CA2022461C

CA2022461C - Method for culturing cells

Info

Publication number: CA2022461C
Application number: CA002022461A
Authority: CA
Inventors: Takao Miyamori; Makoto Uchida; Kanehiko Enomoto; Akihiro Sakimae; Ryozo Numazawa
Original assignee: Mitsubishi Rayon Co Ltd
Current assignee: Mitsubishi Rayon Co Ltd
Priority date: 1989-08-04
Filing date: 1990-08-01
Publication date: 1995-02-07
Anticipated expiration: 2010-08-01
Also published as: CA2022461A1; EP0411658A2; US5149649A; JPH03164169A; EP0411658A3

Abstract

Abstract of the disclosure A method for culturing cells by supplying oxygen to a culture medium through a multilayer composite membrane consisting of a porous layer(s) and a nonporous layer(s) having a thickness of less than 10 micrometers laminated one after the other. By using this multilayer composite membrane for supplying oxygen to the culture medium, oxygen can be supplied to the culture medium and/or the culture broth without causing foaming in the culture medium, cell growth inhibition or reduction in productivity of cellular products.

Description

-1- 2~æ,~

SPECIFICATION

~itle of the Invention METHOD FOR CULTURING CELLS

Background of the Invention l. Field of the Invention The present invention relates to a method for culturing animal, plant and microbial cells effectively.

2. Description of the Prior Art Recently, production of beneficial substances by culturing animal, plant and microbial cells is exten~ively carried out in fields of pharmaceutical, food and chemical industries and the like. In culturing these eells, an effective method for supplying oxygen to a culture medium or a culture broth is of importance.
Conventionally, in culturing cells, oxygen is supplied by a method in which gas containing oxygen, such as air, is directly introduced into a eulture by sparging, namely by a method in which an orifice or a sintered filter is provided at a tip of an air blowing nozzle so that the size of gas bubbles is minimized as much as possible while stirring. Furthexmore, a method in which oxygen partial pressure in a gas supplied or innex pressure in a culture device is increased so as - 2 - ~2 to increase a rate of oxygen absorption into a culture has been also developed.
However, in these sparging methods, excessive foaming in a culture causes a problem. In particular, in culturing animal cells, excessive foaming in the culture medium or culture broth is caused by sparying because a culture medium frequently includes serum which contains proteins acting as a surface active agent;
as a result, the cells growth is markedly inhibited.
Furthermore, in culturing microbial cells, a volume of culture per culture bath has to be reduced because of foaming, which reduce productivity relative to the size of the culture bath. If the forming is excessively vigorous, culturing itself cannot be carried out.
Proposed methods for supplying oxygen more efficiently to cells in order to solve these problems include a method in which an aerobic cultivation of cells is carried out using porous hollow fiber ~Japanese Patent Publication No. 7558/1982) and a method in which oxygen is supplied to a culture indirectly via a non-porous separating membrane made of silicone (Japanese Patent Publication No. 20261/1983, Japanese Patent Laid-open No. 309177/1988).

, [Problems that Present Invention At-tempts to Solve~

However, the porous membrane a~ disclosed in _ 3 _ ~?,~

Japanese Patent Publication No. 7558/1982 is mechanically strong to some extent, but when used for a long period of time, air bubbles may leak from the membrane due to change in blowing pressure, or the membrane becomes hydrophillc by due to the presence of surface active substances so that the culture may permeate into a gas supplying par-t.
Fuxthermore, the method disclosed in Japanese Patent Publication No. 20261/1983, in which a nonporous separating membrane is used, has an advantage that a culture medium or a culture broth does not permeate into the inside of the hollow fibers; however, it is neces-sary to enlarge a membrane area enormously or to use a extremely thin membrane having low diffusion resistance in order to supply sufficient oxygen for the growth of cells, because the oxygen diffusion resistance of the membrane is high. However, the method to supply enough , oxygen for the cell gorwth by enlarging the membrane area is not economically advantageous because the amount of the membrane to be used increases excessively.
Furthermore, in the method disclosed in Japanese Patent Laid-open No. 309177/1988, in which a thin silicone-made nonporous separating membrane is used, it was necessary to employ a complicated method of making the silicone-made nonporous separating thin membxane having weak mechanical strength in a form of 2~2~ ~

net, in order to maintain i-ts mechanical strength and to protect the hollow fiber membrane not to be torn by unexpected force.
Furthermore, when used as a membrane module, the membrane has to be immobilized at the end using a potting agent; however, adhesion of the nonporous separatiny membrane to the potting agent is weak because the surface of the membrane is not rough enough.
Therefore, the surface of the membrane has to be treated in advance to make the surface rough, which complicates a process for producing the membrane module. Further-more~ when the membrane is practically used in cultiva-tion, the thin membrane which has substantially effective oxygen permeability is mechanically weak and easily smashed by pxessure due to cell growth, which results in reduction in gas permeability or destruction of the membrane.

Summar~_~f the Invention An object of the present invention is to solve the problems described above; that is to provide a method for culturing cells effectively by using an oxygen permeable membrane capable of supplying necessary oxygen sufficient for cell growth to a culture medium or a culture broth, which prevents forming in the culture medium or the culture broth, growth inhibition of cells - s and reduction in productivity of cellular products.
As a result of intensive study, the present inventors have found that the bove-mentioned problem can be solved by using a multilayer composite membrane consisting of a nonporous layer(s) and a porous layer(s), and thus completed the present invention.
Namely, the present invention provides a method for culturing cells by supplying oxygen to a culture medium and/or a culture broth via a membrane structure, a multilayer composite membrane, in which a porous layer(s) and a nonporous layer(s) having a -thickness of 10 micrometers or less are laminated one after the other.
According to the present invention, oxygen can be supplied to a culture medium or a culture broth without causing foaming in the culture medium or the culture broth, inhibition of cell growth and reduction in productivity of cellular products; thereby markedly effective cultivation of aerobic microorganisms or animal or plant cells can be carried out.

Brief Description o-f the Drawings Figs. 1, 2, 3 and 5 are graphic drawings illustrating examples of culture baths and culture devices to be used according to the present invention.
Figs. 4 and 6 are cross-sectional views taken on line ~o~

A-A' of Fig. 3 and line line B-B' of Fig. 5, respectively.

Detailed Descri tion of the Invention and Preferred P
Embodimen~s A method for culturing cells according to the present invention comprising a step of culturing cells by supplying oxygen to a culture medium and/or a culture broth via a membrane, said membrane being a multilayer composite membrane in which a porous layer(s) and a nonporous. layer(s) having a thickness of 10 micrometers or less are laminated one after -the other~
Cells to which a cultivation method of the present invention is applicable include animal, plant and insert cells, cells of aerobic microorganisms and protozoan which grow aerobically.
Furthermoxe, a culture medium in the present invention denotes generally a liquid containing nutrient sources necessary for growth of the cells and production of substances~ and a culture broth denotes a liquid comprising above-mentioned culture medium and cells to be cultured.
A multilayer composite membrane denotes a membrane in which a nonporous layerls) and a porous layer(s) are laminated one after the other so that the porous layer(s) fortify the mechanical. strength of the nonporous layer(s). In the multilayer composite membrane, ~?,~

the number of layers in the laminated construction are not specifically limited if the above-mentioned conditions are satisfied; however, it is preferable that at least one side of the outer surface of the multilayer composite membrane is a porous layer.
Representative examples include a three-layered construction in which a nonporous layer is placed between two pr~us layers and a two-layered construction in which a nonporous layer and a porous layer are laminated. Furthermore, the nonporous layer may consist of two or more layers and thus a laminated construction having four or more layers as a whole may be constructed.
In case that the multilayer composite membrane having a porous layer at least on one side of its surfaces is set into a membrane module by ummobilizing the membrane at its end with a potting agent, the multilayer composite membrane can be closely adhered with the potting agent so that the pxocess for making the membrane module is simplified. The thickness of the porous layer may be the same or different one another; however, the thcikness of the multilayer composite membrane is preferably 10 micrometers or more in view of mechanica] strength and desirably 100 micro-meters or less in view of the necessity to enlarge the membrane area relative to the unit volume.
Furthermore, it is preferable to use a multilayer composite membrane having a rate of oxygen permeability of 1 x lO [cm (STP)/cm sec-cmHg3 or more.
Any materials having excellent oxygen permeability can be used in making the nonporous layer in the multilayer composite membrane. Examples include various kinds of polymers such as, e.g. silicone gum homopolymers; polydimethylsiloxane; silicone-poly-carbonate copolymers; polyolefin polymer such as poly-4-methylpentene~l and low density linear polyethy-lene; fluoride polymers such as perfluoroalkyl polymers;
cellulose polymers such as ethylcellulose; polyphenylene oxide; poly-4~-vinylpyridine; segmented polyurethane;
copolymers of these polymer materials; and blends thereof.
The thickness of the nonporous layer is lO micrometers or less in consideration of oxygen permeability, more preferably 5 micrometers or less, or preferably l micrometer or less.
Furthermore, the thickness of the porous layer in the above-mentioned multilayer composite membrane is preferably l to 30 micrometers in consider-ation of mechanical fortification and protective function.
Materials for *he porous layer include hydrophobic polymers such as polyolefin polymers such as polyethylene, polypropylene, poly-3-methylbutene-l and poly-4-methyl~

~ ~3 pentene-l; fluoride polymers such as polyvinylidene fluoride and polytetrafluoroethylene; polystylene, and polyetheretherketone. However, material to be used are not limited to hydrophobic polymers but hydrophilic polymers may also be used. Among these materials~
polyolefin polymers are preEerably used.
An example of the combination of the materials is polyethylene or polypropylene for the porous layer and segmented polyurethane for the nonporous layer, or poly-4-methylpentene-1 for the porous layer and silicone-polycarbonate copolymers for the ~lonporous layer.
It is preferable to use heat resistant materials such as poly-4-methylpentene-1 for the porous layer in consideration that a cell culture vessel is sterilized for use repeatedly by steam sterilization.
The shape of the multilayer composite membrane may be in a form of a flat membrane, a hollow fiber or the like; however, the membrane in a form of a hollow fiber is particularly preferable.
The multilayer composite membrane can be obtained by various methods; however, it is preferable to be obtained by processes of melt-spinning and stretching process because these processes can provide thin layered nonporous layers and porous layers having high mechanical strength. An example of these processes is disclosed in Japanese Patent Laid~open No. 1404/1987 , z ~

or US Patent No. 4,713,29~.
According to the above-mentioned method, a multilayer composite hollow fiber membrane having slit like pores in the porous layer and the slit like poxes are interconnected so as to make tortuous path from one surface to the other surface ~nd have major axis extended in the axial direction o-f the fiber~ -In the use of the above-mentioned multilayer composite membrane, a membrane module which is generally used in an artificial kidney, lung or the like is advantageously used; namely, the membrane may be laminated in several layers in case of a flat membrane, or several fibers may be bundled and immobilized at the end in case of a hollow fiber membraneO Furthermore, the resulting membrane module can be brought into contact with a gas containing oxygen at one surface of the membrane and with a culture medium or a culturè broth at the other surface so as to supply oxygen to the culture medium or the culture broth. In case of the hollow fiber membrane, it is possible to supply oxygen to the culture medium or the culture broth outside the hollow fiber by feeding gas into the hollow parts, i.e. inside, of the hollow fiber, or supply oxygen to the culture medium or the culture broth inside o-f the h~llow fiber by feeding gas to the outer surface of the h~llow fiber.
As for a method for supplying oxygen to a -- 11 ~

culture medium or a culture broth using the multilayer composite membrane, various methods are applicable.
For example, a multilayer composite membrane to supply oxygen is installed apart from a cell culture bath so that oxygen is supplied by passing a culture mediwn or a culture broth through the membrane module. It is also possible to install the membrane module inside the culture bath so that the membrane module is sintered in the culture broth and thus oxygen i5 supplied to the culture broth. Furthermore, the membrane module using a multilayer composite membrane is made in various forms in compliance with the culture method or the shape of cultu~e bath to be used~
Further, in culturing cells using the multilayer composite membrane module, the membrane module is preferably sterilized by gas sterilization using ethylene oxide, steam sterilization or the like.
Furthermore, any kinds of gas which contain oxygen can be used in culturing cells; a gas mixture containing oxygen, air, nitrogen, carbon dioxide gas or the like at an arbitrary ratio can be used.
The present invention will be explained hereinafter more in detail by reference to the accompanying drawings. Fig. 1 shows an example of a cell culture device to practice the present invention.
The number 1 is a culture bath into which a culture - 12 - 2~

medium and suspending cells are put; the number 2 is a stirring propeller to stir the culture broth;
the number 3 is a multilayer composite hollow fiber membrane module to supply oxygen -to the culture broth, which is connected to pipes at both ends each leading to a gas inlet 4 and a gas outlet 5. The nubmer 6 is an inlet for CO2 gas supply and the num~er 7 is an outlet for exhaust gas. This device is so constructed that cells in the culture broth are evenly suspended by the stirring propeller, and oxygen gas necessary for growth of the cells is introduced into the inside (h~llow parts) of each the multilayer composi-te hollow fiber membrane via the gas inlet 4 and then transferredto the culture broth -through the membranes. ~hereby, suf~icient oxygen for the cell growth can be supplied to the culture broth without foaming.
Fig. 2 shows a cell culture device in which a multilayer composite membrane module and a cell culture bath are installed independently~ In this device, a culture medium is circulated in the system by a pump 14. The culture medium flows inside each the multilayer composite hollow fiber membrane, i.e. each hollo~ part~
and oxygen introduced from the gas inlet 4 is supplied to the culture medium. CO2 is supplied through inlet 6'.
A porous hollwo fiber membranes 15 are placed in a culture bath 1', cells are inoculated from a cell ~O~æ~

inoculation port 12 outside the porous hollow fiber membranes, whereas the culture medium flows hollow parts (inside) of -the porous hallow fiber membranes, supplying nutrients and oxygen to the cells outside the porous hollow fiber membranes through their porous walls.
Figs. 3 and 4 individually show a cell culture device in which the shape of the multilayer composite hollow fiber membrane module suitably fits to the shape of the cul~ure bath. These membrane modules are formed in the forms of top-chopped cone and cylidner with a hollow in the center, respectively; a condenser lO for exhausting gas is installed upward the culture bath.
Application of the oxygen supplying method according to the present invention is not limited to the ordinary cultivation of aerobic microorganisms, animal and plant cells and the like but expandèd to c~ll cultivation in which cells are immobilized on the surface of a carrier or cell cultivation in which cells grow inside a carrier using microcarriers and microcapsules, so as to produce beneficial substances.
The present invention will be further explained referring to the following examples.

Example l A membrane module having an effective membrane 2~2~

area of 200 cm was constructed by bundling multilayer composite hollow fiber membranes having a three-layer structure as shown in Table 3 was installed in a one liter volume culture bath shown in Fig. l; mouse myeloma cells, MPC-ll strain (ATCC CCL167), were incubated in the bath under the following conditions:
A medium (hereinafter referred to as RDF medium~
was prepared by adding 10% (w/v) newborn calf serum, 4 mM
glutamine, 25 micrograms/ml streptomycin, 25 U/ml penicillin, 16 mM HEPES buffer solution and 0.01% ~w/v) sodium hydrogencarbonate to a basal medium in which RPMI 1640 medium (a product of Nissui Seiyaku Co.~, DME
medium (Dulbecco Modified Eagle's medium, a product of Nissui Seiyaku Co.) and Ham F12 medium (a product of Flow Laboratories~ are mixed at a ratio of 2:1:1 (v/v), pH
being adjusted to 7.0, 450 ml of the medium was put into -the above~mentioned culture bath, and then 50 ml of precultured cells of MPC-ll strain (5.0xlO cells/ml, having 93% viability) was inoculated into the medium.
Subsequently, air is introduced at a rate of 100 ml/min into the inside of the hollow part of each multilayer composite hollow fiber membrane from the gas inlet 4, and incubation was carried out for 72 hours while stirring using the stirring propeller at 60 ~pm~ Further, during the incubation, CO2 gas was blown from the gas supplying inlet 6 to maintain pH of the culture broth ~2~

at 7.0~
The cell density at the end of the cultivation was 6.8x106 cells/ml and the viability of the cells was 95~. Furthermore, no foaming was observed during the incubation.

Comparative Example 1 Cultivation was carxied out in ~he same manner of Example 1 except that a membrane module having an effective membrane area of 200 cm2 and using the silicone gum hollow fibers having the properties shown in Table 2, was used in place of the multilayer composite hollow fiber membrane module having the three-layer construc-tion. After 72 hours, the cell density was 2.3x106 cells/ml and the viability was 85~.

Example 2 Cell cult.ivation was carried out in the same manner as described in Example 1 except that multilayer composite hollow fiber membranes having the three-layer construction shown in Table 1 were used in place of the multilayer compos.ite hollow fiber m~mbranes having the three-layer construction shown in Table 3.
At the end of the.cultivation, the~cell density was 5.6x105 cells/ml and the viability of the cells was 87~. Furthermore, no foaming was observed during the cultiva-tion.

Example 3 Cells of MPC-ll strain cultured in the medium having -the same medîum composition as in Example 1 was suspended in a physiological saline solution containing 2% sodium alginate to obtain the cell density of 6.0xlO cells/ml. The suspension was put in-to a syringe and dropped into a physiological saline solution containing 2% calcium chloride. In this way, cells were incorporated into spherical capsules (microcapsules, about 600 micrometers in diameter) made of sodium alginate. The cell density in the capsules was 5.4x105 cells/ml and the viability of the cells was 88.0~.
The capsules were filtered and washed with RDF medium supplemented with 0.1% ~w/v) poly-L-lysine~
Subsequently, 50 ml of the content of the above mentioned washea capsule and 450 ml of RDF medium were put into the culture bath as in Example 2 and incubation was carried out at 37C for 14 days. During the incubation air was blown into the bath at 100 ml/min and CO2 gas was supplied to maintain the p~ at 7Ø
At the end of the cultivation the cell density in the capsules was 2.0x108 cells/ml and viability of the cells was 76%.

Example 4 The composite hollow fiber membranes having 2 ~

the three-layer construction shown in Table l were placed in a polycarbonate-made cylindrical vessel to make a multilayer composite membrane module (oxygen enriching de~ice with a container having an effective membrane area of l,000 cm2).
Separately, a porous polyethylene hollow fiber membranes (a product of Mitsubishi Rayon Co., Ltd., EHF 270T, 270 micrometers in diameter, 55 microme*ers thick and 72% in proposity) was placed into a poly-carbonate-made cylindrical vessel (culture bath) to make a cell culture device ha~ing a outer voluem of the hollow fiber membranes of lO0 cm3.
The oxygen enriching device and the cell culture device are sterilized and then assembled with a culture medium resexvoir 13 in a clean box into a system as shown in Fig. 2 and then the system was placed in an incubator at 37C.
Subsequently, 3 liters of a culture medium consisting of Ham F12 ~90%) and fetal calf serum (10%) was put into a culture medium reservoir 13 and circulated in the system using a pump 14. Furthermore, lx107 clls of CHO-Kl (ATCC CCL-61, derived from chinese hamster ovary cells) which had been precultured was asceptically inocualted from a cell inoculation opening 1~2 and then incubation was started.
During the incubation, air was supplied from ,:

2~2~

a gas inlet 4 at a rate of 100 ml/ml and, when a pF~
value measured by a pH sensor 8 exceeded 7.4, carbon dioxide gas was supplied from a CO2 gas supplying inlet 6' and mixed with the supplying gas to maintain the pH 7.4 or less.
The culture medium was circulated in the system at a rate of 100 ml/min and after day 4, the whole medium was exchanged with a fresh medium every 2 days. The incubation was carried out for 10 days.
During the incubation, forming was not observed. After 10 days, the amount of cells in the culture bath 1' was 1.4xlO9.

Comparative Example 2 Incubation was carried out in the same manner as in Example 4 except that polypropylene~made porous hollow fiber membranes (a product of Mitsubishi Rayon Co., Ltd.~ XPF l90M~ were used instead of the three~layer composite hollow fiber membranes. From day 3, foaming was observed in the culture of the oxygen enriching device 3', and on and after day 5, air bubbles were accumulated in the culture bath 1' (cell culture device) and culturing efficiency was decreased;
as a result, the amount of the cells after 10 days of cultivation was 8.5x108.

2 ~ 2 2 ~

Example 5 The cells were incubated in the same manner as described in Example 4 except that the oxygen enriching vessel was made by filling a multilayer composite hollow fiber membranes having the three-layer construction described in Table 3 in a polycarbonate cylindrical vessel so as to make a membrane area of 1000 cm2. After 10 days, the number of the cells in the culture bath 1' was 2.9x10 .

Example 6 The surface of a bulk of a Lili~n auratum was sterili~ed with a 70% ethyl alcohol aqueous solution and a 10% sodium hydrochlorite aqueous solution and then cut into pieces of 5 ~n - 15 ~n squires. One piece each was placed in a test tube (2.5 cm in diameter and 12.5 cm in depth) containing 10 ml of Muràshige-Skoog solid agar medium (pH 6.2) as shown in Table 4 and cultured at 25C under irradiation of 2,500 luxes for 60 days. After the incubation 1-5 calluses per one piece differentia-ted from the bulb was obtained.
150 pieces of ramenta thus obtained was collected and sterilized with sodium hydrogenchlorite for 15 minutes and then thorouyhly washed.
Separately, a top-chopped cone shaped membrane module having a hollow in the middle having an effective 2~2~

membrane area of 1 m2, which was constructed using the multilayer composite hollow fiber membranes shown in Table 1, was placed in the culture bath 1 as shown in Fig. 3, and then 2 liters of a culture medium having the composition shown in Table 4 without agar, supple-mented with abscisic acid (0.2 mg/l), was put into the culture bath and further the above-mentioned ramenta were transplanted.
Cultivation was carried out at 25C for 50 days under the continuous irradiation of 5,000 luxes.
During the cultivation air was supplied from the gas inlet into the hollow part of each the multilayer composite hollow fiber membrane at a rate of 1 liter/min while stirring by a stirring propeller at 30 rpm. A-t a result about 2,000 bulbs were obtained.

Example 7 Cells of Psudomonas ~utida (ATCC 8209) was inoculated into 100 ml of a liquid culture medium ~pH 6.8) consisting of 0.5% meat extract, 0.75% peptone, 0.25~
NaCl, 0.5% glucose, 0.15% malto-extract and 0.15% yeast extract, and incubation was carried out at 30C for 1 day with shaking. A mixed culture consisting of 25 ml of the resulting culture broth and 1975 ml of 30-fold diluted corn steep liqueur (a product of Oji Corn Starch Inc.) was obtained and further supplemented with 4.0~

2~22~

glucose, 0.75% ammonium sulfate and 0.1% yeast extrack.
Separately, a cylindrical membrane module (having an effective area of 1 m ) was constructed using the multilayer composite hollow fiber membranes shown in Table 1 and placed in the culture bath 1 as shown in Fig. 5; 2 liters of the above-mentioned culture broth was puk into the culture bath and then incubation was set up.
During the incubation, air was supplied from 1~ the gas inlet at 1 liter/min while string with the stirring propeller at 300 rpm and maintaining the temperature of the fluid at 30C and ~he pH of the fluid at 7.0 using a 10% NaO~I solution or a 10% H2SO4 solution.
After 24 hours, glucose was added at a concentration of 4% and the incubation was continued for another 24 hours. No anti~forming agent wàs added to the culture medium but forming was not obser~ed during the incubation. The cell concentration at the end of the incubation was 18 g/l in dry weight.

Comparative Example 4 Incubation was carried out in the same manner as in Example 6, except that the polypropylene-made porous hollow fiber membranes (a product of Mitsubishi Rayon Co., Ltd., KPF 190M) were used in palce of the 2~2~

module using the multilayer composite hollow fiber membranes having the three-layer construction. After 20 hours, air forming begun from the hollow fibers in the culture broth. At 24 hours af-ter the beginning oE
the incubation, glucose was added at a concentration of 4~ and the incubation was continued. A~ter 30 hours, foaming was vigorous and the condenser 10 and the bacteria repellent filter 9 got wet because of the foaming, so that the incubation was terminated at 35 hours after the beginning of incubation. A cell concentration at this time was 11 g/l in dry weight.

Example 8 Cultivation was carried out in the same manner as in Example 4 except that the polycarbonate-made cylindrical vessel was filled with the multilayer composi-te hollow fiber membranes having the three-layer structure shown in Table 5 so as to make an oxygen enriching device having an effective area of 1,000 cm2.
After 10 days of the incubation, the cell numbers in the culture bath was l.9xlO .

2 ~

Table 1 Porous layer (outer and Imedlan layer) lnner layers) Material High-density Segmented polyethylene polyurethane Thickness 30 micrometers 0.5 micrometer Inside diameter200 micrometers Rate of oxygen permeation ~cm3/cm2-sec-cmHg)1.2 x 10 .

Table 2 MaterialDimethlsiloxane silicone gum Thickness 100 micrometers Inside diameter200 micrometers Rate of oxygen6.0 x 10 6 permeation (cm /cm .sec.cmHg) 2~2~

a ~^
5~ ~ ~ O
a)~ o ~1 S~ R ~ r~
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.~ g~ ~
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rn ~ o ~1 ~I H o a) I o ~1 ~ a~
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rn~rl ~ O ~ O
O
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o rd ~ o a)o S~ ~r o ~ x o I S~ O ~ r~
R. S~
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a,) a) ~ ~s ed ~ (U
r X ~ rn rn ~rl O O -~1 0 ~ ,1 r,~
rd a) ~ ~) e -,1 ~ O O rd ~1 a Q ~ ~ s-i ~
rd ~a ~ ~ ~a ~ o ~1 ~

2~22f~3~

- 25 ~

Table 4 Ammonium nitrate 1650 mg/l Potassium nitrate 1900 mg/l Calcium chloride 2H2O440 mg/l Magenesium sulfate~7H2O370 mg/l Potassium dihydrogenphosphate 170 mg/1 Na2-EDTA~2H2O 37,3 mg/l Iron (II) sulfate-7H2O 27.8 mg/l acid 6.2 mg/l Borlc Manganese sulfate-4H2O 22.3 mg/l Zinc sulfate-7H2O 8.6 mg/l Sucrose 30 g/l Potassium iodide 0.83 mg/l Sodium molybdate 0.25 mg/l Copper (I) sulfa-te 0.025 mg/l Cobalt chloride 0.025`mg/1 Vitamin Bl 0.40 mg/l Inositol 100 mg/l Pyridoxine chloride . 0.5 mg/l Nicotinic acid 0.5 mg/l Glycine 2.0 mg/l Naphthaleneaceti acid 0.1 mg/1 Agar 10 , g/1 _ ~2~ ~

o o S~
-- o ~ ~ a ,t~
S~ O ~
(d O ~ ~ O
U7 ~1 ~ 5-1 o ~ o--a) rJ
h r~) Q~
o-,l ~ O
a) a) c~ u~
~: o ~ k ' o ~ o ~ ~`
z ~_ t) ~1 ~
.,~ o ~ ~
,1 o 4~
U~C~
o ~
~ X
_~ o ,1 o C~l I o ~d a) u s~ ~ ,1 o~
a) o ~
P~ ~ o ~ ,~ ~ a) o O
tQ r~ ~ O ~ X
~ ~ ~ :~ o o ~ ~ ~ ' , h ~: ~ o ~3 X 1 O ~ ~

S~ ~r o ~ ~-a) I s~
O
:~
_ o ~

a) ~ c~
,1 0 0 ~1 U~ ~ ~1 t~
Lr~ ~ o (I) -1 O o rl (D ~ ~
,4 ~ rl U~ ~' (~ Id E-l ~ E-l H K P.l --

Claims

1. A method for culturing cells by supplying oxygen to a culture medium and/or a culture broth through a membranous structure, wherein the membranous structure is a multilayer composite membrane in which a nonporous layer(s) having a thickness of 10 micro-meters or less and a porous layer(s) are laminated one after the other.

2. A method as set forth in claim 1, wherein a porous layer composes at elast one of the surfaces of the multilayer composite membrane.

3. A method as set forth in claim 1, wherein the multilayer composite membrane consist of two porous layers on the both surfaces and a nonporous layer in a median layer.

4. A method as set forth in claim 1, 2 or 3, which is characterized in that a rate of oxygen permeation of the multilayer composite membrane is 1x10-5 [cm3(STP)/
cm2.sec.cmHg] or more.

5. A method as set forth in claim 1, 2 or 3, wherein the nonporous layer of the multilayer composite membrane is 5 or less micrometers thick.

6. A method as set forth in claim 1, 2 or 3, wherein the multilayer composite membrane is formed in the form of a hollow fiber membrane.

7. A method as set forth in claim 1, 2 or 3, wherein a material for the porous layer of the multi-layer composite membrane is a polyolefin polymer or fluoride polymer.

8. A method as set forth in claim 1, 2 or 3, wherein a material for a porous layer of the multilayer composite membrane is poly-4-methylpentene-1.

9. A method as set forth in claim 1, 2 or 3, wherein the multilayer composite membrane is formed in the form of a hollow fiber membrane having slit-like pores in the porous layer.

10. A method as set forth in claim 1, 2 or 3, wherein a material for the nonporous layer in the multilayer composite membrane is selected from the group consisting of segmented polyurethane, poly-phenylene oxide, poly-4-vinylpyridine, silicone gum polymers, silicon polycarbonate copolymers, polyolefin polymers and fluoride polymers.

11. A method as set forth in claim 1, 2 or 3, wherein the cells are aerobic microorganisms or plant or animal cells.