GB2103239A - Improvements in cell tissue culture - Google Patents

Abstract

Cell tissue is produced, particularly in monolayers, on the plates of a plate heat exchanger through which a growth medium is circulated with air or oxygen in solution. The medium is circulated from a tank (2) by a pump (4) through the heat-exchanger (1) and returned via a gas removal unit (6) with sterile filter (15) to the tank (2). The temperature is controlled by circulating, by means of a pump (35) heated or cooled water through the other side of the heat exchanger (1). <IMAGE>

Description

SPECIFICATION Improvements in cell tissue culture This invention relates to cell tissue culture.

In the production of cell tissue, the cells have traditionally been cultured in Roux bottles or roller bottles. In order to achieve bulk quantities of tissue, large numbers of such bottles need to be used and this is highly labour intensive. Many different systems have been proposed to effect the scale-up of anchorage-dependent cell culture, for animal, plant and fungal cells growing in a surface-attached monolayer. The present invention is intended to provide a method of culturing cell tissue and apparatus for carrying out this method.

Many cells lines show, as a requirement for growth, "anchorage-dependence" on a suitable surface, and as cell/cell contact inhibits further growth, there is established a confluent monolayer culture, covering the available surface.

Therefore, for high production rates of biochemicals and, for example, vaccines such as mumps, rubella, Mareks, measles, influenza, parainfluenza and respiratory syncytia from cell lines such as chick embyro fibroblasts, bovine kidney cells, green monkey cells, and human diploid lung or skin fibroblasts, a large surface area is required.

The production of many viral vaccines and biochemicals such as interferon, human growth hormone, somatastatin, alphathymasin-l and human insulin depends on the successful culturing of animal cells in a surface attached monolayer. Similar products may be obtained from plant cell and fungal cell tissue culture.

As the techniques of molecular biology have produced new cell lines, increased scales of production have been sought and process devices offering large surfaces have been developed.

In particular, there is available a processor comprising a series of metal discs mounted for rotation with a shaft. These discs are mounted in a vessel which has supply and discharge lines for liquid and gaseous media. To operate, the vessel is filled with serum and held with the discs in horizonal planes to allow cells to settle on the plates. The vessel is then rotated so that the discs are in vertical planes, and partially empties to provide a head space for aeration. The discs are then slowly rotated for propagation of the cells and for subsequent treatment of them. In another form of apparatus the cells are cultured and treated on a series of tubes.

Apparatus of this type is expensive to manufacture and cannot readily be modified according to production requirements. Also accurate control of the process is not easy since the culture is essentially a batch process and its temperature and other variables are not readily susceptible to outside regulation.

The present invention overcomes the difficulties.

In accordance with the first aspect of the invention, there is provided a method of culturing animal cell tissue in which cells are caused to settle on plates of a plate heat exchanger and a growth medium is passed in continuous circulation through flow spaces defined by the said plates.

Conveniently, a virus or other additive is added to the circulating growth medium during the circulation.

By innoculating the growth medium with a virus, in situ infection of the cells may be established leading to the production of a vaccine.

The virus may also be used to transform the cell type. Alternatively a transposon or plasmid may be added to the culture. In certain circumstances other additives may be required, such as antibiotics or an additive such as sodium butyrate for the stimulation of interferon production.

Preferably, air or oxygen is dissolved in the circulating growth medium as it enters the heatexchanger.

More preferably, means is provided for removal of air oxygen or carbon dioxide from the growth medium after it leaves the heat-exchanger.

In an embodiment only alternate flow spaces are used for culturing and the intervening flow spaces are used for circulation of water or other medium at a controlled temperature.

In an embodiment a selected variation in the effective area over which the animal cell tissues are grown is obtained by diverting inoculum and growth medium through selected flow spaces of the heat-exchanger.

By separating the heat-exchanger into two or more separate growth regions the initial inoculum may be grown on a small scale before aseptic transfer to the entire heat exchanger. An advantage of such a method is that the subculture, growing at a reduced scale, may be tested for changes in morphology, karyology and life span before a larger culture is established.

Should the sub-culture be mutated or contaminated it may be abandoned.

The selection may be by means of a connector grid.

In an embodiment the velocity of the inoculum and growth medium is substantially constant in all process flow spaces.

By "velocity" is meant both the direction and speed of the flow. It has been found that certain cell lines grow better when the growth-medium flows over a vertical-surface attached monolayer in an upward direction, and that other cell lines have a preference for a descending medium. By ensuring that the velocity of flow is substantially constant, the preferential flow for a particular cell line may be maintained over the whole plate surface.

In an embodiment the effective area over which the cell tissues are grown is increased by means of an insert in any one or more process flow space of the heat exchanger.

By increasing the surface area by means of an insert the total number of cells which may be cultured in the heat exchanger may be increased.

The insert may be a polypropylene grid, a stainless steel wire mesh grid or a perforated metal plate.

By inserting any of the above-mentioned inserts between the plates of the process flow space, the total surface area available for growth may be increased by up to 65% for the same volume inside the heat exchanger. The polypropylene grid is less expensive than a stainless steel grid metal or plates, however it is less amenable for use with media with a solids content or any other fouling component, as the mesh is more difficult to clean and sterilise.

The insert may be a plate of a plate heat exchanger.

The effect of adding the inserted plates to the pack is to increase the surface area of the process flow spaces, by 100% or more, by having a number of process flow spaces between any pair of service flow spaces.

In an embodiment the adhesion of the cell tissues to the surfaces of the process flow space is enhanced by a pretreatment of these surfaces.

The pretreatment may comprise sand blasting or glassblasting of the surfaces of the process flow spaces. The subsequent pitted surface will provide a suitable environment for both initial entrapment and good long-term adhesion for those cells which do not readily adhere to a smooth surface.

The pretreatment may alterntively comprise the application of a surface coating to the surfaces of the process flow spaces.

By coating the surfaces of the process flow spaces with, for example; polylysine, histones, protamine, collagen, gelatin, glass or polystyrene piastic with protons discharged from its surface aromatic groups, the affinity of the cells for the surface is increased.

To effect release of the cells from the surface, proteolytic agent such as trypsin or pronase may be employed. An advantage of the plate heat exchanger as a culture vessel, is that the temperature may be lowered to under 1 50C easily, before treatment with the proteolytic agent. At temperatures below 1 C, access of trypsin and pronase to critical cell components is greatly reduced. Furthermore, the low temperature decreases the degree of adhesiveness to the surface at the lateral edges of the cells and increases exposure of the remaining attachment sites. These effects combine to reduce both the trypsin or pronase activity units required and the exposure time for release.

In an embodiment the adhesion of the cell tissues to the surfaces of the process flow space is reduced by a pretreatment of these surfaces.

The pretreatment comprises the application of a thin fiim coating of PTFE to the surface of the process flow spaces.

This surface is suitable for cells which require to be only lightly adhered to the surface and which must be easily removed.

In an embodiment a duplex gasket assembly is employed.

Where the cells or product from the cells is of particularly hazardous nature the plate exchanger may be constructed in such a form so as to prevent leakage of any hazardous material to the atmosphere, or to the temperature control medium through the sealing gaskets. This refers to our U.K. Patent Application No. GB 2062833A in which is described an inherently safe system of plate sealing to prevent any escape of dangerous fluids either to the atmosphere or to the service fluid.

A detection system is also described for the early warning of any leakage through the primary gasket. This may be most important when culturing highly pathogenic organisms.

In an embodiment lysis of the surface attached cells is carried out in situ.

By lysing the surface-attached cells in situ the content of the cells may be recovered. Lysis may be caused by a number of processes, for example viral infection, temperature change or osmotic shock.

In accordance with the second aspect of the invention, there is provided apparatus for culturing animal cell tissue comprising a plate heat exchanger, means for circulating a temperature control medium through one set of flow spaces of the heat-exchanger, means for circulating a growth medium through the other set of flow spaces, means for removing gases from the circulating growth medium after it leaves the heat-exchanger, means for injecting air or oxygen into the circulating medium, means for injecting inoculum into the circulating medium, and instrumentation to measure the operating parameters to enable control of the process.

In certain circumstances, such as the growth of a pathogenic cell line, cell line inoculated with a pathogenic or a cell line growing in the presence of a toxin or carcinogen it may be desirable to employ a heat-exchanger in which adjacent plates forming the flow spaces for the process medium are welded together in pairs around the peripheries of the plates and around the through holes forming the ports carrying the service medium through the welded pairs of plates, whereas the flow spaces for the service medium are sealed by flexible gaskets.

Use is thus made of the comparatively large surface area available in a plate heat-exchanger, and it will be appreciated that by addition or removal of plates the available surface area may be readily adapted. Also, by circulating a controlled temperature heating or cooling medium in the intervening flow spaces, the culturing temperature can be controlled. If a corrugated plate surface is used then the initially introduced cells are able to settle without the need for reorientation of the surfaces on which the cells are to be cultured. Since only a small proportion of the total growth medium circulated is in contact with the cells at any one time, control of the process can be achieved by treatment of the inedium outside the heat-exchanger, e.g. for pH value nutrient level, dissolved oxygen content and so forth.

The invention will be further described with reference to the accompanying diagrammatic drawings in which: Figure 1, is a schematic diagram of a cellculture apparatus which embodies the present invention, Figure 2 is a schematic diagram of a further cell-culture apparatus which embodies the present invention, Figure 3 is an exploded view of a plate heat exchanger, and Figure 4 is a schematic diagram of two possible inserts by which the surface area available for cell growth may be increased.

Turning firstly to Figure 1. The drawing illustrates a form of apparatus for monolayer tissue culture using a plate heat exchanger 1A and 1 B as a high surface area bio reactor. As is normal in a plate heat-exchanger 1A and 1B contains a pack of corrugated plates (not illustrated) in spaced face-to-face relationship. In order to provide anchorages for cells, the plates have contact points between adjacent plates formed either by the corrugations crossing and abutting, or by the provision of localised pips on the corrugations which normally provide interplate support. These contact points provide secure anchorage from which cells may spread across the plate surface as they propagate.

A holding tank (2) a nutrient growth medium has an outlet line (3) connected to a pump (4) which passes the medium from the tank 2 via one set of flow spaces (illustrated at 43) of the heatexchanger (1). From the heat-exchanger (1) the medium passes back via a de-gassing device (6) to a return line (27) to the tank (2).

Between the pump (4) and the heat-exchanger 1A and 1 B there is provided an injection line (8), which may be used to supply air or oxygen into the circuit. A withdrawal line (28) is also provided downstream of the pump (4). A storage tank (5) is arranged in parallel with the tank (2) to enable a secondary medium to be circulated by the pump (4) through the heat-exchanger (1A and 1 B).

Medium is supplied to the tank (5) via line (42).

Steam inlets (9, 10, 1 1, 12, 13, 14, 15, 16) are provided for in situ steam sterilisation of the system with condensate lines (17, 18, 19, 20, 21, 22, 23, 24, 25, 26). The de-gassing device 6 may for instance be in the form of a vacuum vessel, and is provided to remove air and/or carbon dioxide and/or other gases from the circulating medium so as to prevent aerobic bacterial contamination and to remove the waste products of the cell growth within the heat-exchanger (1A and 1 B). The air or carbon dioxide drawn off is removed via a sterilising filter (7), thus preventing escape of pathogenic organisms into the environment.

If no de-gassing device is required the excess air from injection point (9) may simply be vented off from tank (2) via a filter (29).

Instrumentation for the control of the process includes a pH meter (30), a pressure gauge (31) and a temperature gauge (32). The pH meter (30) controls injection of correcting acid or alkali via a line (33), and pressure gauge (31) controls a pressure'valve (34).

The flow spaces between those occupied by the circulating medium have heating or cooling water circulated through them by means of a pump (35). The water may for instance be circulated through a heat-exchanger (36) where it is heated by steam fed in via a line (37) through a valve (38) controlled by the temperature gauge (32). If cooling is desired, fresh or even chilled water may be provided via a line (39) pumped through the heat-exchanger and then drawn off through a drain valve 40.

Prior to start-up, the equipment as illustrated must be sterilised by either chemical sterilants or preferably by steaming at 121 OC for 30 minutes via steam inlets (9, 10, 1 1, 12, 13, 14, 15, 16).

Sterile growth medium containing the appropriate cell line inoculum may then be aseptically introduced into the tank (2). This inoculum is then charged into the plate heat exchanger section 1 A in sufficient quantity to fill its side of the plate heat-exchanger 1A and hence flood the plate surface area. Alternatively sterile growth medium may be introduced from the tank (2), and innoculated with the appropriate cell line to be propagated at the injection port (44). Sterile air or oxygen may be bubbled through the inoculum from the injector (8) if required.

Alternatively the inoculum may be charged into both sections of the plate heat-exchanger ( 1 A and 1 B). Because of the corrugated nature of the plates the cells in the inoculum will naturally settle onto the horizontal component of the plate surface and hence no re-orientation or other cell attachment mechanism is necessary as with flat plates to discs. The many plate to plate or pip to pip contact points provide a secure anchorage from which the cells may spread across the plate surface. In any embodiment which employs an insert in any one or more flow spaces, the cells will also become attached to the surface of the insert.

Once cell adhesion is achieved the inoculum medium may be drained. The system is then recharged with fresh medium which may then be recirculated through section (1A) or (1A and 1 B) in order that the adhered cells may grow to the required density. Air or oxygen may be injected through port 8 if required. The air/CO2 removal device (6) may be activated in order to prevent aerobic bacterial contamination and to remove the waste products of cell growth. The medium is recirculated back to the tank (2).

During the cell growth phase, conditions of culture have to be maintained at optimum level.

The sterile air supply via injector 8 must be closely controlled as must the pH of the medium.

The air or oxygen is dissolved in the circulating medium. Very close temperature control can be maintained by recirculating warm or cool water on the service side (41) of the plates using a pump (35).

If the inoculum section 1 A only of the plate heat-exchanger is being used, then once cell growth is complete the cells may be removed by draining the growth medium and recharging the system with a trypsin solution to loosen the cells from the plate surface and increasing the flow rate so as to induce turbulence in the flow plates of section 1 A in order to detach the cells from the plate surface. The cell suspension may then be charged into both sections 1 A and 1 B of the plate heat-exchanger so as to allow settling of cells on the total available plate surface. Once reattachment is complete the trypsin solution may be drained and the system recharged with growth medium. The procedure is then repeated as before until cell growth is complete.

It is then possible to inject any further inducing agent such as a virus or an additive such as sodium butyrate for the stimulation of interferon production.

Removal of extracellular product is achieved by completely draining the medium from the system.

If the cells themselves are to be harvested then the same procedure as before is carried out and the cell suspension finally removed via line (28).

An alternative to the injection of air or oxygen for aeration purposes is to supersaturate the medium with oxygen as described in our patent application No. GB 2034750A.

Turning now to Figure 2 there is illustrated a further form of apparatus for culture of cells using a plate heat-exchanger (50).

A growth medium reservoir (51) equipped with a stirring means (52), temperature sensor (53) and pH sensor (54) may be sterilized by steam injection through port (56). Growth medium may be admitted into the reservoir through port (55).

Corrections to the pH etc. may be made through addition port (74).

A three way valve (57) connects the reservoir (51) with a harvest solution inlet port (58) and pump (59). The pump (59) circulates the growth medium from the reservoir past the inlet and outlet ports (60) into the heat exchanger process flow spaces (61). The growth medium returns to the reservoir via a flow meter (62).

The headspace of the reservoir (51) may be gassed through line (63) metered at a flow meter (64). A sterilizing filter (65) may be incorporated into the gas line (63). A sample may be withdrawn at the sample port (70).

The heat exchanger process flow spaces are sterilized by steam admitted at a port (68) and drained as condensate through a port (69).

Inoculum is normally introduced into the system through a port (60) to fill the process flow spaces (61) of the heat-exchanger (50). The temperature of process and service fluids may be sensed by sensors (66) and (67). Excess gas in the system may be bled off through sterilizing filter (71). The service fluid circulating in flow spaces (72) may be chilled or heated by a unit (73).

The advantages of both these systems include: 1. Extended surface area for cell growth in a small volume, to a maximum of 1500 m2.

2. Corrugated surface for good cell adhesion.

3. Variable surface area by plate removal or addition.

4. Good process control due to the low medium hold up volume and recirculating flow.

5. Rapid equilibration of temperature enables optimisation of product formation and minimises cell losses during transfer between seed and production stages.

6. Aseptic growth and product recovery.

Turning now to Figure 3 there is illustrated an exploded view of a plate heat exchanger, in which plates (80, 84) are compressed between a head (81) and a follower (82). The connector grid (83) may be employed to separate the flow of process liquid and service fluid over plates (80) and (84), effectively placing two heat-exchangers within the same frame.

By employing a substantially similar arrangement the flow of nutrient through the section (80) (1A in Figure 1), shown chain dotted in Figure 3, may be separated from the flow of nutrient through the section (84) (1 B in Figure 1) shown as a solid line in Figure 3, while the service fluid, shown as a dashed line in Figure 3, flows through both sections (80 and 84).

Turning now to Figure 4, there are illustrated some of the possible inserts which may be placed between the plates of the process flow space to increase the surface area available for cell growth.

The perforated metal plate (90) has holes (91) in which the contact points of the adjacent plates bordering the flow space may locate. The plate may be of, for example, stainless steel, titanium, or another suitable metal.

The grid (92) may be of stainless steel wire mesh or of polyproplyene.

For cleaning, the cells are dislodged from the plates and/or as hereinbefore described and washed out of the system. Steam cleaning and sterilising follows. Burning is thus avoided.

In order that the invention may be further understood there are provided the following examples.

Example 1 The apparatus as described above having an available surface area for cell culture of 2 mZ provided by 24 stainless steel plates was used to culture human fibroblasts as follows: The heat exchanger and part of the associated pipework connecting it to the growth medium reservoir was sterilised by direct steam injection and held at 1 5 psi for 60 minutes. The reservoir vessel, comprising a glass and stainless steel fermentation vessel of 3 1. working capacity, together with the remaining pipework and the pump head to be used for circulation of the growth medium were sterilised by autoclaving for 140 minutes at 1 5 psi.

The heat exchanger was cooled by passing cold water through the jacket side and then the temperature was stabilised at 36 to 370C by circulating controlled temperature water through the jacket side. The reservoir, the remaining pipework and pump were connected to the heat exchanger aseptically.

An inoculumof 1.8x108MRChuman fibroblast cells suspended in 2.7 1. growth medium was introduced upwards into the cell culture chamber of the heat exchanger until the chamber was completely full. The growth medium used was Eagle's medium containing Earle's salts, supplemented with 8% bovine serum, nonessential amino acids and peniciliin/streptomycin.

The medium was buffered with 2.2 g sodium bicarbonate per litre overlaid with 5% carbon dioxide in air and contained the pH indicator, pheno-red.

An additional 3.2 11 growth medium was introduced into the reservoir vessel aseptically.

This was stirred, the headspace of the vessel gassed with 5% carbon dioxide in air, heated to and maintained at 370C.

The system was then left undisturbed at 30C for 24 hours to allow the cells to attach to the surface of the heat exchanger plates.

At the end of the attachment period the growth medium was circulated around the loop comprising the reservoir vessel, the pump and the cell culture side of the heat-exchanger at a rate 100--130 ml per minute for a period of 7 days such that the flow was upward through the heat exchanger concurrent with the flow of water through the jacket side. The growth medium was then drained completely from the apparatus and 5.81. fresh medium introduced aseptically. The fresh growth medium was circulated at 130-280 ml/per minute for a further 3 days. ~ Circulation of the growth medium was stopped. Phosphate buffered saline (3 1,) was introduced to displace the growth medium downwards through the heat exchanger. The phosphate buffered saline was in turn displaced by 0.25% trypsin in phosphate buffered saline (1 I,).The cell culture chamber was drained completely and maintained at 36-370C by the water passing through the jacket side for 1 hour.

Three aliquots (2 1.) of serum-free growth medium were then introduced in succession upwards into the cell culture chamber and 5% carbon dioxide in air was pulsed through each aliquot before draining the refilling chamber with the next aliquot.

The number of cells inoculated and harvested were determined by a Coulter Counter and checked with a haemocytometer.

Total cells inoculated 1.8x108 Cells harvested (i) in drained growth medium 2.2x108 (ii) saline washbuffer and trypsin solution 8.7 x 108 (iii) pulsed serum-free medium 29.1x108 Total 29.2x108 Example 2 Using the method of Example 1 human fibroblasts were cultured as follows: A heat exchanger containing 24 stainless steel plates which provided 2 m2 surface available for cell culture was inoculated with 1 .35x 108 MRC 5 human fibroblast cells. The cells were introduced as a suspension in growth medium comprising minimum essential medium (Eagle) containing Earle's salts supplemented with 8% bovine serum, non-essential amino acids, penicillin/streptomycin and 2.2 g/l sodium bicarbonate.

The inoculated vessel was held at 360C for 17.5 hours. The volume of growth medium was increased to 5.8 1. and the growth medium was then circulated through the cell culture chamber at a rate of 100--200 ml per minute for a period of 5 days such that the flow was upward through the heat-exchanger and countercurrent to the flow of water through the jacket side. The growth medium was drained and replaced with 5.8 fresh growth medium which was circulated for a further 5 days. The circulating medium was maintained at a temperature of 34-360C. and oxygenated by passing 5% carbon dioxide in air through the head space of the reservoir vessel. pH of the growth medium was maintained by addition of sodium bicarbonate.

The growth medium was flushed from the cell culture chamber with phosphate buffered saline and the cells prepared for removal from the growth surface by washing with 0.25% trypsin dissolved in phosphate buffered saline. After all solutions were drained from the cell chamber the cells were maintained at 35-370C for 1.5 hours in the presence of residual trypsin solution. Cells were removed from the apparatus with three aliquots of serum-free growth medium which were agitated by the injection of 5% carbon dioxide in air when each was held within the cell culture chamber.

Total cells inoculated 1 .35x 108 Cells harvested (i) in drained growth medium 1.2x106 (ii) in saline wash buffer and trypsin solution 1.5x106 (iii) pulsed serum-free medium 16.1x108 Total 16.2x108 Further monitoring devices such as glucose, dissolved oxygen and carbon dioxide analysers may be added.

Various modifications may be made within the scope of the invention.

Claims (26)

Claims

1. A method of culturing cell tissue in which cells are caused to settle on plates of a plate heat exchanger and a growth medium is passed in continuous circulation through flow spaces defined by the said plates.

2. A method as claimed in claim 1, in which a various or other additive is added to the circulating growth medium during the circulation.

3. A method as claimed in claim 1 or 2, in which air or oxygen is dissolved in the circulating growth medium as it enters the heat-exchanger.

4. A method as claimed in claim 1,2 or 3, in which means is provided for removal of air oxygen or carbon dioxide from the growth medium after it leaves the heat-exchanger.

5. A method as claimed in any of the preceding claims, in which only alternate flow spaces are used for culturing and the intervening flow spaces are used for circulation of water or other medium at a controlled temperature.

6. A method as claimed in any of the preceding claims in which a selected variation in the effective area over which the animal cell tissues are grown is obtained by diverting the inoculum and growth medium through selected flow spaces of the heat-exchanger.

7. A method as claimed in claim 6, in which the selection is by means of a connector grid.

8. A method as claimed in any of the preceding claims, in which the velocity of the inoculum and growth medium is substantially constant in all process flow spaces.

9. A method as claimed in any of the preceding claims, in which the effective area over which the cell tissues are grown is increased by means of an insert in any one or more process flow space of the heat-exchanger.

10. A method as claimed in claim 9, in which the insert is a polypropylene grid.

11. A method as claimed in claim 9, in which the insert is a stainless steel wire mesh grid.

12. A method as claimed in claim 9, in which the insert is a perforated metal plate.

13. A method as claimed in claim 9, in which the insert is a plate of a plate heat-exchanger.

14. A method as claimed in any of the preceding claims in which the adhesion of the cell tissues to the surfaces of the process flow space is enhanced by a pretreatment of these surfaces.

1 5. A method as claimed in claim 14, in which the pretreatment comprises sandblasting or glassblasting of the surfaces of the process flow spaces.

16. A method as claimed in claim 14, in which the pretreatment comprises the application of a surface coating to the surfaces of the process flow-spaces.

1 7. A method as claimed in any of claims 1 to 12, in which the adhesion of the cell tissues to the surfaces of the process flow space is reduced by a pretreatment of these surfaces.

18. A method as claimed in claim 17, in which the pretreatment comprises the application of a thin film of PTFE to the surfaces of the process flow spaces.

1 9. A method as claimed in any preceding claim in which a duplex gasket assembly is employed.

20. A method as claimed in any of the preceding claims, in which lysis of the surface attached cells is carried-out in situ.

21. A method as claimed in any of the preceding claims in which the composition of the inoculum and growth medium, temperatures of the process and service fluids and rates of flow are monitored and controlled by an electronic control means.

22. A method of culturing cell tissue substantially as hereinbefore described with reference to the accompanying diagrams and examples.

23. Apparatus for culturing cell tissue comprising a plate heat exchanger, means for circulating a temperature control medium through one set of flow spaces of the heat exchanger, means for circulating a growth medium through the other set of flow spaces, means for removing gases from the circulating growth medium after it leaves heat exchanger, means for injecting air or oxygen into the circulating medium, means for injecting inoculum into the circulating medium, and instrumentation to measure the operating parameters to enable control of the process.

24. Apparatus for culturing cell tissue substantially as hereinbefore described with reference to the accompanying drawing.

25. A method of culturing cell tissue in which cells are caused to settle on plates of a plate heat exchanger and a growth medium is passed in continuous circulation through flow spaces defined by said plates and in which a selected variation in the effective area over which the cell tissues are grown is obtained by diverting the inoculum and growth medium through selected flow spaces of the heat exchanger.

26. A method of culturing cells tissue in which cells are caused to settle on plates of a plate heat exchanger and a growth medium is passed in continuous circulating through flow spaces defined by said plates, and adjacent plates forming the flow spaces for the process medium are welded together in pairs around the peripheries of the plates and around the through holes forming the ports carrying the service medium through the welded pairs of plates, whereas the flow spaces for the service medium are sealed by flexible gaskets in which the holes forming the ports for the process medium are sealed from the flow spaces for the service medium and from the ambient space by a duplex gasket arrangement with a sealed space between the gasketing.