CA1321159C - Method of cultivating living cells and bioreactor therefor - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
In a bioreactor, oxygen is supplied to a suspen-sion of living cells using a carrier liquid in which the oxygen is soluble. The carrier liquid is immiscible in the culture medium and has a specific gravity greater than that of the culture medium, so that when the oxygen bearing carrier liquid in injected into the culture medium, it will give up its oxygen and coalesce at the bottom of the reactor. The carrier liquid is oxygenated through a membrane to avoid contamination. The oxygenator may be temperature controlled to control the temperature of the carrier liquid and thus the culture medium. The concentration of carbon dioxide may also be controlled to ensure that it does not become excessive. The reactor may carry out either batch processes or continuous perfusion.

Description

1 32~159 METHOD OF CULTIVATING LIVING CELLS AND BIOREACTOR THEREFOR

BACKGROUND OF THE INVENTION
Bioreactors or fermentors in which a product is formed by an aqueous suspension of living cells oxidizing an organic substrate are usually equipped with a device which introduces and disperses a gaseous phase containing the oxygen necessary for the growth and respiration of the cells.
The princ~ples of these devices are varied, but most share the characteristic of submitting the aqueous phase to considerable turbulent and shear forces, e.g.
stirring, agitating. The gaseous phase may be injected or entrained into the aqueous phase or it may be the contin-uous phase into which the aqueous phase is sprayed or otherwise injected. In both cases, the exchange of oxygen and carbon dioxide occurs across the resulting gas-liquid interface. The forces required to create an interface of sufficient surface area for an active aerobic culture are unfortunately also sufficient to rupture many types of cells which could otherwise be used in bioreactors.
In systems where oxygen is supplied by in~ecting and dispersing air, which is 79% nitrogen, a gross excess of air is required because of the inefficiency and incom-,.

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~32~9 pleteness of oxygen absorption into the aqueous phase.The result very often is foaming as certain substances in the aqueous phase form f:ilms around the air bubbles retarding their disengagement from the culture. Foam formation reduces bioreactor volumetric productivity by retaining part of the culture in a spent-gas environment, ie. oxygen-poor and nitrogen and carbon dioxide rich, where the cells are less active and/or altered metaboli-cally. Foam also migrates and increases the risk of the culture coming into contact with the external environment and becoming contaminated. Finally, any air injected into a bioreactor obviously comes from a non-sterile environment and must be filtered and sterilized for an aseptic culture and the exit gas must be similarly treated because of the hazard it may in turn represent to the environment. This adds to the overall cost and complexity of the installation. There is therefore a need in the biotechnological industries for bioreactors which are adequately oxygenated by a mechanism which neither subjects the culture to significant turbulent and sheer forces nor brings it into direct contact with the environment.
One way of reducing the agitation required for certain cultures is to create an emulsion of the culture ~3211~9 in another non-miscible liquid phase. Oxygen may then be supplied by contacting the continuous carrier phase with air or oxygen gas. The culture obtains oxygen via the liguid-liquid interface thus created. Such a technique is described in Canadian Patent No. 1,164,376, granted to the firm Henkel ~ommanditgesellschaft Auf Aktien of Germany, which claims both a hydrocarbon liquid suitable as the continuous phase and a process for producing highly viscous Xanthomonas biopolymers in the emulsified agueous phase. The purpose of the invention is primarily to reduce the viscosity of the reactor contents, which will not be greater than the viscosity of the continuous phase which contains no biopolymer. This technigue is difficult to apply to cultures of eukaryotic cells, because emulsi-fied droplets must be very small in order to be stable, i.e. below 10~ diameter. and these cells are normally close to this size, imposing severe diffusional limita-tions on the substrate and product. Moreover, the mechan-ical energy reguired initially in order to form such an emulsion would rupture cells such as spheroplacts, lymph-ocytes, or other mammalian tissue derived cells, whose membranes are also sensitive to the emulsifying surfac-tants used. None of this affects bacteria significantly, making this technigue possible with Xanthomonas.

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13211~9 The recent development of totally inert liquids in which gases are highly soluble while solids and most other liquids, especially water, are practically insoluble raises the possibility of oxygenating a bioreactor without actually injecting a gaseous phase into the culture or subjecting it to any significant turbulent and shear forces. These liquids, of perfluorinated hydrocarbon composition, more specifically perfluorinated methyl and di-methyl cyclohexanes are marketed under the trade name of Flutec in the U.K. Similar products in the form of aqueous emulsions such as Fluosol-DA (trademark) have received attention as blood extenders. This use is based on the ability of the emulsion to dissolve oxygen and supply it to living tissues and is the subject of various patent applications. Flutecs, however, are not emulsions, are totally immiscible with water, and dissolve up to twenty times as much oxygen as does water under identical conditions. Their viscosity is close to that of water, while th~ir density is substantially greater. These characteristics, plus their total chemical and physiological inertness appears to make them suitable as an oxygen carrier in bioreactors.
In European patent application 0164813, published 18 December 1985, of Teijin Ltd., there is _ 5 132~

described a method of cultivating animal or plant cells which involves feeding a liguid fluorocarbon with molecular oxygen dissolved in it into a cultivator vessel containing a continuous phase of cultivation liguor with suspended cells. The fluorocarbon is allowed to settle, and a heavy phase, primarily fluorocarbvon, is withdrawn, re-oxygenated and returned to the vessel.
There are some significant difficulties in the practical application of such a system. One of these is dispersion of the gas carrying inert liquid in the culture medium. With two fine a dispersion. carrier liquid droplets remain in suspension for too long and become oxygen starved. Another difficulty is incomplete coalescence of the carrier liguid at the bottom of the vessel. This may result from an excessively fine dispersion of the carrier liquid or gas adsorption on the surface of the droplets. The clustering without coalescence of the carrier liquid droplets can entrap culture in the bed of droplets.
Other desirable improvements in the known system include the elimination of direct contact between the carrier liguid and any gaseous phase. This addresses two problems, evaporation of the volatile carrier liguid as sterility of the airation process. For some cultures it ~321~9 may be desirable to provide independent control of the carbon dioxide of partia] pressure in the reactor independently of the oxygen partial pressure. It is also desirable to provide a mixing action within the reactor, without resorting to a mechanical action on the culture.
The use of an hermetic reactor allows perfusion culture using suction from the downstream and of the system. A
further desirable characteristic is allowing a continuous, steady state culture. Aseptic sampling of the reactor contents is also desirable.
The present invention, in various of its aspects, addresses one or more of these areas of potential improvement.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a method of supplying oxygen to a suspension of living cells in a liquid culture medium using a carrier liquid in which oxygen is soluble, the carrier liquid being immiscible in the culture medium and having a specific gravity greater than that of the culture medium, the method comprising:
passing the carrier li~uid through a passage free from direct contact with a body of oxygen containing gas and in contact with one side of an oxygen-permeable t , , ' '. , `. , :
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_ 7 _ ~3 21~ 59 membrane;
exposing the opposite side of the membrane to a body of oxygen containing gas whereby oxygen permeates through the membrane and dissolves in the carrier liquid;
injecting the carrier liquid into the aqueous suspension; and allowing the carrier liquid to separate from the aqueous suspension.
Thus, an inert perfluorocarbon or other carrier liquid with sufficient dissolved oxygen may be injected into an aqueous cell suspension in a reactor which may be in the form of a tall cylindrical column. In the time required for this liquid to descend to the bottom of the column, the oxygen will be absorbed from it into the aqueous phase for consumption by the respiring cells. The liquid coalesces readily and forms a continuous bulk phase which can be recirculated to the top of the reactor and re-injected. After separation rrom the aqueous phase, and before reinjection, carbon dioxide may be removed and fresh oxygen added to the oxygenating liquid.
By oxygenating the carrier liquid through a membrane, free from contact with a body of gas, evaporation of the carrier liquid is substantially eliminated, sterility problems are minimized and the . , , ................ ~ :

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oxygen is fully dissolved ln the carrier.
According to another aspect of the invention there is provided a reactor for carrying out bioreactions in aqueous suspensions of living cells, said reactor com-prising;
a vessel for containing a body of the aqueous suspension;
injector means for injecting a carrier liquid into the aqueous suspension;
carrier liquid cycling means for collecting coalesced carrier liquid from a selected location in the vessel and r~cycling the carrier liquid to the injector means; and oxygenator means for oxygenating the carrier liquid as it is being recycled, comprising a membrane permeable to oxygen, means for passing the carrier liquid over one side of the membrane out of contact with any body of gas, and means for passing an oxygen carrying gas over the other side of the membrane.
The dispersed dense inert liquid is preferably confined to one part of the reactor vessel by one or more upright partitions placed inside the reactor. This will create a density differential between the culture on the two sides of the partition, which will in turn cause :. . :

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g continuous mixing of the culture inside the reactor. This is important if conditions such as pH and temperature are to be properly monitored and controlled.
The advantages of a bioreactor based on this principle include a greatly reduced risk of introducing microbial and other contaminants into the culture, a reduction in foaming of the culture, and the elimination of shear forces applied to the agueous phase, all achieved while permitting ade~uate oxygenation of aerobic cultures.
This is of particular interest in the production of high-purity substances of biological origin using biotechnological methods especially when the transforming catalyst is an aerobic single cell, shock-labile living organism or when the product itself is shock or shear-liable. This is the case in many modern pharma-ceutical and medical applications, eg. monocolonal anti-body production, synthetic human hormone production, viral antigen and vaccine production.
In conventional fermentors, aeration serves not only to supply oxygen to the culture but to remove the carbon dioxide produced as well. The volumetric solubility of carbon dioxide in water is nearly 30 times greater than that of oxygen at the same partial pressure.
Where the d:issolved carbon dioxide may reach toxic levels, :

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- lo ~321~9 a culture must be adequately interfaced with a phase in which the carbon dioxide partial pressure is very low.
Air bubbled through a bioreactor at a sufficient rate and properly dispersed provides the necessary contact wi~h such a phase thereby permitting the transfer, and hence removal of carbon dioxide from the culture.
In the present system, however, where oxygen is introduced into the recirculating inert fluid by permeation through a membrane, the primary exit route for carbon dioxide may be the culture surface. Under these conditions carbon dioxide could reach saturation conditions in both the culture and the inert fluid before leaving the vessel and become toxic to the cells.
A device which contacts the inert liquid with some surface area through which carbon dioxide may be removed prior to pumping the liquid through the oxygen dissolution tube is therefore desirable. Such a device may have the following characteristics:
i. a large surface area for gas exchange;
ii. a surface membrane highly permeable to carbon dioxide but impervious to liquids, e.g. a silicone membrane on a porous support;
iii. low carbon dioxide partial pressure on the 2~9 gaseous side of the membrane.
BRIEF DESCRIPTION OF THE DRA~INGS
In the accompanying drawings which illustrate exemplary embodiments of the present invention:
Figure 1 illustrates one embodiment of the reac-tor;
Figure 2 is a sectional view of an oxygenator outlet;
Figure 3 is a cross-section of a gas partial pressure monitoring device; and Figure 4 illustrates an alternative embodiment of the reactor.
DETAILED DESCRIPTION
Referring to the accompanying drawings, especially to Figures 1, 2 and 3, there is illustrated a batch process bioreactor 10. The reactor includes cylindrical Pyrex (trade mark) glass vessel 12 supported on a base 14. The large mouth 16 of the vessel is closed with a rubber stopper 18. A carrier liquid withdrawal tube extends from the stopper along the centre of the vessel 12 to near its bottom, where it is open to the interior of the vessel. A draft tube 22 is mounted concentrically on the withdrawal tube 20. The draft tube is a polypropylene cylinder with an open mouth ~4 at the - . ,.
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132~1~9 bottom and a generally flat panel 26 at the top. Large openings 28 are formed in the side wall and the panel 26 of the draft tube to allow free communicat~on between the interior of the draft tube and the remainder of the vessel 12. The base 26 has a small hole (not illustrated) that receives the carrier liquid withdrawal tube 20. The draft tube 22 is held in place on the withdrawal tube 20 with two small rubber stoppers 30 one above and one below the panel ~6.
As illustrated most particularly in Figure 3, the carrier liquid withdrawal tube 20 extends through a hole in the large rubber stopper 18 and then through a transverse bore in a stopper 3~. Within the stopper 32, the tube has an opening 34 (Figure 3) confronting a longitudinal bore 36 in the stopper. The bore 36 is closed with a polypropylene tube 37 wrapped with polytetrafluoroethylene tape 38 which closes the tube end.
An electrode 40 projects into the tube 37 and is used to monitor the partial pressures of oxygen and carbon dioxide as desired.
Within the reactor is a carrier liquid diffuser 46. This is a short length of soft polyurethane tubing 48 with a series of small holes 50 through which a liquid in the tube may be dispersed into a culture within the react-~. ~ .: :- ..,. ... . ... ..: .
., .,, .. .:. :., -- 13 - 132~9 or. With a carrier liquid flow rate through the tubing of 100-200 millimeters per minute, a holds approximately 0.2 to 0.3 millimeters in diameter will give a suitable dis-persion. One end of the dispersion tube 48 is connected to an inlet tube 52 passing through the stopper 18.
Positioned above the reactor vessel is a sampler 56 which is an inverted tube 58 closed with a plug 60. A
polypropylene tube 64 extends through the stopper 18 and the plug 60 and supports the inverted sampling tube 58 in its upright position. The lower end of the tube 64 is connected to a flexible sampling tube 65 that extends through one of the openings 28 into the dra~t tube 22.
A sample extraction tube 66 extends through the plug 60 and to the closed top end of the tube 58. Outside of the tube 58, the extraction tube 66 is connected to a syringe 68 and is equipped with a clamp 69 between the sampler and the syringe.
The carrier liquid withdrawal tube extends from the stopper 32 to a plug 74 at the upstream end of a breather 7B. This includes a silicone elastomer tube 80 several metres long with a 6.4 millimeter inside diameter and a 0.8 millimeter wall thickness. Within the tube 80 is an inner tube 82 of silicone elastomer with a 1.5 millimeter inside diameter and 0.5 millimeter wall .. . .. . . .. ..

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thickness. A single inner tube is illustrated in the drawing for the sake of clarity but additional inner tubes may be used where required. The combined tubes are housed within a temperature control chamber 84. This may be an incubator or a water bath.
At the outlet end of the breather, the tubes 80 and 82 are connected to a plug 86 as illustrated most par-ticularly in Figure 2. The plug has a centre hole carry-ing a tube coupling 88 with inner and outer barbed ends 90 and 92 respectively. The tube 80 is fitted on the end 90, while the end 92 is connected to the end of the inlet tube 52 leading to the end of diffuser tube 48. Within the plug 86 and fitting 88, the inner tube 82 is connected to a polytetrafluoroethylene (PTFE) capilliary tube 93. The tubing arrangement in the plug 74 is similar to that illustrated in Figure 2. In both cases, the ends of the inner tube 82 are open to atmosphere through the PTFE
capilliary tubes. Where desired, the inner tube 82 can be connected to a small air pump or to an oxygen supply, especially where the temperature contol chamber 82 is a water bath and gas exchange through the outer tube 80 is minimized.
Between the plug 86 and the stopper 18, the in-let tube 52 passes through a peristaltic pump 94 that 1321~9 draws oxygenated carrier liquid from the breather and pumps it to the diffuser 46 that is submerged within a body of culture 96 within the vessel 12. The carrier liquid is dispersed into the culture outside of the draft tube 22. The effective density of the medium outside the draft tube is then greater than that within the draft tube, so that as the dense carrier liquid settles to the bottom of the vessel, a natural circulation is set up with the culture medium ascending sending through the draft tube and descending outside of the draft tube. At the bottom of the vessel, the carrier liquid coalesces as a body of carrier liquid 98 and is withdrawn through the carrier liquid withdrawal tube 20, through the sampling chamber 32 where the oxygen and carbon dioxide content of the carrier liquid can be monitored and thence to the breather for re-oxygenation.
Within the breather, the outer and inner tubes 80 and 82 respectively act as membranes over which the carrier liquid passes. These form a sterile barrier per-meable to oxygen so that oxygen will permeate through both tubes and into the carrier liquid. In those embodiments where the temperature control chamber 84 is a water bath, oxygen is supplied through the inner tube or tubes 82 and it may be desirable to complement the oxygen supply ~32~ ~59 with a pump or the use of pure oxygen as mentioned above.
The culture sampler 56 uses the gas carrying inert liquid to reduce the risk of contamination. The tube 58 and the tube 66 contain the carrier liquid when the sampler is not in use. This prevents micro-organisms from growing into the vessel. Suction from the syringe 68 draws culture from within the draft tube 22, through tubes 65 and 64 into the inverted test tube 58. The culture collects at the top of the inverted test tube from where it may be withdrawn through tube 66 by the syringe 68.
Referring now to Figures 4, 5, and 6, there is illustrated a bioreactor system for continuous perfusion culture. The system includes a perfusion reactor 100 con-sisting of a cylindrical glass vessel 102 supported on a base 104. The mouth 106 of the vessel is closed with a large stopper 108. Extending through the stopper, along the centre of the vessel to a position near the bottom is a carrier liquid withdrawal tube 110. This supports a flexible partition 112 that extends across the tank and is supported on the tube 110 by two short flanges 114 and 116. The partition serves as a baffle separating the cul-ture into clenser and lighter sides to promote mixing in the same way as the draft tube 22 of the batch process reactor.

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~ 3 ~ 9 The carrier liquid withdrawal tube extends through the stopper 108 to a monitoring unit 118 like that illustrated in Figure 3. Sampling electrodes 134 monitor the oxygen and carbon dioxide partial pressures of the carrier liquid leaving the reactor vessel. They are connected to an oxygen partial pressure monitor 138 and a carbon dioxide partial pressure monitor 1~0.
Carrier liquid leaving the monitoring unit 118 is lead to the peristaltic pump 156 to be pu~ped through an inlet plug 158 of a breath.r 160. The breather is housed with a temperature controlled chamber 162. The construction and operation of this breather is the same as that of the breather 78 in the embodiment of Figure 1 and will not be further described.
The breather serves for the removal of excess carbon dioxide by permeation of the carbon dioxide through the membranes into air, which has a low carbon dioxide partial pressure.
After leaving the breather 160, the carrier liquid passes through an outlet line 176 leading through the stopper 108 to the diffuser 178 within the reactor.
The diffuser produces a fine dispersion of oxygenated carrier li~uid on one side of the partition 112. As the carrier li~uid settles, it gives up its oxygen to the - ~-:

, - 18 _ 132~9 culture and produces a circulating flow of culture, down-ward on the diffuser side of the partition and upwards on the other side. Between the breather and the diffuser 178, the line 176 for the carrier liquid is connected to a bleed line 180 equipped with a clamp 182. The bleed line is used for inoculation of the vessel 102 with a starter culture. When the bleed line 180 is not in use, it must remain filled with the carrier liquid and clamped shut with the clamp 182.
Between the bleed line 180 and the diffuser 178, the carrier liquid line 176 carries a clamp 184. Upstream of the bleed 180 is a branch line 186 extending through stopper 108 into the reactor vessel. The branch line 186 is equipped with a clamp 190. Within the reactor vessel it is connected to one end of a hollow ceramic rod 192.
Two rods 192 in the reactor are connected in series, be-tween the branch line 186 and a supernatant outlet 188 that extends through the stopper 108 for connection to a collector 193. The line 188 is branched with a line 194 connected to the carrier liquid withdrawal tube 110. Line 188 is equipped with a clamp 195 and branch line 194 with a clamp 196.
The ceramic rods 192 for withdrawing supernatant are located on the opposite side of the partition 112 from 132~ ~9 the diffuser 178. Also on that side of the partition is a culture collection tube 206 that leads through the stopper 108 and to the collector 193. The tube 206 has a clamp 208.
The collector 193 i.s similar to the sampler il-lustrated in Figure 1. In this case the inverted tube 226 has a conical end 228 to provide a better concentration of the collected material. A withdrawal tube 229 runs from the conical end of the tube 226, through the stopper 224 then to a peristaltic pump 210. A sampling tube 230 is branched off the tube 229 and connected to a syringe 234.
The sampling tube 230 is equipped with a clamp 232.
Downstream of the pump 210, the withdrawal tube 229 branches into two clamped tubes 236 and 238, leading to vented collection bottles 237 and 239 respectively. Tubes 236 and 238 have respective clamps 241 and 242.
The system also includes a culture medium reser-voir 240, which is a bottle closed with a stopper 242. A
line 244 from the bottle leads through the stopper 108.
The stopper 242 is also equipped with a vent 246 carrying a filter 248. The culture medium is drawn into the vessel from the reservoir 240 when supernantant and culture are withdrawn into the collectors 198 and 210.
A pH probe 252 extends through the stopper 108 .. ... ...

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- 20 - ~ 3~ 9 into the culture medium. This is connected to a pH moni-tor 254. Where desired the monitor can be used to control a pH adjuster 256. This is associated with two beakers 258 and 259 which may contain hydrochloric acid and sodium hydroxide solutions. These solutions are added to the reactor in controlled quantities through the use of pumps 260 and 262 connected to an addition port 264.
Although it could be argued that the bioreactor contents are brought into indirect contact with the ambient air through the lines leading to the inlet 264 microbial contaminents entering this way would not survive the passage.
The reactor is also equipped with a thermometer 266 that projects through the stopper 108 into the culture within the reactor. The thermometer output may be used to control a heater 268 controlling the breather temperature chamber 162.
The reactor is equipped with a vent 270 that includes a filter and a clamp for closing the vent when it is not in use.
In use of the reactor illustrated in Figure 4 carrier liquid is withdrawn through the tube 110 to the monitoring unit 118 where its oxygen and carbon dioxide content may be monitored. The carrier liquid then passes . . ~ ~ , , ....

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t32~1~9 to the breather where it is oxygenated and then back to the reactor through the nlQrmally open clamp 184 and diffuser 178. The branch line 186 is normally closed by clamp 190.
Culture is withdraw~n from the vessel through the culture collection tube 206 to the collector 193. From the collector the culture is drawn by the pump 210 to culture collection bottle 237. Similarly, supernatant may be withdrawn from the vessel through the ceramic rods lg2 and collector 193 to bottle 239. The clamps 195, 208, 241 and 242 controls which flow is withdrawn. The suction created by culture or supernatant withdrawal draws in culture medium from the reservoir 240.
Periodically, the clamp 184 is closed and the clamp 190 is opened. This causes carrier liquid to be injected into the reactor through the ceramic rods 192, to backwash the rods from the inside to dislodge accumulated debris.
The reactor vent is normally closed. It is only opened for autoclaving the system, filling it with carrier liquid and medium and for inoculation. Otherwise, the system works by suction created by the withdrawal of supernatant and cultured concentrate.
While specific embodiments of the present inven-,:
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1321~9 tion have been described in the foregolng, it is to be understood that other embodiments are possible within the scope of the invention. The invention is to be considered limited only by the scope of the appended claims.

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Claims (15)

1. A method of supplying oxygen to a suspension of living cells in a liquid culture medium using a carrier liquid in which oxygen is soluble, the carrier liquid being immiscible in the culture medium and having a specific gravity greater than that of the culture medium.
The method comprising:
passing the carrier liquid through a passage free from direct contact with a body of oxygen containing gas and in contact with one side of an oxygen-permeable membrane;
exposing the opposite side of the membrane to a body of oxygen containing gas whereby oxygen permeates through the membrane and dissolves in the carrier liquid;
injecting the carrier liquid into the aqueous suspension; and allowing the carrier liquid to separate from the aqueous suspension.

2. A method of controlling gas concentrations in a suspension of living cells in a liquid culture medium using a carrier liquid in which oxygen and carbon dioxide are soluble, the carrier liquid being immiscible in the culture medium and having a specific gravity greater than that of the culture medium, the method comprising:

contacting the carrier liquid with one side of a carbon dioxide-permeable membrane;
exposing the opposite side of the membrane to a reduced partial pressure of carbon dioxide whereby carbon dioxide permeates through the membrane from the carrier liquid;
dissolving oxygen in the carrier liquid;
injecting the carrier liquid into the aqueous suspension; and allowing the carrier liquid to separate from the aqueous suspension.

3. A reactor for carrying out bioreactions in aque-ous suspensions of living cells, said reactor comprising:
a vessel for containing a body of the aqueous suspension;
injector means for injecting a carrier liquid into the aqueous suspension;
carrier liquid cycling means for collecting coalesced carrier liquid from a selected location in the vessel and recycling carrier liquid into the injector means; and oxygenator means for oxygenating the carrier liquid as it is being cycled, comprising a membrane perme-able to oxygen, means for passing the carrier liquid over one side of the membrane and means for passing an oxygen carrying gas over the other side of the membrane.

4. A reactor according to Claim 3 wherein the membrane comprises a tube.

5. A reactor according to Claim 3 wherein the oxy-genator means comprise an outer tube, an inner tube posi-tioned within and extending along the outer tube, means for passing the carrier liquid through the outer tube and around the inner tube, the membrane comprising at least the inner tube.

6. A reactor according to Claim 5 wherein membrane further comprisese the outer tube.

7. A reactor according to Claim 3 including means for enclosing the oxygenator means in a controlled temper-ature environment.

8. A reactor according to Claim 5 including means for supplying gaseous oxygen to the interior of the inner tube.

9. A reactor according to Claim 3 including parti-tion means in the vessel separating the body of aqueous suspension into at least two zones, the partition means extending vertically from a position below the top of the body of aqueous suspension to a position above the bottom of the body of aqueous suspension, the injector means com-prising means for injecting a dispersion of the carrier liquid into the aqueous suspension on one side only of the partition.

10. A reactor according to Claim 9 wherein the par-tition is a baffle extending across the vessel.

11. A reactor according to Claim 9 wherein the par-tition is a vertically oriented tube with open top and bottom ends.

12. Apparatus according to Claim 3 wherein the re-actor is hermetically closed and including a culture medi-um reservoir having an outlet tube leading to the interior of the reactor, and product removal means for removing reaction products from the reactor under suction.

13. A reactor according to Claim 3 including means for sampling the aqueous suspension within the reactor comprising a substantially closed container substantially filled with carrier liquid, culture sampling means includ-ing a tube extending into the reactor for withdrawing cul-ture therefrom and into the container for injecting cul-ture into the container and sample withdrawal means com-prising a tube extending into the container and having an inlet end adjacent the closed upper end thereof whereby culture coalesced at the closed upper end of the container and floating on the carrier liquid may be withdrawn through the sample tube.

14. A reactor according to Claim 3 including moni-toring means for monitoring the concentration of gases dissolved in the carrier liquid.

15. A reactor according to Claim 14 wherein the mon-itoring means comprise a sampling chamber, means for withdrawing coalesced carrier liquid from the reactor into the sampling chamber and means for withdrawing carrier liquid from the sampling chamber to the carrier liquid cycling means, a gas permeable membrane closing one side of the chamber, a non-sterile monitoring chamber on an opposite side of the membrane and a gas concentration mon-itoring electrode projecting into the non-sterile chamber.