells, comprising:

[0191] a pre-culture vessel including structures for adherent cell growth;

[0192] a vibrator adapted to vibrate the bioreactor to separate the cells from the structure; and

[0193] A bioreactor downstream of the pre-culture vessel is provided for receiving the separated cells.

[0194] 50. The system according to item 49, further comprising a pump for pumping fluid into or out of the pre-culture container so as to move the liquid level relative to the structure.

[0195] 51. The system according to item 50, further comprising a controller for controlling the pump.

[0196] 52. The system of item 51, wherein the controller is adapted to control the vibrator.

[0197] For the purposes of this disclosure, the following terms have the following meanings:

[0198] Unless the context clearly dictates otherwise, "a," "an," and "the" include singular and plural referents. By way of example, "a compartment" means one compartment or more than one compartment.

[0199] As used herein, "about," "substantially," "generally," or "approximately" in reference to a measurable value such as a parameter, amount, duration, or the like is intended 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 or from the specified value, as such variations are suitable for performing the disclosed methods. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0200] As used herein, “comprise,” “comprising,” “comprises,” and “comprised of” are synonymous with “include,” “including,” “includes,” or “contain,” “containing,” and are inclusive or open-ended terms that specify the presence of the following, for example, “comprises” does not exclude or preclude the presence of additional, unrecited parts, features, elements, components, steps known in the art or disclosed herein.

[0201] Although preferred embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes, and substitutions will occur to them. It should be understood that the different alternatives of the embodiments of the present invention described herein may be used to practice the present invention. The following claims are intended to define the scope of protection under applicable law, and therefore cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A cell harvesting device, characterized in that: The cell harvesting device comprises: an agitator adapted to receive the bioreactor; and A connector is provided for connecting the bioreactor to the agitator.

2. The cell harvesting device according to claim 1, characterized in that The cell harvesting device includes the bioreactor.

3. The cell harvesting device according to claim 1, wherein The agitator comprises a deck upon which the bioreactor is placed.

4. The cell harvesting device according to claim 1, wherein The connector includes mechanical structure for coupling the agitator to the bioreactor.

5. The cell harvesting device according to claim 1, wherein The connector is sufficiently rigid to transfer mechanical energy to the bioreactor.

6. The cell harvesting device according to claim 1, characterized in that The connector comprises an annular part for engaging the lid or cover of the bioreactor with the overhang.

7. The cell harvesting device according to claim 6, characterized in that The overhanging portion is releasably connected to the support.

8. The cell harvesting device according to claim 1, wherein The agitator further includes at least one support attached thereto.

9. The cell harvesting device according to claim 8, characterized in that At least one of the supports is directly attached to the agitator.

10. The cell harvesting device according to claim 2, characterized in that The connector is assembled to the bioreactor.

11. The cell harvesting device according to claim 1, characterized in that The agitator includes a vibrator or a shaker.

12. The cell harvesting device according to claim 1, wherein The cell harvesting device forms part of a docking station for the bioreactor.

13. The cell harvesting device according to claim 1, wherein The cell harvesting apparatus further comprises a controller adapted to control the agitator.

14. The cell harvesting device according to claim 2, wherein The bioreactor comprises a fixed bed bioreactor.

15. The cell harvesting device according to claim 2, characterized in that The bioreactor comprises a structured fixed bed bioreactor.

16. The cell harvesting device according to claim 2, wherein The bioreactor includes structures for cell retention / adhesion and growth.

17. The cell harvesting device according to claim 16, wherein The structure for cell retention / adhesion and growth includes a fixed bed.

18. The cell harvesting device according to claim 16, wherein The structure for cell retention / adhesion and growth includes a fixed bed having multiple cell immobilization layers.

19. The cell harvesting device according to claim 18, wherein The multiple cell-immobilization layers are arranged in a stacked or spiral configuration.

20. The cell harvesting device according to claim 18, wherein The cell-immobilizing layer is arranged to directly contact an adjacent layer or with a space between the adjacent layers.

21. The cell harvesting device according to claim 17, wherein The structure for cell retention / adhesion and growth includes a fixed bed which is a 3D printed fixed bed.

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