GB2163453A - Vessel for culturing cells on microcarriers or in capsules - Google Patents

Abstract

Disclosed is a vessel (8) for culturing suspensions of animal cells or of microcarrier-immobilized animal cells. The vessel features three output ports: one (34) adjacent the bottom for draining the entire contents of the vessel; one (58) adjacent the bottom covered by a screen or the like which permits passage of fluids but precludes passage of cells and/or microcarriers; and one (48) spaced above the bottom with a similar screening element which permits continuous feeding of the cells. The vessel also includes a stirring device (112) having a stirring rate acceleration controller and an integral device for making microcarriers or capsules in situ. <IMAGE>

Description

SPECIFICATION Vessel for culturing cells on microcarriers or in capsules This invention relates to large volume culture vessels useful for growing animal cells. More particu larly, it relates to a sterilizable large volume apparatus within which an animal cell culture may be microencapsulated or immobilized on or in water-insolubie carrier particles, fed batchwise or continuously, aerated, gently stirred, and harvested.

If the potential of the new recombinant DNA and hybridoma technologies is to be exploited to mass produce commercial quantities of valuable proteins, it is necessary to develop methods and apparatus for culturing fragile animal cells on at least a semi-automated basis and in large volume. Eucaryotic animal cells which produce lymphokines, hormones, monoclonal antibodies or other valuable proteins or glycosylated proteins typically are extremely fragile and have strict oxygen and nutrient requirements. In recent years significant progress has been made in immobilizing such cells on or in microcarrier beads or within semi-permeable capsules. Such systems can aid in protecting the cells from shear forces and can provide a microenvironment well suited for cell growth. See, for example, U.S. 4,352,883, U.S. 4,409,331, and U.S. 4,399,219.

Eucaryotic animal cells, if they are to be grown to high density, require a sterile environment in which nutrients are available for ingestion and wastes are removed. The pH of the medium and the partial pressures of the oxygen and carbon dioxide must be controlled to near optimum levels.

The preparation of immobilized seed cultures is typically conducted separately from the mass culturing, and the cells must be transported to the culture vessel increasing the risk of contamination by stray virus or microorganisms. The mass transfer requirement of growing cells requires that the medium and cell microcarriers be stirred. This can lead to cell damage or disintegration of the microcarrier if a conventional paddle-type stirring device commonly used in bacterial fermenters is employed.

The growth of animal cell cultures in microcapsules has many advantages as disclosed in the aforementioned U.S. Patent No. 4,409,331 and copending patent application Serial No. 579,491 filed February 13, 1984 entitled Cell Culturing with Gas Sparging. In the culture of large quantities of such encapsulated animal cells, it would be desirable to employ a sterilizable cell culture vessel designed to facilitate collection of spent medium or medium containing the protein of interest separately from the capsules, washing the encapsulated culture, and collection of the capsules for harvesting or further treatment.

Devices for culturing animal cells are known. Examples include the devices disclosed in U.S.

4,355,906, U.S. 4,203,801, U.S. 4,377,639, U.S.

4,204,042, U.S. 4,343,904, U.S. 4,087,327, and U.K.

2,059,436A. Recognizing the fragile nature of ani mal cells, Harker et al disclose in U.S. 2,958,517 a magnetic stirring bar device driven by a motor fit ted with a rheostat so that the speed of stirring can be controlled. Gavin discloses in U.S. 3,013,950 a culture vessel including a fluid outlet valved to per mit continuous feeding and collection of animal cells. Tolbert et al, in U.S. 4,184,916 disclose a culture apparatus for mamallian cells which permits continuous media flow while retaining the cells or carrier particles within the culture vessel by means of a filter unit.

It is an object of this invention to provide a steri lizable vessel for culturing free animal cells or cells adhered to the surface of or disposed within solid or gelled porous microcarriers, or contained within permeable capsules (hereinafter "microcarrier-immobilized cells"), which permits continuous or batch feeding, provides agitation without substantially damaging the microcarriers or cells, facilitates washing and separation of the microcarrierimmobilized cells from the medium, permits the manufacture of capsules or other microcarriers in situ, and automatically controls the volume of culture.

The present invention is apparatus for culturing animal cells disposed in a medium, said apparatus comprising a vessel having a bottom wall and side walls defining a volume for holding said medium and said animal cells, means for introducing culture medium into said vessel, means, adjacent the bottom wall of said vessel, defining a first output port of a size sufficient to permit removal of medium and intact cells, means defining a second output port spaced above said bottom wall for draining excess medium from said volume, and first porous means defining openings of a size sufficient to preclude passage of said cells but to permit passage of said medium, said first porous means being interposed between said second output port and said volume.

With such an arrangement, it is possible to continuously feed medium into the vessel at any desired rate while maintaining the total volume of fluid and maintaining the microcarriers or the cells within the vessel. Optionally, the second output port is adjustable or replaceable so that the volume of fluid held in the vessel during culturing may be varied.

In preferred embodiments, the apparatus further comprises a third output port adjacent the bottom wall of the vessel and a screen or the like having suitable sized opening to preclude passage of the microcarriers or cells. Preferably, both the second and third port are disposed within a cylindrical porous tube extending upwardly from the bottom wall of the vessel.The third output port permits draining the entire fluid contents of the vessel while retaining the cells, microcapsules, or other microcarriers. It is useful, for example, if the cells are to be washed during processing.

In preferred embodiments the apparatus includes a paddle for stirring the animal cells and medium, a prime mover such as an electric motor for rotating the paddles, and a control for the prime mover for regulating the rotational acceleration of the paddle.These components in combination permit slow acceleration of the stirring rate which has been found to minimize damage to the microcarriers and animal cells. Preferably, the controller accelerates the paddle gradually over a period of at least one minute.

The vessel may also include an input port for introducing microcarrier-immobilized animal cells and an integral device for forming plural shape-retaining matrices containing dispersed animal cells.

These can act as microcarriers per se or may be further treated in situ to form permeable capsules.

In the latter case, the vessel further comprises a port for introducing fluid containing components for coating the shape-retaining matrices such as a polycation of the type used to form membranes in the encapsulation procedure disclosed in U.S.

4,352,883.

The device for forming the shape-retaining matrices preferably comprises a modification of a known "jet head" droplet forming device. It features a plenum for holding a solution of a gellable substance such as sodium alginate containing plural animal cells, a plurality of conduits, i.e., hollow needles, extending downwardly from the plenum, each of which define an opening disposed below the plenum, and a pump or other means for forcing the gellable substance through the conduits.

The device also includes structure defining air passageways adjacent the openings of the conduits for interrupting the flow of gellable substance, thereby separating the flow into plural drops.Lastly, the drops after formation fall into a reservoir for holding a gelling solution such as calcium chloride to convert the drops to shape-retaining matrices. The matrices, disposed in the calcium chloride solution, are then transported through a conduit directly into the vessel where they may be subsequently treated with various reagent of the type disclosed in U.S. 4,352,883 and in copending U.K. Application 8503247, Serial No. 2,153,780.

Thus, the shape-retaining, calcium cross-linked matrices may be washed several times in saline and subsequently exposed to a polycation solution such as a solution of polylysine or polyornithine to form a permeable membrane about the gelled matrices. Also, a dilute solution of sodium alginate may be added after membrane formation to prevent microcapsule clumping or for other purposes, and the interior matrix of the thus formed capsules may be reliquified by the introduction of a solution of sodium citrate or a chelating agent.

Thus, the apparatus of the invention permits in situ manufacture of microcapsules or other microcarrier-immobilized animal cells, enables growth of suspension cell cultures or immobilized cell cultures to high density with either batch or continu ous feeding without the necessity of plural metering pumps, and facilates collection of me dium containing substances of interest produced by the cells, washing of the cells, or collection of the microcarriers for cell harvesting or harvesting of proteins of interest retained within the microcar riers.

Embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which: Figure 1 is an exploded perspective, partially cutaway view of a microcarrier-immobilized cell culture vessel embodying the invention; Figure 2 is a perspective detailed view of the cover of the vessel of Figure 1 illustrating an exemplary input port configuration; Figure 3 is a detailed view of a portion of the vessel of Figure 1 illustrating the vessel's output ports for collecting microcarriers or cells, draining medium for harvest or replacement, and maintaining automatically a selected volume of medium within the vessel; and Figure 4 is a detailed view of integral apparatus for forming shape-retaining gel spheres or other shapes containing immobilized cells.

Like reference characters in the respective drawn figures indicate corresponding parts.

Referring to the drawing, Figure 1 depicts a culture vessel 8, its cover assembly 12, and its paddle drive and controller assembly 14, and its optional integral droplet forming apparatus 9.

The vessel 8 comprises a cylindrical 316L stainless steel structure comprising sidewalls 10, an open top 16, integral handles 18 and 20, and supports 22 extending from a bottom wall 32. Open top 16 defines a sterilizable, elastomeric seal 24 and a circumferential flange 26 for mating with the cover assembly 12. Handles 18 and 20 comprise hollow cylinders for receiving the lift bars of a hydraulic lift or the like. As illustrated in Figure 1, vessel 8 in use is filled with a multiplicity of cells, microcapsules, or other microcarriers depicted at 28 disposed in a culture medium or other fluid 30.

The bottom wall 32 of vessel 8 defines a centrally disposed port 34 serviced by a conduit 36 controlled by a valve 38 having a male, tapered tubing attachment segment 40. Fixed to vessel bottom 32 is an upwardly extending, cylindrical, perforated stainless steel screen 42 having a cap 44. The tube defines openings of a size small enough to preclude passage of microcarriers or of cells, depending on the intended use of the vessel. Within the tube is a standpipe 46, extending upwardly parallel to tube 42 and having an upper open end 48 which can determine a maximum volume of culture the vessel can contain. Standpipe 46 is removably connected, e.g., by a threaded coupling 49, to a port 50 passing through the bottom 32 of vessel 8 within the perforated cylinder 42. A standpipe of different height (not shown) may be substituted for standpipe 46 if a different maximum volume of fluid is desired. The output port 50 leads to conduit 52, its associated valve 54, and tapered tubing attachment segment 56.Adjacent port 50 within perforated cylinder 42 is a second port 58 passing through bottom wall 32 of the vessel 8 which again is serviced by a conduit 60, its associated valve 62, and a male, tapered tubing attachment segment 64.

Disposed on the left side of the cylindrical wall 10 of vessel 8 is an input port 66 having a threaded connector 68 for attaching the outlet pipe 70 of a microcarrier-forming apparatus 9 described in more detail hereinafter.

Cover 12 comprises a hexagonal sealing plate 100 having sealing hool assemblies 102 at each corner. To seal the vessel, the cover is placed atop cylindrical vessel 8, the sealing hooks 104 of the respective assemblies 102 are turned to engage the underside of the flange 26 with seal 24 pressing against the bottom surface of plate 100, and the knobs 106 are turned to urge plate 100 against seal 24. Plate 100 may be fabricated from clear plastic, e.g., polycarbonate, to permit visual inspection of the interior of vessel 8.

Disposed centrally in plate 100 is a magnetic driven element 108 comprising a magnet and a conventional rotatable, bearing-mounted shaft 110 fitted with a paddle 112. The paddle comprises a pair of angled paddle blades 114, 116 disposed 180 degrees apart which define planes 90 degrees apart. The plate 100 includes plural threaded shafts 118 for attaching paddle drive and controller assembly 14 to cover 12 by means of bolts (not shown). Disposed radially about driven element 108 in plate 100 are six ports designated 110, 112, 114, 116, 118 and 120. Each port passes through plate 100 and is provided with a fitting (not shown in Figure 1) for receiving various tubes and control devices useful for manufacturing microcarriers in situ, introducing various wash solutions, supplementing the medium, monitoring oxygen partial pressure, pH, etc., and for other purposes.

Port 110 is serviced by medium inputfeedline 122 fitted with a filter housing 124, and a male, tubing attachment segment 126. Port 112 and its attached tubing 128 and filter housing 130 serve as a vent. Port 114 has an elongate tube 132 passing through it and is fitted with a coupling 134 for receiving a conventional oxygen probe, pH meter probe, or other device for monitoring the condition of the medium or other fluid disposed in vessel 8.

Port 116 is fitted with tubing 135 and female connector 136. This structure serves as an input for cells or for previously formed microcarriers, or for the introduction of various fluids used in fabricating capsules or microcarriers in situ. Ports 118 and 120 are fitted with tubes 138 and 140 and their associated filter elements 142, 144, and male tubing attachment segments 146, 148. Port 118, tube 138, filter element 142, and segment 146 may be used to provide gases of a defined composition in the head space disposed in vessel 8 above the level of fluid 30. Port 120, tube 140, filter element 144 and segment 148 may be connected to a source of air, oxygen, or an oxygen/carbon dioxide mixture.

Gases passed through port 120 are directed by conduit 150 to a microporous sparger head 152 to oxygenate the medium as disclosed in copending application Serial No. 579,491 referenced above.

Paddle drive and controller assembly 14 comprises a base plate 306 with a support shaft 308.

An electric motor 300 is mounted on base plate 306 with its drive shaft (not shown) passing through plate 306 coupled to a conventional annular magnetic drive element (not shown) within a drive housing 310. When assembly 14 is positioned on cover assembly 12, the annular drive element is disposed coaxially around magnetic driven element 108 on cover assembly 12. Rotary motion of motor 300 is thus transferred to shaft 110 and paddle 112 through a magnetic couple. Electric power for motor 300 is supplied through a controller 302 containing circuitry well known to those skilled in the art which serves to control the rotational acceleration of motor 300 and thus of paddle 112. Controller 302 may contain circuitry controllable by knob 304 which sets the rate of change of rotary motion of the paddle 112 to one of several fixed rates.Alternatively, knob 304 may simply actuate circuitry which steadily increases the rotational velocity of paddle 112 such that it reaches its optimum rotational velocity, e.g., 20 rpm, in a given time period, e.g., 5 minutes. Knob 305 sets the maximum rotational velocity of paddle 112.

Referring to Figure 4, a gelled matrix forming apparatus 9 for use in connection with culture vessel 8 is shown in detail. Apparatus 9 comprises a housing 72 defining a reservoir 200 fitted with a reservoir input port 202 used to fill reservoir 200 with a gelling solution. Port 202 may also serve as a vent. A valve 204 disposed in outlet line 206 regulates the flow of microcarriers fabricated in apparatus 9 through conduit 70 and into vessel 8. The solution in reservoir 200 may be, for example, a dilute solution of calcium chloride used to gel sodium alginate droplets formed as disclosed hereinafter and containing eucaryotic animal cells.

Atop reservoir 200 is a drop forming apparatus generally depicted at 208. It comprises a structure defining a plenum 210 and a separate compartment 212. Plural hollow needles 214 extend from plenum 210 through space 212 and pass through the bottom wall 216 of droplet forming apparatus 208 where they emerge within conical openings 218 disposed above reservoir 200. Between each each of the hollow needles and the bottom wall 216 is an annular space providing an air passageway between compartment 212 and conical openings 218. Plenum 210 is fed by a conduit 220, optionally serviced by a pump (not shown) for delivering an animal cell culture disposed in a gellable solution, e.g., sodium alginate.Space 212 is fed by an air input tube 222 and an associated filter housing 224. Droplet forming apparatus 208 may be fabricated from clear plastic such as polycarbonate or polysulfone.The inside diameter of the hollow needles 214, the air pressure introduced through tube 222, and the viscosity of the gellable solution contained in plenum 210 together determine the size of the droplets made in apparatus 9.

The above-described apparatus may be used to manufacture microcarriers comprising animal cells and to culture immobilized or non-immobilized cells to high density while providing fresh medium on a continuous or an intermittent basis. The apparatus 9 for forming shape-retaining gelled matrices containing eurcaryotic animal cells is an optional component.lf desired, it may be omitted from the apparatus. The microcarriers or conventional suspension cultures may be prepared separately and introduced into the culture vessel, for example, through input port 116.

A typical sequence of operation of this embodiment of the invention will now be described. Before sterilization of the device in an autoclave or the like, cover assembly 12 is sealed to top 16 as described above. After sterilization paddle drive and controller assembly 14 are bolted to plate 100 on cover assembly 12, and isotonic saline is pumped into the vessel, for example, through input port 116. With valves 38 and 62 closed, the saline may be filled to a level, for example, just below input coupling 66.

A solution of, for example, 1.2% sodium alginate in an animal cell culture, e.g., a hybridoma culture which produces monoclonal antibodies, is introduced through conduit 220 where it fills plenum 210 and passes through hollow needles 214.0ne suitable feed rate is 6 litres of gellable solution per hour. Air or another nontoxic gas, for example, at a rate of 70 cubic feet per hour, is introduced through line 222 where it fills space 212 and passes about hollow needles 214through the annular openings in wall 216. The effect of the conical openings 218 in combination with the air flow is to cut off drops of sodium alginate, depicted at 230, on the order of, for example, 500-1,000 microns in diameter. Reservoir 200, which previously has been filled with a 1.2% calcium chloride solution via input conduit 202, receives the droplets.

On contact with the solution, the sodium alginate in the droplets 230 is gelled by interaction with the calcium ions in reservoir 200 to form a multiplicity of shape-retaining matrices which can act as microcarriers. On a continuous or intermittent basis, the shape-retaining calcium alginate gel spheres or other shapes pass through outlet port 206, valve 204, conduit 70, and coupling 66 and enter the saline solution in vessel 8 where, as disclosed in copending U.K. application Serial No. 2153780, they are expanded.The gel spheres may be washed several times in fresh batches of saline by opening valve 62, draining a portion of the previous saline wash solution, and then introducing fresh saline, for example, through input port 116.At this point, the vessel may be filled with medium designed for growing the cells in which case the calcium alginate gel spheres containing the animal cells act as microcarriers. However, it is preferred to form semipermeable membranes about the gel spheres and thereafter to reliquify the gel so as to promote mass transfer and cell growth as disclosed in the application referenced immediately above and in U.S. Patent 4,352,883.

To form the membranes, a portion of the saline in which the gel spheres are disposed is drained through valve 62. Because of perforated screen 42, no gelled spheres are lost during this process.

Thereafter a solution of a polycation such as polylysine or polyornithine is introduced into vessel 8, for example, through port 116. The polycation interacts with negatively charged groups on the sur- face of the calcium alginate-gelled, shape-retaining matrices to form a semipermeable membrane about each. After one or more treatments involving different concentrations of the same polycation or different polycations, the liquid phase may again be removed via output port 58, conduit 60, and valve 62. Preferably, a dilute solution of sodium alginate is then introduced into the vessel to neutralize positive charges on the microcapsules. It is also desirable to wash the microcapsules containing animal cells with a solution of citrate or a chelating agent to remove calcium ions from the interior of the capsules, thereby reliquifying the gel.

After removing the charge-neutralizing solution and washing the capsules several times in saline, the medium is introduced through port 110 with valves 62 and 38 closed and valve 54 open. When the medium has filled vessel 8 up to the opening 48 of standpipe 46, medium passes through the perforated screen 42, descends down standpipe 46, and passes through valve 54, tubing attachment 56, and tubing (not shown) connected thereto. The vessel is accordingly filled with, for example, 5 litres of microcapsules in 20 litres of medium to make a total volume of 25 litres.

Alternatively, previously manufactured microcarriers containing cells, or a conventional suspension culture may be introduced directly into vessel 8 through a suitable input port in cover plate 100.

At this point controller 302 is actuated as acceleration control knob 304 and rotational velocity control knob 305 are set to actuate motor 300. The magnetic drive element under the control of controller 302 and driven by motor 300 starts slow rotation of driven element 108 and shaft 110 so that paddle 112 slowly picks up rotational velocity. This minimizes damage to the microcapsules and/or the animal cells contained in the vessel. During the course of the culturing procedure, medium may be periodically drained from the culture, through output port 58 and the open valve 62, and replaced with fresh medium via input port 110. Pressure within the vessel is maintained at or close to atmospheric by vent 112. An oxygen probe or pH probe may be inserted through port 114 and tube 132 to monitor the partial pressure of gases or other conditions in the medium.The composition of the gas in the head space within the vessel may be controlled by introducing a flow of air, carbon dioxide, oxygen, etc. through 118. If desiredd, when growing encapsulated cell cultures, the culture may be oxygenated via port 112 and associated sparging head 152 as disclosed in copending U.S. application Serial No. 579,493.

Continuous replenishment of culture medium may be conducted simply by metering in culture medium through portll0. As the fluid level in vessel 8 rises, excess medium will be drained through standpipe 46 and valve 54. Microcarriers or free cells contained in the medium cannot pass through the openings in the perforated screen 42 and accordingly are retained within thr vessel.

If the porosity of the microcapsules is such that the protein of interest produced by the encapsulated cells can traverse the capsule membranes, the protein may be purified from the medium. In this case, the cells may be batch fed during their protein production phase, or continuously fed, and the protein may be collected from the spent me dium drained from the vessel either through port 58 and valve 54 . If as preferred, the porosity of the membranes is controlled so as to retain all or substantially all of the protein of interest within the microcapsules, at the end of the culture period, valve 38 is opened and the entire contents of the vessel is collected through a tube (not shown) attached to male tubing segment 40. The protein may then be harvested by disrupting the microcapsules and separating it from the cells and other components contained therein.

Claims (19)

1. Apparatus for culturing animal cells disposed in a medium, said apparatus comprising a vessel having a bottom wall and side walls defining a volume for holding said medium and said animal cells, means for introducing culture medium into said vessel, means, adjacent the bottom wall of said vessel, defining a first output port of a size sufficient to permit removal of medium and intact cells, means defining a second output port spaced above said bottom wall for draining excess medium from said volume, and first porous means defining openings of a size sufficient to preclude passage of said cells but to permit passage of said medium, said first porous means being interposed between said second output port and said volume.

2. Apparatus as claimed in claim 1, wherein said cells are immobilized in microcarriers, said first output port is of a size sufficient to permit removal of intact microcarriers, and said openings are dimensioned to preclude passage of said microcarriers.

3. Apparatus as claimed in claim 1, further comprising means adjacent the bottom wall of said vessel defining a third output port for draining fluid from said volume, and second porous means defining openings of a size sufficient to preclude passage of said cells but to permit passage of said medium, said second porous means being interposed between said third output port and said volume.

4. Apparatus as claimed in claim 2, further comprising means adjacent the bottom wall of said vessel defining a third output port for draining fluid from said volume and second porous means defining openings of a size sufficient to preclude passage of said microcarriers but to permit passage of said medium, said second porous means being interposed between said third output port and said volume.

5. Apparatus as claimed in any preceding claim, further comprising a paddle disposed within said volume for stirring said animal cells and said medium, drive means for rotating said paddle, and control means for regulating the rotational acceleration of said paddle so as to minimize damage to said cells or to said microcarriers.

6. Apparatus as claimed in any of claims 1 to 4, further comprising a first input port for injecting microcarrier-immobilized animal cells into said volume.

7. Apparatus as claimed in claim 6, further comprising integral means for forming plural shape-retaining matrices containing animal cells and conduit means in communication with said first input port for transporting said shape-retain ing matrices into said volume.

8. Apparatus as claimed in claim 7, further comprising a second input port for introducing into said vessel fluids containing components for coat ing said shape-retaining matrices to form mem branes about said matrices in said vessel.

9. Apparatus as claimed in any of claims 1 to 4, wherein said means defining a second output port includes means for adjusting the level of said sec ond output port within said vessel.

10. Apparatus for manufacturing microcarrier immobilized animal cells and for culturing said animal cells, said apparatus comprising a vessel hav ing a bottom wall and side walls defining a volume for holding fluids, means for introducing fluids into said vessel, means adjacent the bottom wall of said vessel defining a first output port of a size sufficient to permit removal of intact microcarriers, integral means for forming plural shape-retaining matrices containing dispersed animal cells,-and conduit means for transporting said shape-retaining matrices into said vessel from said matrices forming means.

11. Apparatus as claimed in claim 10, further comprising a second input port for introducing into said vessel fluids containing components for coating said shape-retaining matrices to form membranes about said matrices in said vessel.

12. Apparatus as claimed in claim 11, further comprising means adjacent the bottom wall of said vessel defining a third output port for draining fluids from said vessel and porous means defining openings of a size sufficient to preclude passage of microcarriers contained in said vessel but to permit passage of fluids, said porous means being interposed between said third output port and said volume.

13. Apparatus as claimed in claim 10, further comprising a paddle disposed within said volume for stirring fluids contained therein, drive means for rotating said paddle, and control means for regulating the rotational acceleration of said paddle so as to minimize damage to microcarriers and cells contained in fluids disposed in said vessel.

14. Apparatus as claimed in claim 10, wherein said integral means for forming shape-retaining matrices containing dispersed animal cells comprises a plenum for holding a solution of a gellable substance containing plural animal cells, plural conduits in communication with said plenum, each of which defines an opening disposed below said plenum, means for forcing a gellable substance through said conduits, means defining air passageways adjacent said openings for interrupting the flow of said gellable substance to separate said flow into plural drops, and a reservoir for holding a gelling solution to convert said drops into shaperetaining matrices.

15. Apparatus as claimed in claim 14, further comprising a second input port for introducing into said vessel fluids containing components for coat ing said shapc-retaining matrices to form membranes about said matrices in said vessel.

16. Apparatus as claimed in claim 14, further comprising means adjacent the bottom wall of said vessel defining a third oLitptit port for draining fluids from said vessel and porous means defining openings of a size sufficient to preclude passage of microcarriers contained in said vessel but to permit passage of fluids, said porous means being interposed between said third output port and said volume.

17. In an animal cell culturing vessel having a paddle for stirring an animal cell culture disposed therein, the improvement comprising means for regulating the rotational acceleration of said paddle so as to minimize damage to cells or microcarriers contained therein.

18. The improvement of claim 17, wherein said means for regulating comprises an electric motor drive controller for accelerating said paddle gradually over a period of at least one minute.

19. Apparatus for culturing animal cells disposed in a medium, substantially as hereinbefore described with reference to, and shown in, the accompanying drawings.