JPS6260074B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a large volume culture vessel useful for growing animal cells. In particular, a sterilizable, large-volume device in which animal cells are microencapsulated or immobilized on or within water-insoluble carrier particles, fed with medium all at once or continuously, aerated, and gently It can be stirred and collected.

If new recombinant DNA and hybridoma technologies are to produce large commercial quantities of useful proteins, it is necessary to develop methods and apparatus for culturing fragile animal cells in large volumes in at least semi-automated operation. . Eukaryotic cells, which produce useful proteins such as lymphokines, hormones, monoclonal antibodies, and glycosylated proteins, are highly fragile and have strict requirements for oxygen and nutrients. In recent years, great advances have been made in immobilizing such cells on or within microcarrier beads or within semipermeable capsules. Such systems protect cells from shear forces and
Provide a suitable microenvironment for cell growth. See, eg, US Pat. No. 4,352,883, US Pat. No. 4,409,331 and US Pat. No. 4,399,219.

BACKGROUND OF THE INVENTION Growing eukaryotic cells to high densities requires a sterile environment to provide nutrients for intake and remove waste products. The pH of the medium and the partial pressures of oxygen and carbon dioxide must be controlled around appropriate levels. Preparation of fixed cells is usually performed separately from mass culture, and the risk of contamination with stray viruses and microorganisms increases when cells are transferred to culture vessels. In order to transfer a large amount of proliferated cells, it is necessary to stir the medium and cell microcarriers. When a spatula-type stirring device used in conventional bacterial fermentation is used, cell damage and microcarrier breakage occur.

The growth of animal cells in microcapsules is described in the aforementioned U.S. Pat. No. 4,409,331 and co-filed patent application no. As you can see, it has many advantages. To culture such encapsulated animal cells in large quantities, the working medium or the medium containing the protein of interest is separated from the capsule, combined, the encapsulated cells are washed, and the capsule is removed for harvest or further treatment. It is desirable to use sterilizable cell culture vessels designed to facilitate the combination of cells.

Devices for culturing animal cells are known. Examples include U.S. Patent Nos. 4,355,906, 4,203,801,
No. 4377639, No. 4204042, No. 4343904, No.
4087327 and British Patent No. 2059436A. Harker 5 recognized that animal cells are fragile and issued US Patent No. 2958517.
described a magnetic stirring bar device driven by a motor equipped with a rheostat to control the speed of stirring. Gavin, in US Pat. No. 3,013,950, disclosed a culture vessel in which a liquid outlet allowed animal cells to be continuously integrated and fed with culture medium. Tolbert 5 published in US Pat. No. 4,184,916 a culture device for mammalian cells in which the culture medium is continuously drained while the cells and carrier particles are retained in the culture vessel by a filter device.

Problems to be Solved by the Invention It is an object of the present invention to solve the problem of free animal cells or cells attached to or enclosed in solid or gel porous microcarriers or contained within permeable capsules. Similarly, capsule microelectrodes can be used to culture microcarriers (hereinafter referred to as "microcarriers-immobilized cells"), which can be fed with a medium continuously or all at once, and can be agitated without substantially disturbing the microcarriers or cells. The objective is to provide a sterilizable container that can be manufactured and automatically control the culture volume. Another objective is to efficiently proliferate fixed cells in a large volume. For example, it is possible to provide equipment that allows high densities of 20 liters or more. Another objective is to provide a cell culture vessel designed to harvest culture media or fixed cells or conventional suspension culture cells. These and other objects of the invention will become apparent from the following description and figures.

Means for Solving the Problem The present invention relates to a device for culturing cells, in particular microcarrier-immobilized cells, in a medium.
The device consists of a container with a bottom wall and side walls for holding medium and cells or microcarriers - immobilized cells, an inlet for introducing the medium into the container, and for removing both the medium, cells and intact microcarriers. an outlet adjacent to the bottom wall of the container. A second outlet is located above the bottom wall for draining excess medium from the container. A central portion, such as a ridge or window, having holes sized to allow the medium to pass through but not the microcarriers or cells is placed between the second outlet and the cell-containing liquid. With such an arrangement, it is possible to retain the entire volume of liquid and keep the microcarriers and cells within the container, while continuously feeding the medium into the container at the desired rate. Operationally, the second outlet is adjustable and replaceable, allowing the volume of liquid in the container to be varied during incubation.

In a preferred embodiment, the device further comprises a third outlet near the bottom wall of the container and a suitably sized porous means to prevent the passage of microcarriers or cells. Preferably, the second and third outlets are located within a cylindrical hole extending above the bottom wall. A third outlet can drain all liquid while retaining cells, microcapsules or other microcarriers. This is useful, for example, when cells must be washed during a process.

In a preferred embodiment, the device includes a web for agitating the animal cells and medium, a motive force such as an electric motor for rotating the web, and a controller for the motive force for adjusting the speed of rotation of the web. . The combination of these constructs allows for slow acceleration at stirring speeds that minimize damage to the microcarriers and animal cells. Preferably, the controller gradually accelerates the webbing over at least one minute.

Further according to the invention, the container comprises an inlet for introducing the microcarrier-immobilized animal cells and an integrated device for forming a plurality of shape-retentive matrices containing dispersed animal cells. These can act as microcarriers themselves or can be further processed to form permeable capsules. In the latter case, the container has an inlet for introducing a liquid containing a component that coats the shape-retaining matrix, such as a polycation of the type used to form the membrane in the encapsulation process described in U.S. Pat. No. 4,352,883. have The apparatus for producing shape-retaining matrices consists of a modification of the well-known "jet head" droplet forming device. It has a space for holding a solution of a gel-like substance such as sodium alginate containing a plurality of animal cells, and a plurality of conduits extending below this space with hollow needles and serving as windows, each located below the space. Features a pump or other means for passing the gel-like material into the conduit. The device also includes structure with an air passageway near the opening of the conduit to block the flow of the gel-like material, thereby dividing the flow into multiple droplets. The formed droplets are then deposited into a reservoir containing a gel-like solution, such as calcium chloride, to convert the droplets into a shape-retaining matrix. The matrix in the calcium chloride solution was transferred directly into a container through a conduit and then subjected to a process of the type disclosed in US Pat. Can be treated with various reagents. The shape-retaining calcium cross-linked matrix can thus be washed several times with saline and subsequently exposed to a polycation solution such as polylysine or polyornithine to form a permeable membrane around the gel-like matrix. After the membrane is formed, a dilute solution of sodium alginate may be added to prevent agglomeration of the microcapsules or for other purposes, and the internal matrix of the capsules thus prepared may be made of sodium citrate or It can be made into a liquid by injecting a solution of the chelating agent.

Operation Thus, the device of the present invention allows the production of microcapsules or microcarriers-immobilized animal cells,
Suspension or immobilized culture cells can be grown to high density by feeding medium all at once or sequentially without the need for multiple metering pumps, and the medium containing substances of interest produced by the cells can be grown to high densities. It is suitable for recovery, washing of cells, recovery of microcarriers for cell collection, or recovery of proteins of interest retained on microcarriers.

Other features and advantages of the invention appear in the following description, drawings,
and will become apparent in the art from the claims.

FIG. 1 is a perspective and partially cross-sectional exploded parts arrangement view of the microcarrier-immobilized cell culture vessel of the present invention. FIG. 2 is a detailed perspective view of the cover of the container of FIG. 1 depicting a typical inlet structure. Figure 3 depicts the outlet portion of the vessel of Figure 1 for collecting microcarriers or cells, ejecting the medium for collection or replacement, and automatically retaining a certain amount of medium within the vessel. FIG. 4 is a detailed view of the entire apparatus for making shape-retaining gel globules or other molded articles containing immobilized cells.

Reference numbers drawn in each figure indicate corresponding parts.

Referring to the figures, FIG. 1 shows the culture vessel 8, its cover part 12, the webbed propulsion and control part 14, and the optional complete droplet forming part 9.

Container 8 is comprised of a cylindrical stainless steel structure comprising side walls 10, an open top 16, handles 18 and 20, and a support 22 extending from a bottom wall 32. Open head 16
includes a sterilizable resilient seal 24 and a cover part 12.
It is bordered by a ring flange 26 for connecting with. Handles 18 and 20 are constructed from hollow cylinders for receiving hoist rods such as hydraulic hoists. As shown in FIG. 1, the container 8 in use has two
A large number of cells contained in the culture medium described in 8.
Microcapsules or microcarriers or other liquids3
Filled with 0. The bottom wall 32 of the container 8 has a central first outlet 34 which is associated with a conduit 36 controlled by a valve 38 having a tapered male tube connection portion 40 .

A cylindrical, porous stainless steel cylinder 42 with an upwardly extending lid 44 is secured to the bottom wall 32. The tube has an opening small enough to prevent the passage of microcarriers or cells depending on the purpose for which the container is used. Within the tube is a conduit 46 which extends upwardly parallel to the cylinder 42 and has a second outlet 48 at its top open end which defines the maximum culture volume that the vessel will contain. The conduit 46 is removably connected, ie, connected by a connection 49 to the outlet 50 through the bottom 32 of the container 8 within the cylinder 42 of the first and second porous means. The connection provides a means for adjusting the position of the second outlet. Conduits of different heights can be substituted for different maximum fluid volumes desired. The outlet 50 is connected to a conduit 52 and an attached valve 5.
4, leading to the tapered tube connection section 56; A third outlet 58 is located within the cylinder 42 adjacent the outlet 50 and passes through the bottom wall 32 of the container 8 and has a conduit 60, a valve 62 and a male tapered tube connection portion 64. Arranged on the left side of the side wall 10 of the container 8 is an inlet 66 with a connection 68 for connection to an outlet pipe 70 of the microcarrier-fabrication device, which will be described in detail below. The cover 12 is comprised of a hexagonal sealing plate 100 with a sealing latch 102 at each corner. To seal the container, place the cover on the cylindrical container 8, press the sealing key 104 of the lock part 102 against the bottom surface of the sealing plate 100 with the seal 24, engage the lower side of the rim 26, and remove the cover 106. Seal 24 against plate 10
Push it to 0. The plate 100 is made of transparent plastic, such as polycarbonate, so that the interior of the container 8 can be viewed.

In the center of the plate 100, a magnetic drive component 108 constituted by a magnet and a conventional rotatable shaft 109 with a bearing attached to a web 111 are arranged. The web consists of a set of 90 degree angled plates 113 spaced apart from each other at 180 degrees.
Consisting of 115. Plate 100 includes a plurality of shafts 118 for attaching web drive and control components 14 to cover 12 by bolts (not shown). 6 entrances 110, 112, 114, 11
6, 118 and 120 are driving parts 108 of the plate 100
located radially around the Each inlet passes through the plate 100 and can be used to create microcarriers, inject various washing solutions, provide culture medium, monitor oxygen partial pressure, PH, etc., or be useful for other purposes. It is equipped with various tubes and control devices.

The inlet 110 is equipped with a medium inlet line 122 with a filter receptacle 124 and a male tube connection 126. The inlet 112 and its attachment tube 128 and filter receptacle 130 serve as outlets. Entrance 1
14 has a long tube 132 passing through it and is equipped with a connection 134 for receiving a conventional oxygen probe, PH measuring probe, or other device for monitoring the condition of the medium or other liquid entering the container 8 . The inlet 116 is equipped with a tube 135 and a female connection 136. This structure serves for the injection of cells or various liquids used in prefabricated microcarriers or assembled capsules or microcarriers. Inlets 118 and 120 are connected to tubes 138 and 140 and their associated filter components 14.
2,144 and male tube connecting portions 146,148. The inlet 118, the tube 138, the filter part 142 and the connecting part 146 are connected to the liquid 30 of the container 8.
It can be used to supply a gas of a defined composition in the headspace above the level of . Entrance 1
20, tube 140, filter part 144 and connection part 148 can be connected to a source of air, oxygen or an oxygen/carbon dioxide mixture. Gas passing through inlet 120 is conveyed through conduit 150 to stoma jet head 152 for oxygenation of the medium as disclosed in US Patent Application No. 579,493 referenced above.

The web drive and control component 14 is a support shaft 30
It is composed of a base plate 306 with a number 8 on it. The electric motor 300 is mounted on a base plate 306 with a drive shaft (not shown) and connected to a conventional annular magnetic drive component (not shown) in a drive box 310.
It penetrates and connects. When the part 14 is placed on the cover part 12, the annular drive part is positioned coaxially on the cover part 12, around the magnetic drive part 108. The rotational motion of motor 300 is transferred to shaft 109 and web 111 through magnetic coupling. Power for motor 300 is provided through controller 302, which includes circuitry well known in the art to control the rotational speed of motor 300 and paddle 111. Controller 302 contains circuitry that can be controlled by a knob 304 that can set the rate of change of the rotational movement of web 111 to one of several fixed speeds. In other words, the knob 304 is the web 111
Activate a circuit that gradually increases the rotational speed of the motor to a suitable rotational speed, e.g. 20 rpm, over a given period of time, e.g. 5 minutes. Knob 305 sets the maximum rotational speed of web 111. Referring to FIG. 4, details of the gel-like matrix production device 9 used in combination with the culture vessel 8 are shown. The device 9 consists of a container 72 containing an aquarium 200 equipped with an aquarium inlet 202 for filling the aquarium 200 with a gel-like solution. Inlet 202 also serves as an outlet. Exit line 20
A valve 204 located at 6 is made in device 9 and regulates flow through pipe 70 to container 8 . The solution in bath 200 is a dilute solution of calcium chloride prepared as hereinafter referred to and used to gel sodium alginate droplets containing eukaryotic cells.

Droplet making device 208 is above the water tank 200.
Structure divided by space 210 and separate compartment 21
Consists of 2. A plurality of hollow needles 214 extend from space 210 through space 212 and connect to aquarium 200 .
It penetrates the bottom wall 216 of the droplet production device 208 where it exits into a conical opening 218 located above.
The annular space between each hollow needle and the bottom wall 216 provides an air passage between the compartment 212 and the conical opening 218. Space 210 is fed through conduit 220, optionally using a pump (not shown), to transport cultured animal cells in a gel-like solution, such as sodium alginate. Space 212 is supplied by an air intake pipe 222 and an associated filter container 224 . Droplet generator 208 is constructed from a transparent plastic such as polycarbon or polysulfone. The inner diameter of the hollow needle 214, the air pressure injected through the tube 222, and the viscosity of the gelling solution contained within the space 210 together determine the droplet size in the device 9.

The above-described device is used to create microcarriers containing animal cells and to culture immobilized or non-immobilized cells to high density while continuously or intermittently providing fresh medium. Apparatus 9 for producing a shape-retaining gel matrix containing eukaryotic cells
is an arbitrary component. This can be removed from the device if desired. Microcarriers or suspension cultures can be prepared separately and, for example, injected into the culture vessel through the inlet 116.

A typical sequence of operation of the present invention will now be described.
Before sterilizing this device in an autoclave, etc.
Cover part 12 is sealed to head 16 as described above. After sterilization, the webbed drive and control parts 14 are attached to the plate 10.
0 and the isotonic saline solution is pumped into the container through the inlet 116, for example. Valves 38 and 62 are closed and the saline solution can be filled, for example after inlet connection 66, to the lower level.

e.g. 1.2 in animal cell culture, e.g. cultured hybridomas producing monoclonal antibodies.
% sodium alginate, which fills the space 210 and passes through the hollow needle 214 through the conduit 2.
Inject through 20. One suitable gel solution rate is 6 liters per hour.
Air or non-toxic gas is injected through tube 222 at a rate of, for example, 70 cubic feet per hour, filling space 212 and passing through hollow needle 214 through an annular opening in wall 216. The conical opening 218 allows a flow of air to form droplets of sodium alginate as noted at 230, e.g.
It has the ability to cut at the 1000 micron level. Calcium chloride is then introduced through the inlet conduit 202.
A water tank 200 filled with 1.2% solution receives the water droplets. Upon contact with the solution, the droplets of sodium alginate react with the calcium ions in the water bath 200 to form a large number of shape-retaining matrices that act as microcarriers. Shape-retentive calcium alginate gel globules or other shapes may be connected continuously or intermittently to outlet 206, valve 204, and inlet 70 as disclosed in co-pending U.S. Patent Application No. 579,494, referenced above. 6 and enters the saline solution in container 8, where it expands. The gel globule opens the valve 62, drains the previous saline solution and introduces new saline solution into the inlet 11, for example.
It can be washed several times with fresh saline by injecting from 6. At this point, the container 8 is filled with a medium for cell growth such that the calcium alginate gel globules containing animal cells act as microcarriers. However, formation of a semi-permeable membrane around the gel globules and liquefaction of the gel promotes bulk transport and cell proliferation as disclosed in the just-referenced U.S. Patent Application No. 579,494 and U.S. Patent No. 4,352,883. As you did, desirable.

For film formation, the saline containing the gel globules is discharged from the third outlet 58 and the first outlet 34 through the valve 62 of the third outlet 58, respectively. No gel globules are lost in this process due to the cylinder 42 being a perforated screen. Thereafter, a polycation solution such as polylysine or polyornithine is injected into the container 8 through the hole 116, for example. The polycations react with the negative charges on the surface of the calcium alginate gelation and shape retention matrix to form a semi-permeable membrane, respectively. After one or more treatments with different concentrations of the same polycation or with different polycations, the liquid phase is again discharged through outlet 58, conduit 60 and valve 62. Preferably, a dilute solution of alginic acid is injected into the container to neutralize the positive charges on the microcapsules. It is desirable to wash the microcapsules containing the animal cells with a solution of citric acid or a chelating agent to remove calcium ions from within the capsule, thereby liquefying the gel.

After removing the charged neutralizing solution and washing the capsule several times with saline, the medium is passed through inlet 110 to valve 6.
2 and 38, and open the valve 54 to inject.
When the medium is filled to the open end 48 of the conduit 46, the medium passes through the cylinder 4, which is a perforated screen.
2 and under the conduit 46 through the valve 54, the tube connection 56, and the tube (not shown) connected thereto. The container is thus filled with, for example, 20 liters of medium and 5 liters of microcapsules, totaling 25 liters.

Alternatively, prefabricated cell-containing microcarriers or conventional suspension cultures can also be directly injected into the container 8 through a suitable inlet in the sealing plate 100.

At this point, the controller 302 is activated, and the acceleration control knob 304 and rotational speed control knob 305 are activated to control the motor 30.
Make 0 work. Under the control of the controller 302, the magnetic drive component is driven by the motor 300 to drive the drive component 108.
Then, the shaft 109 starts to slowly rotate, and the web 111 slowly increases the rotation speed. This minimizes disturbance to the microcapsules and/or animal cells contained in the container. During the cultivation process, the medium is periodically discharged through the outlet 58 by opening the valve 62 and replaced with fresh medium through the inlet 110. The pressure within the container is maintained near atmospheric pressure by outlet 112. Oxygen or PH probes are inserted through inlet 114 and tube 132 to monitor gas partial pressure or other conditions in the medium. The composition of the gas in the head space of the container is
controlled by the injection of airflow through 18, such as air, carbon dioxide and oxygen. When growing encapsulated cultured cells, the culture medium can be oxygenated, if desired, through the inlet 112 and associated spout head 152, as disclosed in co-filed US patent application Ser. No. 579,493.

Continuous replenishment of culture medium can be easily accomplished by supplying culture medium through inlet 110. As the volume of liquid in container 8 increases, excess medium is drained into conduit 46.
from there through outlet 50 and through valve 54. Microcarriers and free cells in the medium do not pass through the openings of the perforated screen (cylinder) 42 and are therefore retained within the container. If the pores in the microcapsule allow the interesting protein produced by the cells within the capsule to pass through the capsule membrane, the protein can be purified from the culture medium. In this case, during the period of production of these proteins, the cells are given medium at once, but continuously, and from the container to the third outlet 5.
8 and outlet 50 via valve 62 or conduit 46.
This protein is merged from the spent medium which is discharged through valve 54. If the pores of the membrane are to be controlled such that all or most of the protein is retained within the microcapsules at the end of the culture, open the valve 38 of the first outlet 34;
Preferably, the entire volume of the container is merged through a tube (not shown) connecting to the male tube connection portion 40.
The protein is collected by destroying the microcapsule and separating it into the cells and other components contained within it.

The invention may also include forms having other specific forms within its spirit and scope.

Accordingly, other implementations are within the scope of the following claims.

[Brief explanation of the drawing]

FIG. 1 is a perspective and partially cross-sectional exploded parts arrangement view of the microcarrier-immobilized cell culture vessel of the present invention. FIG. 2 is a detailed perspective view of the cover of the container of FIG. 1 depicting a typical inlet structure. Figure 3 depicts the outlet portion of the vessel of Figure 1 for collecting microcarriers or cells, ejecting the medium for collection or replacement, and automatically retaining a certain amount of medium within the vessel. FIG. 4 is a detailed view of the entire apparatus for making shape-retaining gel globules or other moldings containing immobilized cells. Explanation of symbols, 8... Culture container, 9... Small droplet forming part (gel matrix production device), 10...
side wall, 12...cover part, 14...webbed propulsion and control part, 16...opening head, 18...handle, 20...handle, 22...support, 24...
Seal, 26...Ring, 30...Multiple cells, microcapsules or microcarriers contained in the culture medium,
or other liquid, 32...bottom wall of container 8, 34...
First outlet, 36... Conduit, 38... Valve, 40
...male tube connection part, 42 ... cylinder (first and second porous means), 44 ... lid, 46 ...
...Conduit, 48...Top opening end (second outlet), 4
9... Connection, 50... Outlet, 54... Valve, 5
6...Pipe connection part, 58...Third outlet, 60...
Conduit, 62... Valve, 64... Male tapered pipe connection part, 66... Inlet, 68... Connection, 70... Pipe (conduit), 72... Container, 100... Sealing plate,
102...Key parts, 104...Key, 108...
...Magnetic drive part, 109...Shaft, 110...
...Entrance, 111...Webbed, 112...Entrance, 1
13... Webbed piece, 114... Entrance, 115...
Webbed piece, 116...Inlet, 118...Inlet, 1
20... Inlet, 112... Culture medium injection line, 124...
...Filter insert, 126...Male pipe connection part, 1
28...Adhesion tube, 130...Filter insert, 1
32...Pipe, 134...Connection, 135...Pipe, 1
36...Female connection, 138...Pipe, 140...Pipe,
142... Filter parts, 144... Filter parts, 146... Male tube connection part, 148... Male pipe connection part, 150... Conduit, 152... Small hole spout head, 200... Water tank, 202... Inlet , 204
... Valve, 206 ... Outlet, 208 ... Droplet production device, 210 ... Space, 212 ... Compartment, 21
4... Hollow needle, 216... Bottom wall, 218... Conical opening, 220... Conduit, 222... Air intake tube, 224... Filter container, 230
... droplet of sodium alginate, 300 ... electric motor, 302 ... controller, 304 ... knob, 305 ... knob, 306 ... base plate, 308
...Support shaft, 310...Drive box.

Claims (1)

[Scope of Claims] 1. A device for culturing animal cells immobilized on microcarriers placed in a medium, comprising: a container having a bottom wall and side walls defining a space; means for introducing a culture medium into said container; a container adjacent to the bottom wall of said container sufficient to effect the removal of the culture medium and cells immobilized on the microcarriers; means having a first outlet sized; means defining a second outlet located on the bottom wall for controlling the volume of culture medium in said container by draining excess medium from said space; means adjacent to the bottom wall of the container defining a third outlet for evacuation of fluid from the space; means large enough to prevent the passage of cells immobilized on the microcarriers but to allow passage of the medium; a first porous means having an aperture located between the second outlet and the space; and means for blocking the passage of cells immobilized on the microcarrier but for preventing the passage of the medium. a second porous means having an opening of sufficient size to permit the second porous means to be located between the third outlet and said space. 2. A web located in the space for stirring the animal cells and the medium, a driving means for rotating the web, and adjusting the rotational acceleration of the web to minimize damage to the cells or the microcarriers. 2. The apparatus of claim 1, further comprising control means for controlling the apparatus. 3. The device of claim 1, further comprising a first inlet for injecting microcarrier-immobilized animal cells into the space. 4 further comprising integrated means for producing a plurality of shape-retaining matrices containing animal cells and conduit means communicating with the first inlet for transporting the shape-retaining matrices into the space; The apparatus according to claim 3. 5. Claim 4 further comprising a second inlet for introducing into said container a liquid containing a component that coats said shape-retaining matrix to create a membrane around said matrix in said container.
Section equipment. 6. The device of claim 1, wherein said means having a second outlet includes means for adjusting the level of the second outlet within said container. 7. An apparatus for forming microcarriers and culturing animal cells immobilized on the microcarriers, comprising: a container having a bottom wall and side walls defining a space for holding a liquid; means for introducing the liquid into the container; means adjacent the bottom wall of the container having a first outlet of sufficient size to allow removal of the medium, the animal cells and the microcarriers; reducing the volume of culture medium by draining excess medium from the space; means for defining a second outlet located on the top of said bottom wall for controlling the flow of liquid; means adjacent to the bottom wall of said container for defining a third outlet for discharging liquid from said space; passage of said microcarriers; a first porous means located between the second outlet and the space, the means having an opening of sufficient size to prevent the passage of the medium, but permit the passage of the cells or microcarriers; a second porous means located between the third outlet and the space, the second porous means having an opening of sufficient size to prevent the passage of the medium, the second porous means being located between the third outlet and the space; containing dispersed animal cells; an integral means for producing a plurality of shape-retaining matrices; and conduit means for transporting the shape-retaining matrices from the matrix-producing means into the container. 8. Further comprising a second inlet for introducing into the container a liquid containing a component for coating the shape-retaining matrix to form a film around the matrix in the container. Equipment of scope 7. 9. Furthermore, a web within the container for stirring the liquid contained therein, a driving means for rotating the web, and a method that minimizes damage to microcarriers and cells contained in the liquid within the container. 8. Apparatus according to claim 7, further comprising control means for adjusting the rotational acceleration of said web. 10 An integrated means for forming a shape-retaining matrix in which animal cells are dispersed includes a space for holding a solution of a gel-like substance containing a plurality of animal cells, and a plurality of conduits communicating with the space. a conduit, each having an opening disposed below the space; means for forcing a gel-like material through the conduit; Claim 7 comprising means having an air passage adjacent said opening for interfering with the flow of material, and a reservoir holding a gel-like solution for converting said droplet into a shape-retaining matrix. Section equipment. 11. The invention further comprises a second inlet for introducing into the container a liquid containing a component for coating the shape-retaining matrix to form a film around the matrix in the container. Equipment of scope 10.