FIELD OF THE INVENTION

The present invention relates to a system for cell culture, and components of that system.

BACKGROUND TO THE INVENTION

As consumer interest in intensive animal agriculture declines, there is growing activity around replacing foodstuffs obtained from animal sources with cultured meat products and cultured meat substitutes. Such cultured foodstuffs may include animal products such as meat, muscle tissue, offal tissue; or they may include ingredients for food use, such as animal fats or proteins. One critical requirement for commercial production of cultured foodstuffs is the ability to grow cells in culture on a large scale.

Various forms of bioreactor for large scale cell culture are known in the art, but current products on the market are not necessarily suitable for food-grade production of cultured foodstuffs such as animal cells or animal products. The present invention addresses the need for such a cell culture system.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided a system for cell culture, the system comprising: a reactor module comprising a plurality of cell culture vessels mounted within a common support frame; each cell culture vessel comprising: i) a flexible gas-permeable membrane forming a closed chamber having opposed ends, and defining an interior and exterior of said chamber; ii) one or more fluid port(s), each allowing fluid communication between the interior and exterior of the chamber; iii) compression means located exterior to the chamber for selectively compressing a portion of the chamber so as to divide the interior of the chamber into separate regions, at least one of said regions having a fluid port; and wherein the compression means is movable with respect to the chamber, so as to alter the size of the separate regions.

The culture vessels of this system have a number of features which provide advantages in cell culture. In particular, the division of the chamber into separate regions allows a first region to be used for cell culture, while a second region can act as a reservoir for culture media and other components to be added to the culture. As the cell mass in the first region increases, the movable compression means may be moved so as to increase the volume of the first region and introduce additional culture media. This can help to keep the cell culture under optimum conditions without the need to introduce additional media into the vessel. However, the fluid port(s) also allow addition or removal of components from the vessel where necessary - for example, introduction of cell differentiation reagents at an appropriate time during the culture process; or removal and harvesting of cells or cell products. The selective compression of the compression means can also be adjusted to control the degree of isolation of the two regions, and hence allow a greater or lesser degree of fluid mixing between the two. In some embodiments, the interior of the chamber is divided into two regions, although in some cases more than two regions may be provided (for example, three, four, five, six or more regions).

Forming the chamber of a flexible gas-permeable membrane allows gas exchange through the membrane to maintain the cells, while the flexible nature of the membrane allows for expansion of the vessel as the cell mass increases. The gas-permeable membrane may be formed from any suitable material, although preferred materials include e.g. polydimethylsiloxane, polyurethane, or fluorinated ethylene propylene.

In some embodiments, each cell culture vessel comprises at least first and second fluid ports, and each of a first and second region of the interior of the chamber has a respective fluid port.

In preferred embodiments, each cell culture vessel comprises first and second fluid ports, and the compression means is located between said first and second fluid ports, such that each of the first and second regions has a respective fluid port. In some embodiments, the first and second fluid ports are located at or toward first and second opposed ends of the chamber; and the compression means is movable along the chamber toward either of the first and second opposed ends.

In embodiments, the movable compression means may comprise paired rigid rods located on either side of the exterior of the chamber. The rods may be rotatable about their long axis (eg, the rods are in the form of rollers) for ease of movement along the chamber. In other embodiments, the rods may be non-rotatable, or move in a prescribed path to control the rotation angle as a function of the position as related to the vessel or other system geometry. The shape of the rod cross-section may be one or more of circular, rectangular, ovoid, elliptical, or other. Different rods may have different crosssections. In some embodiments more than two rods may be present; for example, “paired rigid rods” may include three rods, one on a first side of the chamber, and two on a second side, which together divide the chamber into first and second regions. The rods may have a deformable or low friction coating (for example, silicone rubber or polytetrafluoroethylene). In preferred embodiments, the rods are movable with respect to one another, to increase or decrease their separation from one another; this can be used to apply compression to the chamber or to release compression when desired. In some embodiments, this movement may be perpendicular to the gas permeable membrane, while in others the relative movement may be parallel to the gas permeable membrane, to increase or decrease the horizontal separation of the rods. Both directions of movement may be combined. This is in addition to the overall movement of the compression means with respect to (eg, along) the vessel or system, which will typically also be parallel to the gas permeable membrane. In a typical embodiment, the rods are retained within a mounting frame; this frame may include motor mechanisms or actuators to adjust the relative position of the rods. Movement of the rods along the chamber can be achieved by moving the mounting frame; while in some embodiments the mounting frame or the culture vessel includes a motor to do this, in preferred embodiments a motor is present in the reactor module to move the mounting frame. As is discussed further elsewhere herein, this arrangement allows multiple mounting frames of multiple culture vessels to be mechanically linked to the same motor and moved in unison.

In yet further embodiments, the compression means need not take the form of paired rigid rods as described. For example, a single moveable rod on a first side of the chamber may be used in combination with a rigid supporting surface on a second side of the chamber, with the rod being urged toward the surface to achieve the desired compression.

It will be noted that the system and culture vessels of the invention are not restricted to two regions; with appropriate arrangement of the culture vessel and compression means, multiple regions may be formed. For example, provision of three separate compression means may allow division of a culture vessel into four separate regions. This may be used to permit, for example, culture of multiple cultures under different conditions or at different stages of growth within the same culture vessel. In some embodiments, each such region may have a respective fluid port. The various compression means in these examples may move and alter the sizes of the regions independently or in a coordinated manner.

Embodiments of the invention may further comprise a gas-impermeable membrane located so as to cover one of the first and second regions. Preferably the membrane is attached to the movable compression means. Conveniently, where the compression means takes the form of rotatable rods, the gas-impermeable membrane may be in part wound around one or each of the rods; this allows the amount of membrane covering one of the regions to adjust as the location of the rods changes, such that the membrane reflects the size of that region. Use of the gas-impermeable membrane provides two benefits: one, it provides additional physical protection to a part of the culture vessel; and two, it can exclude air from being able to pass a portion of the gas-permeable membrane. This can help to reduce degradation of components within the vessel from exposure to air when such components do not require oxygen (that is, where there are no living cell cultures, such as in the reservoir region). Any suitable material may be used for the gas- impermeable membrane; examples of such suitable materials include PET (polyethylene terephthalate), LDPE (low density polyethylene), PE (polyethylene), PVC (polyvinyl chloride), Kapton® films or polyimide films, and metallised films.

The culture vessel preferably further comprises first and second rigid supporting rods, which may be located at or towards the opposed first and second ends of the chamber. The rods are formed so as to have the strength to support the weight of a culture vessel and its contents (cell culture, growth media, etc). The rods may be bonded or otherwise fixed to the gas-permeable membrane. In preferred embodiments, the rods are primarily formed from the same material as the gas-permeable membrane; this simplifies the bonding process, as well as recovery of materials for recycling. Additional rods may be present in some embodiments.

In preferred embodiments, one or more (and preferably each of) the supporting rods extend between the interior and exterior of the chamber, and form the fluid port(s). This may be achieved by providing each rod forming a fluid port with an internal fluid channel and corresponding openings which communicate between the interior and exterior of the rod, with at least one opening being external to the chamber, and at least one opening being internal to the chamber. This allows fluid to be delivered to or removed from the chamber via the supporting rods. The rods may be provided with one or more endpieces which are designed to cooperate with corresponding external devices, for example, devices for introducing or removing fluid.

In some embodiments, the culture vessel may further comprise reinforcing or supporting bands or mesh surrounding the exterior of the chamber. This may further strengthen the chamber, while allowing expansion to accommodate increases in cell culture mass.

The reactor module itself is preferably wheeled or is otherwise movable, to facilitate movement of the module between locations. The support frame of the reactor module may comprise one or more fluid manifolds which are connectable to, or which connect to, the fluid ports of culture vessels held in the reactor module. This allows fluid addition to or removal from multiple culture vessels in parallel, so maintaining the culture vessels. Preferably the reactor module further comprises a fluid transfer port in connection with the fluid manifold(s); the fluid transfer port is preferably so as to permit sterile fluid transfer from an external source to the fluid manifold(s), or vice versa. Such systems are commonly referred to as alpha beta transfer ports, rapid transfer ports, or similar. Such ports include two parts, alpha and beta ports on separate modules, and in general prevent full opening of the port until the two ports are connected.

The support frame may also comprise one or more frame members for connecting to and engaging with the movable compression means of multiple culture vessels. This frame member may itself be movable, and its movement may drive synchronised movement of multiple compression means. The reactor module may include a motor to drive movement of this frame member.

The reactor module may further comprise means for rocking, canting, or otherwise agitating the culture vessels therein. In some embodiments, the support frame may be constructed so as to permit relative movement of elements of the frame; a motorised drive may therefore provide for movement of one or more elements such that the frame as a whole deforms, resulting in rocking or movement of the culture vessels. In some embodiments the support frame may be located on a platform which itself may be rocked, or subjected to orbital motion. In other embodiments a separate canting surface may be provided, and the reactor modules transported to and from the canting surface at appropriate times.

The reactor module may further comprise one or more additional reservoirs for containing materials to aid cellular maintenance or direct the phenotype (for example, differentiation factors). In embodiments, one or more additional reservoirs may contain a defined atmosphere, to provide a desired gas balance to the cell culture.

The system as a whole may comprise a plurality of reactor modules as described. Such a plurality of reactor modules (or indeed a single reactor module) may be located within an environmentally-controlled location. The environmentally-controlled location may be an environment chamber. Relevant considerations for such a location include interior circulation of atmosphere; insulation to prevent heat loss to environment; gas inlets (air, carbon dioxide, and/or oxygen) positioned around the location; venting to maintain consistent pressure; and potentially monitoring in multiple locations to control atmospheric composition. Where a single reactor module is concerned, the environmentally-controlled location may be defined by a rigid or flexible cover enveloping the reactor module; this envelope may be used to define a preferred atmosphere within the module for cell culture.

The system may further comprise a process module for selective connection to a reactor module. In general, a process module may comprise sterile fluid transfer ports for connecting to a corresponding port on a reactor module, holding vessels, filters, and pumps to facilitate sterile mass transfer, cleaning and sterilisation, and instruments for monitoring of culture within reactor modules. Separating these functions from the reactor module allows for efficient use of resources, as the reactor modules can be constructed with minimum additional components (eg, sensors, pumps, etc) which may not always be needed in the reactor module. These additional components can then be placed in the process module. The inventors envisage that one process module may serve multiple reactor modules, and the movable nature of the separate modules allows reactor modules to be efficiently arranged within an environmentally-controlled location until retrieved to use with the process module. Transport of modules may be performed manually, but is intended for automation systems including, but not limited to, autonomous mobile robots, mobile guided robots, mobile stacking robots, and/or automated pallet shuttles paired with Cartesian gantry robotics. In other embodiments, a distinct process module may not be used, and the various functions performed by additional separate elements. However, this is not preferred. In some embodiments, sensors, pumps, and so on may be provided as part of the reactor module.

In some embodiments, the process module and the reactor module comprise cooperating alpha and beta transfer ports, wherein the alpha port includes a port seal and sealing gasket on an exposed face, which in use engages with an opposed face of the beta port to provide a seal; and both alpha and beta ports include fluid pipes which together define a fluid flow path between the process module and the reactor module. Each alpha and beta transfer port may further comprise a motorised iris arranged to selectively open and close each fluid pipe. Each alpha port may further include a chamber sterilisation port communicating between the exterior of the port and a void surrounding the alpha fluid pipe; this allows sterilisation materials (eg, hydrogen peroxide) to be introduced to the fluid flow path once alpha and beta ports are mated.

The culture vessel may comprise a cell culture. The cells may be any suitable cell; but may include vertebrate cells (for example, avian cells, including chicken cells; mammalian cells, including bovine, porcine, caprine, or ovine cells; piscine cells) or invertebrate cells (for example, insect or molluscan cells). The cells may be cells of a specific tissue type, for example, muscle cells, blood cells, liver cells, or the constituent types of white adipose tissue, beige adipose tissue, or brown adipose tissue; and/or may be cells of a specific cell type, for example, myocytes, adipocytes, preadipocytes, or satellite cells. In embodiments the cells are multipotent or pluripotent cells. In some embodiments, the cells are initially pluripotent or multipotent cells (for example mesenchymal stem/stromal cells), and are induced to differentiate during cell culture.

The culture vessel may comprise cell culture medium. The particular composition of the cell culture medium will differ depending on the cells to be cultured, but the skilled person will be able to select an appropriate composition for the culture medium.

The culture vessel may further comprise scaffolding for supporting cell culture. By scaffolding here is meant a solid support to which cells may adhere to promote growth. In embodiments the scaffolding may be in the form of particles. In preferred embodiments, the scaffolding is an edible material; this is advantageous when the cultured cells or cell products are intended for food use. Examples of edible scaffolding include plant-derived proteins or polysaccharides. Alternatively, or in addition, cells may be cultured in suspension, without scaffolding. A particulate scaffolding may provide a combination of the two; the particles may be maintained in suspension, while forming a scaffold for cell adhesion and growth. The preferred approach will depend on the nature of the cells and the desired products.

The culture vessel may further comprise means for promoting mixing of vessel contents; for example, pumps, impellers and the like.

Also provided as an aspect of the invention is a culture vessel as described herein.

A further aspect of the invention provides an alpha-beta transfer port system as described herein. Such a system may comprise cooperating alpha and beta transfer ports, wherein the alpha port includes a port seal and sealing gasket on an exposed face, which in use engages with an opposed face of the beta port to provide a seal; and both alpha and beta ports include fluid pipes which together define a fluid flow path between the ports. Each alpha and beta port may comprise a motorised iris to selectively open or close the respective fluid pipe. The alpha port may further include a chamber sterilisation port communicating between the exterior of the port and a void surrounding the alpha fluid pipe.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a culture vessel in accordance with an embodiment described herein Figure 2 shows a close up view of a part of the culture vessel of Figure 1

Figure 3 shows a reactor module in accordance with an embodiment described herein Figure 4 shows a further reactor module in accordance with an embodiment described herein; close up views of a rocking mechanism on the rollers are also shown

Figure 5 illustrates the movement of the rollers along culture vessels linked in a reactor module

Figure 6 shows a cutaway view of alpha and beta fluid transfer ports suitable for use with the present system

DETAILED DESCRIPTION OF THE INVENTION

Described herein is a system for cell culture; although primarily intended as a system for use in preparation of meat-free food replacement products, it will be apparent that it may be applied to other situations where large-scale cell culture and collection of cell mass and/or cell products is desired (for example, including manufacturing of advanced therapy medicinal products).

In general terms, the system is intended to allow expansion of multipotent or pluripotent cells (for example, mesenchymal stem cells, preferably porcine mesenchymal stem/stromal cells), and differentiate them into adipocytes (that is, fat storage cells). The final adipocyte mass is intended for incorporation into products in place of animal fat or solidified plant oils (eg in food products including but not limited to meat analogues, baked goods, dairy alternatives or confectionery; or to replace tallow in cosmetics, etc.).

The system is devised as a modular system, to allow for easy scaling and expansion. Cells are grown and their cellular phenotype controlled in a plurality of identical culture vessels fluidically-linked into discrete reactor modules. Modules are housed in an environmental chamber with a controlled atmosphere and temperature. Gas exchange into the culture vessels is achieved through semi-permeable membranes. Modules travel to, and return from, an ancillary device for mass transfers (e.g. inoculation or harvest). That ancillary device is referred to herein as a process module. Travel may be performed manually, but is intended for automation systems including, but not limited to, autonomous mobile robots (AMR), mobile guided robots (AGR), mobile stacking robots, and/or automated pallet shuttles paired with Cartesian gantry robotics.

An overview of an individual culture vessel is shown in Figure 1. The culture vessel 10 comprises a gas-permeable membrane 12 which is sealed along the edges to form a closed chamber defined by the membrane. Opposed ends 14, 16 of the chamber each include a hollow support rod 18, 20 running along the edges of the chamber.

The hollow support rods 18, 20 include a number of cutouts (not shown) in the portions within the chamber to form a fluid flow path between the hollow interior of the rod, the interior of the chamber, and the exterior of the chamber.

One of the support rods 18 is shown in more detail in Figure 2. The rod 18 is generally made from the same material as the gas-permeable membrane 12, and is heat-welded thereto. A profiled wedge portion 24 allows for secure welding between the rod 18 and the membrane 12 at the transition between the interior and exterior of the chamber. The rod 18 includes an open end including a connector 22 with features such as a groove or clip to allow secure attachment of the end to piping or tubing to allow fluid transfer. The connector 22 of the rod 18 may be formed from the same material as the rest of the rod and the membrane, although in embodiments it may be formed from, for example, stainless steel, to permit the connector to be removed and reused in the event that the remainder of the culture vessel is taken out of commission or otherwise recycled. The support rods are of sufficient strength to support the weight of a culture vessel when filled with cell culture.

Referring once again to Figure 1 , it can be seen that the exterior of the culture vessel also carries a number of support bands 26 encircling the vessel, intended to provide additional support and strength to the vessel, particularly as it expands when filled with culture medium.

Finally, disposed within the support bands 26 but exterior to the chamber are a pair of opposed rollers 28, mounted on a common frame (not shown). The frame includes actuators to move the rollers 28 into (or out of) closer alignment, to thereby compress opposed walls of the chamber together. When compressed, the rollers 28 effectively divide the chamber into two separate fluidic regions; a first region 30 is referred to as the culture chamber (in which cell culture takes place, and cells are expanded and differentiated), while the second 32 is referred to as the vessel reservoir (in which culture medium and other reagents are retained). The rollers 28 are movable along the length of the culture vessel, with the effect that the relative sizes of the culture chamber 30 and vessel reservoir 32 are adjusted, and culture medium or other reagents transferred from the reservoir 32 to the culture chamber 30.

Multiple culture vessels 10 are assembled into a common frame, to form a reactor module 34, shown in Figure 3. The frame 36 includes a number of mounting bars 38, 40 to retain each culture vessel 10 in place by receiving the ends of the support rods 14, 16. To the open ends of the support rods protruding through these mounting bars is mounted a fluid manifold 42, 44. Each fluid manifold connects to a fluid transfer port (not shown here, but illustrated in Figure 6) to allow sterile transfer of fluid to and from the culture vessels; the presence of the manifolds 42, 44 connects each culture vessel 10 into a common fluid circuit, such that cell culture proceeds in parallel. Also part of the reactor module 34 is a movable mounting bar 46 which has pins which run in tracks along the length of the module. The movable mounting bar 46 receives and retains the paired rollers 28 of each culture vessel 10. The movable mounting bar 46 can be moved along the module by a motor (not shown), which will keep each pair of rollers 28 in synchronised movement.

This particular reactor module 34 is constructed to permit some relative movement of the mounting bars 38, 40, such that moving these results in relative movement of the respective culture vessels. This movement can be automated to achieve periodic agitation of the culture vessels and the cells being cultured therein. See the lower part of Figure 3.

An alternate reactor module 134 is shown, schematically, in Figure 4. This module is generally similar to that shown in Figure 3, in that it includes multiple culture vessels and rollers, but has been adapted to provide dual axis pivoting to agitate the culture vessels. The rollers 128 of each culture vessel are retained in a multi-axis hinged retainer 147 allowing pivoting of the retainer 147 with respect to the culture vessel. Multiple retainers 147 can be linked together to provide an equivalent of the movable mounting bar 46 of Figure 3, serving to keep all rollers synchronised in their movement.

Figure 5 shows schematically the movement of the rollers 28 along the culture vessel to enlarge the culture chamber and reduce the size of the vessel reservoir. The rollers 28a, b begin initially in an aligned position (upper figure) such that the chambers of the culture vessel are fluidically isolated from one another. To begin movement, an actuator (not shown) in the movable mounting bar 46 separates the two rollers by advancing the upper roller 28a slightly with respect to the lower roller 28b (second figure). This opens a gap between the two rollers, so allowing fluid to move between the two chambers. The mounting bar 46 is then advanced forward with the two rollers 28a, b in their offset configuration, to enlarge the culture chamber while allowing fluid (culture medium and other components) to flow from the vessel reservoir into the culture chamber. Finally, once the mounting bar has stopped moving (lower figure) the actuator in the mounting bar 46 advances the lower roller 28b so that it is again aligned with the upper roller 28a, isolating the two chambers. The system may include alpha and beta fluid transfer ports to allow transfer of sterile fluids and cultures in non-sterile environments. Various brands of suitable ports are available, but one example of a port is shown in Figure 6. The transfer port includes an alpha port 50 and a beta port 52. In general, the system is designed with interlocks which prevent a fluid passage being opened between the two ports until the interior is sealed, so as not to break sterility. The alpha port 50 is mounted in the process module, while the beta port 52 is mounted in the reactor module. The alpha port 50 includes a port seal 58 and sealing gasket 60 on an exposed face, which in use will engage with an opposed face of the beta port 52 to provide a seal. Both ports include a fluid pipe 54, 56, which together define a fluid flow path between the process module and the reactor module. Each fluid pipe 54, 56 is initially closed by means of a motorised iris 60 to prevent dust or other materials entering the fluid pipes. The alpha port fluid pipe 54 is laterally movable to engage with the beta port fluid pipe 56. The alpha port further includes a chamber sterilisation port 62 communicating between the exterior of the port and the void surrounding the alpha fluid pipe 54.

In use, the two ports are aligned and docked, and the port seal 58 and sealing gasket 60 of the alpha port seat against the beta port to provide an initial seal. At this stage, the interior of the two ports is not sterile, but the interior of the fluid pipes is. The chamber sterilisation port is used to flush the port interior with a sterilising agent (for example, vaporised hydrogen peroxide, ethanol v/v water 70%). The motorised iris dust covers 60 are then opened, and the alpha port fluid pipe 54 advanced so as to fully engage with the beta port fluid pipe 56. The sterilisation port is flushed once more, and a sterile fluid connection is formed between the two ports. Given that the iris dust covers 60 are not in the sterile fluid path, it is possible to produce these (or elements of these) as replaceable or semi-replaceable components, and to produce these from materials such as sustainable plant-based components (paper, cellulose, wood, fabrics) rather than stainless steel. Such materials may degrade during repeated sterilisation cycles, but the cost of replacement will be offset by the reduced overall cost of such materials compared with stainless steel etc.

The foregoing describes in general terms the arrangement and structure of the culture vessels and reactor modules. The system as a whole may also include one or more process modules, holding vessels, filters, and pumps to facilitate sterile mass transfer, cleaning and sterilisation, and monitoring of culture within reactor modules. The process module can be connected to a reactor module via corresponding fluid transfer ports provided in each of the reactor modules and in the process module. The process module may be in a fixed location, and individual reactor modules may be retrieved from an environmental chamber in which atmosphere, temperature, and humidity are controlled. This process is ideally largely automated, using robotic transfer.

We now describe in more detail one particular workflow process for preparing the system and cultivating cells. The following abbreviations are used in the workflow:

CV - Culture vessel

RM - Reactor module

PM - Process module

CR - Canting rack, used to tilt or rotate the RM

EC - Environmental chamber

In brief, the workflow consists of ten core steps: 1) prepare reactor modules; 2) inoculate from cell bank; 3) expand cell population at [N-2] stage; 4) passage; 5) expand cell population at [N-1 ] stage; 6) passage; 7) expand cell population at [N] stage; 8) prepare for differentiation; 9) differentiate; 10) harvest. After harvest, portions of the workflow may be repeated to obtain further harvested product. These are described in more detail below.

Process steps

1 . Prepare reactor modules a. Assemble CV and RM in workshop i. Equip select modules with sensors (e.g. metabolites, pH, dissolved oxygen, dissolved CO2) as representatives of a batch ii. Each RM contains a quantity of CVs to match the optimised cell split ratio Q iii. CV working volume min:max ratio is not dependent on Q, but is in turn dependent on mechanical constraints and media cell support capacity

(as an alternative to this step 1 .a, pre-assembled CV and RM may be obtained from outside third parties) b. Transport each new RM to the PM c. Clean and sterilise d. Set CV rollers to the minimum working volume, to divide interior into culture chamber and reservoir e. Disconnect RM from PM aseptically f. Transport RM to QC hold area during sterility validation ulate from cell bank a. Transport RM to PM b. Connect RM to PM aseptically c. Inoculate CV culture chambers with cells, media, and (optional) scaffolding d. Fill CV reservoirs with culture media and (optional) scaffolding e. (optional) Fill RM reservoir with growth supplement f. Disconnect RM from PM aseptically g. Transport RM to designated culture position in the EC. The module capacity of the chamber may be adapted to the available facility volume.and cell population [N-2], Modules remain in position for the duration a. Rollers increase CV working volume and transfer reservoir mass (e.g. culture media or scaffolding) to meet the increasing metabolic demands of the cell population. If necessary, additional factors may be supplied by RM reservoirs. b. Agitation may be applied in rocking or orbital motion to enhance gas transfer or incorporation of the additional mass. sage a. Option 1: Using a canting rack, to remove the need for aseptic connections (culture remains inside the reactor module) i. Transport RM to CR ii. Divide the contents of inoculated CV, now at maximum working volume, amongst the remaining CVs iii. Return RM to culture position in EC b. Option 2: Using the process module i. Transport RM to PM ii. Connect RM to PM aseptically iii. Transfer contents of inoculated CV, now at maximum working volume, to the holding vessel iv. Transfer holding contents to remaining CVs in RM v. Disconnect RM from PM aseptically vi. Return RM to culture position in EC and cell population [N-1], as in [N-2] expansion sage (1 to Q split) a. Transport RM to PM b. Connect RM to PM aseptically c. Transfer contents of all CVs, now at maximum working volumes, to the holding vessel d. Reset CV rollers to minimum working volume e. Inoculate all CV culture chambers with cells, media, and (optional) scaffolding f. Fill CV reservoirs with culture media and (optional) scaffolding g. (optional) Fill RM reservoir with growth supplement h. Disconnect RM from PM aseptically i. Return RM to culture position in EC j. Repeat inoculation for Q-1 additional RM until holding vessel is empty i. RM may originate from the holding area, which may be populated with new assemblies from Step 1 or cleaned assemblies from harvest Step 10 k. Designate RM1 as [N-1] for continual reseed l. Designate RM2-Q as [N] for expansion and cell population [N], as in [N-1] expansion pare for differentiation a. Transport RM to PM b. Connect RM to PM aseptically c. Perform one or more of the following: i. Transfer culture mass into PM to concentrate solids by means of in-line filtration or centrifugation ii. Coat cell-laden scaffolding (if scaffolding used) with a material (e.g. hydrogel or other polymeric matrix or protein matrix, polysaccharide matrix or combination thereof) to prevent cell liftoff during differentiation iii. Remove fluid from CVs to decrease concentration of waste products. Waste fluid to be retained for reuse, repurpose, or byproduct exploitation. iv. Decrease culture volume and increase reservoir volume with CV rollers v. Transfer culture mass back to CV, with fresh media vi. Fill CV reservoirs with differentiation supplement vii. Fill RM reservoirs with differentiation supplement d. Disconnect RM from PM aseptically e. Return RM to culture position in EC f. Repeat inoculation for Q-1 additional RM until holding vessel is empty rentiate a. Rollers increase CV working volume and transfer reservoir mass (e.g. differentiation supplement) to meet the increasing metabolic demands of the cell population. If necessary, additional factors may be supplied by RM reservoirs. b. Agitation may be applied (eg, in rocking, sinusoidal, or orbital motion) to enhance gas transfer or incorporation of the additional mass. vest a. Transport RM to PM b. Connect RM to PM aseptically c. Transfer culture mass into PM to concentrate solids by means of in-line filtration or centrifugation. Waste fluid to be retained for reuse, repurpose, or byproduct exploitation. d. Flush RM to reduce protein buildup, or perform full CIP/SIP routine e. Disconnect RM from PM aseptically f. Transport RM to QC hold area, or inspection area g. Concentrate solids further to desired specification by means of filtration (including for example as a gravity-driven settling bed, standing wave filtration, centrifugal filtration, etc). Critical quality attributes are monitored during the process. h. Transfer mass to long-term storage containers i. Repeat inoculation for Q-1 additional RM until holding vessel is emptycess repeats from step 6: passaging of the N-1 expansion ection and maintenance of reactor modules a. Remove modules from circulation for inspection at Step 10: structural integrity, wear in moving components, protein buildup on interior surfaces. It will be apparent that the present invention provides a number of advantages compared with prior art systems. A vessel’s range of working volume is a critical factor in determining the number of mass transfers to a larger vessel. Each mass transfer increases cost and contamination risk. In stir tank reactors, the typical range is 30-75% total vessel volume (2.5x increase) . The vessels in this invention may reach 10-95% total vessel volume (9.5x increase).

The inclusion of a culture volume and reservoir volume in the same vessel facilitates in situ process intensification and reduces mass transfers.

The ability to decouple the reactor modules and store at any location eliminates the need for piping to scale linearly with the total facility culture volume. Instead, piping scales with the number of process modules. Further, plumbing, pneumatic, and/or hydraulic lines are limited to the process module, rather than the reactor module; and reactor modules may be stored at relatively high density to make maximum use of available space.