JPH04281784A - Incubation of cho cell and production of cell-grown product - Google Patents

Abstract

PURPOSE:To obtain the title product in high efficiency by incubating gene recombinant CHO cells in high density. CONSTITUTION:Using a microcarrier having numerous vacuoles >2/mum, in size partitioned by membranes with the vacuoles mutually communicating open cell structure through the openings of the membranes partitioning adjacent vacuoles, gene recombinant CHO cells are incubated, and from the resulting culture solution, the objective cell-grown product is collected. The present method eliminates the drawbacks in the previous CHO cell incubation methods, markedly improving the culture concentration for the cells and the yield of the objective product.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for culturing genetically modified CHO cells and a method for producing cell growth products thereof. More specifically, the present invention relates to a method for culturing genetically modified CHO cells at high density using a porous microcarrier having a continuous pore structure, and an efficient method for producing cell growth products thereof.

0002

[Prior art] Compared to conventional monolayer culture methods using flasks and roller bottles, which have small culture surface area/volume values, culture methods using microcarriers expand the culture surface area with a small volume, and at the same time increase the culture volume. CH, which is an adhesion-dependent cell, can maintain a high cell density per cell.
It is currently widely used in O cell culture.

[0003] V was the first to report that adhesion-dependent cells could be cultured on suspended beads by agitation.
An Wezel. [A. L. van Wez
el, “ Growth of Cellstrai
ns and primary Cells on M
icrocarriers in homogeneous
Culture”, Nature, 216, 6
4 (1967)]

[0004] He is a member of Pharmac.
Diethylaminoethyl (DEAE)-substituted dextran beads (DEAE-Sephadex A-5
We showed that attachment-dependent cells, such as human diploid fibroblasts, can be cultured in large quantities in a homogeneous suspension microcarrier system by using 0 ) at a concentration of 1 mg/ml. However, DEAE-Sephadex
It has been found that when A-50 is used at concentrations above 1-2 mg/ml, toxicity appears and cell production does not increase proportionately.

[0005] After that, MIT's D. W. Levine
, D. I. C. Wang, W. G. Tilly
is the charge capacity of microcarriers from 0.1 to 4.5 me
It was discovered that many types of adhesion-dependent cells could be grown satisfactorily by controlling the ratio within the range of q/g (Japanese Patent Publication No. 57-3
5953, Special Publication No. 56-14270, Special Publication No. 57-55
14).

[0006] The thus improved microcarrier is manufactured by Pharmacia Fine Chemical
It is manufactured industrially under the name Cytodex I by s. Chemically, this microcarrier is manufactured from a crosslinked dextran matrix with a charge capacity of approximately 1.5 meq/g by having diethylaminoethyl groups covalently attached to the dextran chains. With the commercialization of this improved microcarrier, the microcarrier culture method has made great progress, and currently 10
It has been shown that zero or more cell types can be cultured on microcarriers.

Microcarrier culture methods have been applied to the production of interferon, various viruses, monoclonal antibodies, embryonic cancer antigens, etc. [Cell culture technology; edited by Saburo Fukui and Yukio Sugino, Kodansha Science. Fik (1985)]. Clark and Hirten
stein uses a microcarrier system,
Optimizing the culture method for cell growth, human fibroblast cells 1
reported that a yield of 3 x 104 units of INF-β was obtained per 06 cells [J. M. Clark and
M. D. Hirtenstein, J. INF Re
s. , 1. 391 (1981)].

[0008] Microcarriers themselves have been further improved, with the aim of high-density culture of cells by increasing the culture surface area/volume, and by making large pores so that cells can penetrate inside the microcarrier beads and proliferate. A microcarrier consisting of a structure has been developed.

[0009] Kjell Nilsson et al.
After granulating microscopic benzene droplets in an aqueous gelatin solution, the benzene was removed and crosslinked to develop gelatin microcarriers with a porous structure. As a result of stirring culture of BHK cells and Vero cells using this porous gelatin microcarrier, it was possible to obtain approximately twice the number of both cells compared to gelatin microcarriers without pores. [ Kjell Nilsson
, Ferenc Buzsaky and Klaus
Mosbach, Biotechnology v.
ol. 4, 11 (1986)] It is clear that microcarriers composed of large-pore porous structures are useful for improving cell culture density, but K.
The method of Jell Nilsson et al. does not sufficiently increase the culture surface area/volume.

[0010] Microcarriers made of a porous structure with large pores improve cell culture density because they increase the culture surface area per microcarrier volume.
With the same opening diameter, the thinner the wall separating the openings, the larger the culture surface area and the effective internal culture volume of the microcarrier, as long as the strength is not compromised. Such microcarriers have a large number of vacuoles with a diameter larger than about 2 μm separated by a membrane, and the vacuoles form a continuous pore structure that communicates with each other through openings in the membrane separating adjacent vacuoles. Developed microcarriers with
, Japanese Patent Application Publication No. 208331/1999).

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for culturing genetically modified CHO cells at high density using microcarriers and efficiently obtaining cell growth products.

0012

[Means for Solving the Problems] The present inventor has proposed the above-mentioned microcarrier developed by the present inventor, which has a large number of vacuoles separated by a membrane and having a diameter of more than 2 μm, and the vacuoles are adjacent to each other. We further investigated microcarriers that form a continuous pore structure that communicates with each other through openings in the membrane that separate the vacuoles, and when we cultured genetically modified CHO cells using these microcarriers, we were surprised to find that Surprisingly, compared to the conventional microcarrier culture method, 10
It was discovered that the target substance, a cell growth product, could be obtained with nearly twice the efficiency, and the present invention was completed.

That is, the present invention has a large number of vacuoles with a diameter larger than 2 μm separated by a membrane, and the vacuoles have a continuous pore structure that communicates with each other through openings in the membrane separating adjacent vacuoles. The forming microcarriers are used to form a suspension in a cell culture medium to which recombinant CH
This is a method for culturing genetically modified CHO cells, which is characterized by inoculating O cells to obtain a cell culture medium and maintaining this under cell growth conditions. Further, the present invention is a method for producing a cell growth product of genetically modified CHO cells, which comprises collecting the cell growth product from the above-mentioned culture solution.

The size of the vacuoles of the large-pore porous microcarrier used in the present invention is larger than about 2 μm in diameter, preferably the majority of the vacuoles are 5 μm or more, and more preferably 10 μm.
m or more. When the size is smaller than this, free movement of cells and culture fluid in the large-pore porous microcarrier cannot be achieved. Although the upper limit of the size of the vacuole is not particularly limited, it is set to 200 μm or less, more preferably 100 μm or less in order to increase the effective surface area than the current microcarrier.

There are no particular restrictions on the thickness or structure of the vacuole septum, except that a connecting port for connecting the vacuoles to each other should be opened, but the size of the opening is as follows: It is preferable that it is not too small compared to the vacuole;
In order to allow entry of cells, the diameter is preferably 1 μm or more or about 1/30 of the vacuole diameter. If the opening is too large, the strength of the particle structure will be insufficient, leading to destruction during use, which is undesirable. Therefore, it is desirable that the opening is about 1/3 or less, particularly about 2/3 or less, of the vacuole diameter. .

[0016] In addition to the above-mentioned large-diameter openings, the diaphragm may also have a finer pore structure, but this is rather desirable in order to promote the circulation of the culture medium. The thickness of the membrane separating the vacuoles of the large-pore porous microcarrier used in the present invention is not particularly limited, but is 1/4 or less of the vacuole diameter or 5 μm or less, preferably 3 μm or less, and more preferably 1 μm. It is as follows.

The average particle diameter of the large-pore porous microcarrier used in the present invention is preferably 100 to 1000 μm. 1
If it is smaller than 00 μm, the number of cells retained per microcarrier will be small and separation from the medium will be difficult. On the other hand, if the diameter is larger than 1000 μm, nutrients, oxygen, etc. will not be sufficiently supplied to the cells in the center of the microcarrier, and the culture environment will deteriorate, making it unsuitable for high-density culture.

Further, the average porosity of the large-pore porous microcarrier is preferably 80 to 99%. More preferably 9
It is 0-98%. When the average porosity is less than 80%,
There is not enough space for holding cells and it is not suitable for high-density culture. In particular, when the specific gravity of the polymeric substance constituting the large-pore porous microcarrier is greater than 1.1, if the polymeric substance accounts for 20% or more, the specific gravity of the microcarrier becomes too large, and gentle stirring is required. Suspension culture becomes difficult. On the other hand, when the average porosity exceeds 99%, the strength of the large-pore porous microcarrier decreases, and there is a risk of deformation and breakage during long-term agitation suspension culture.

The membrane constituting the large-pore porous microcarrier used in the present invention is substantially made of a polymeric substance. Here, polymers include rubber, polysaccharides, proteins,
Water-soluble synthetic polymers and organic solvent-soluble synthetic polymers are preferred, but there are no particular limitations, and inorganic polymers are also included within the scope of the present invention. Specific examples of polymeric substances include natural plant compounds such as cellulose and its derivatives, gum arabic, gum tragaganth, carrageenan, agar, guar gum, gum karaya, locust bean gum, pectin, galactan, mannan, pullulan or xanthan gum, natural Animal compounds such as gelatin, casein,
caseinate potassium salt, caseinate sodium salt, or chondroitin sulfate sodium salt, collagen, eslatin, fibroin, chitosan, chitin, hyaluronic acid,
Starch-based semi-synthetic polymer compounds, such as carboxymethyl starch, methylhydroxypropyl starch, dextrin,
Dextran, alginic acid-based semisynthetic polymer compounds, such as alginate propylene glycol ester or alginate, synthetic polymer compounds, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylethyl ether, carboxyvinyl polymer, polyacrylic acid,
polyacrylamide, polystyrene, polyvinyl chloride,
Examples include polyvinyl acetate, polyacrylonitrile, etc. or copolymers of their respective monomers with other vinyl monomers, condensation polymers and epoxy reactants such as polyamide, polyester, polyaramid, and addition polymers of polyurethane and others. It also includes derivatives, salts, crosslinked products, and blends of polymeric substances as specific examples listed above.
The polymer is not particularly limited, but those derived from cellulose and cellulose derivatives are particularly preferred.

[0020] The average molecular weight of the polymer forming the large-pore porous microcarrier is not particularly limited. Furthermore, the presence of metals, low molecular weight inorganic substances, low molecular weight organic substances, and gases in the polymer is permissible as long as this does not impair the purpose of the present invention. The shape of large-pore porous microcarriers is usually selected from spherical, prolate spherical, or oblate spherical shapes, but special shapes such as cylindrical, cylindrical, cubic, rectangular, and saddle shapes are also allowed. It will be done.

[0021] The large-pore porous microcarrier used in the present invention can be prepared by, for example, forming a polymer solution into a desired shape and cooling it to below the solidification temperature of the solution to freeze it, and then extracting and removing the solvent. Manufactured by a method that eliminates solubility.

[0022] According to the above manufacturing method, there is no need to add foreign matter such as a porous material to the polymer solution during manufacturing, so it is possible to easily create minute droplets with a uniform diameter, and the particle size can be controlled arbitrarily. can be done. The diameter and shape of the vacuole are
Basically, it is determined by the size and shape of the crystals formed when the solvent in the solution freezes and solidifies. Therefore, the shape and pore diameter of the vacuole can be adjusted by changing the freeze-solidification conditions such as the type of polymer solution and the temperature.

[0023] The large-pore porous microcarrier used in the present invention can be subjected to various surface modifications in order to improve the adhesion and proliferation of genetically modified CHO cells. Examples include coating with collagen, gelatin, fibronectin, etc., controlling the hydrophilicity of the surface by introducing positively charged groups, negatively charged groups, introducing hydrophobic groups or hydrophilic groups, and various cell adhesion factors, such as RGD.
(Arg-Gly-Asp tripeptide) may be introduced through a covalent bond, but this is not particularly limited.

[0024] In principle, microcarrier culture is a very flexible technique and the culture can be carried out in virtually any cell culture vessel. Microcarriers include, for example, Petri dishes, plastic bags, tubes, hollow fibers, spiral films, Jensen spiral films, gyrogens, columns, multiwell trays, multiplates, culture bottles, roller bottles, etc. [M. W. Glacke
n et al. , Trend in Biotec
h. , 1, 105 (1983), etc.], can be used to increase the surface area of conventional containers. However, when used in agitated suspension culture, the advantages of the microcarrier method aimed at high-density culture can be fully demonstrated by uniformly controlling the cell culture environment for each microcarrier. In all cases, culture vessels must be non-toxic and sterile.

[0025] A container for microcarrier culture is selected depending on the purpose of culture and the amount of culture. Laboratory-scale microcarrier cultures are typically 5 liters or less and a variety of vessels are used. Large-scale microcarrier culture is approximately 5
They range from liters to several thousand liters and require specially designed vessels that allow monitoring of parameters such as gas pressure and pH. In such appropriate culture, the amount of microcarriers used may range from several microcarriers to several hundred microcarriers/ml.

[0026] An example of a culture tank for microcarriers used for agitation suspension culture is Bellco's 5
Examples include 00 ml spinner flasks. This spinner flask includes a temperature sensor, pH electrode, dissolved oxygen electrode,
It is sufficient to assemble and use a device equipped with a porous Teflon tube, a medium supply/discharge nozzle, a microcarrier precipitation tube, a sample collection port, etc. as necessary. At the same time, a cell culture controller (System Ferment)
or MODEL AIS-12, ABLE Inc.), the pH can be controlled with CO2 gas or NaHCO3 solution, and the dissolved oxygen can be controlled by supplying air or pure oxygen through a porous Teflon tube.

A gene for producing a cell growth product to be produced was introduced into the cells. Genetically modified CHO cells are used. Culturing is usually carried out by the following method, but is not particularly limited.

[0028] Microcarriers are placed in a suitable glass bottle.
Du excluding calcium and magnesium ions
Add lbecco's phosphate buffer solution (hereinafter referred to as PBS) and stir gently from time to time for several minutes. Gently remove the supernatant and wash the microcarriers with fresh PBS with gentle agitation. Discard the PBS again, add 100 ml of new PBS to 1 g of microcarriers, and sterilize the microcarriers by autoclaving using pure water (120° C., 20 minutes). Depending on the surface modification treatment method, sterilization with 70% ethanol may be better than autoclaving. In this case, the above PBS
In the same manner as washing, after washing three times with 70% ethanol, it is left at room temperature overnight, and then washed three times with PBS.

Prior to use, the sterilized microcarriers are allowed to settle and the supernatant is removed, and the sterilized microcarriers are incubated in a serum-free solution for approximately 30 minutes.
Add 100 ml of culture medium warmed to 7°C to 1 g of microcarriers and wash (Pharmacia, Mi
crocarrier cell culture p
principles and methods).

[0030] There are various methods for adhering cells to microcarriers, but the microcarriers, cells, and culture medium are placed in a culture tank, and methods such as intermittent stirring or the Ficoll method [Kobayashi Tissue Culture 15 (1989)] are used. It is important to adhere as uniformly as possible. The concentration of cells to be prepared is, for example, 1 x 107 ~
Using cells appropriately prepared as 5 x 10 cells/ml, the cell concentration at the time of adhesion is, for example, 1 x 10 cells/ml.
It may be used so that the concentration is about 1×10 6 cells/ml.

[0031] In the agitation suspension culture, the agitation speed is set at a speed necessary and sufficient to keep all the microcarriers in suspension after completion of cell adhesion. After the start of culture, the culture solution is exchanged intermittently or continuously as necessary, and the temperature, pH, dissolved oxygen concentration, etc. are controlled to appropriate conditions, for example, around 37°C and pH around 7.4. , the cells are maintained under favorable growth conditions, and the desired cell growth product is collected from the culture solution. Collection and purification of this product may be carried out by conventional methods by appropriately selecting and combining gel filtration, ion-exchange chromatography, reversed-phase chromatography, and the like.

Depending on the culture conditions, even if the number of cells increases, the desired cell growth product may not be obtained as expected. Even in this case, the culture conditions are optimized, the amount of cell growth products in the replaced culture solution is checked, and the product is collected when the desired amount has been produced. The desired production amount varies depending on the purpose.

[0033]

Effects of the Invention The method for culturing genetically modified CHO cells and the method for producing the cell growth products of the present invention use large-pore microcarriers with a diameter of more than 2 μm, so they have good cell adhesion and proliferation, and Since the cells are cultured inside the carrier, the shearing force caused by stirring does not damage the cells. Moreover, by effectively utilizing the internal porous surface as a cell adhesion surface, it is possible to dramatically increase the cell culture concentration and, at the same time, greatly improve the yield of the cell growth product that is the production target. In short, according to the method of the present invention, the drawbacks of conventional CHO cell culture methods can be overcome, and the cell culture concentration and yield of cell growth products can be significantly improved.

[0034]

[Examples] The present invention will be specifically described below with reference to Examples. The diameter of the particles was measured by taking a photograph using an optical microscope at an appropriate magnification. The undried microcarriers are rapidly cooled with liquid nitrogen and frozen while preserving their structure.
After freeze-drying in a vacuum of 0.1 Torr, the sample was subjected to gold sputtering treatment, enlarged to an appropriate magnification using a scanning electron microscope, and the pore diameter was observed and measured. The pore size and film thickness inside the microcarrier were determined by freezing with liquid nitrogen as described above, dividing the microcarrier at the same temperature, drying it in a vacuum, and then observing and measuring it with a scanning electron microscope. Ta. Regarding the particle size and pore size, if they were not a perfect sphere or a perfect circle, they were defined as the shortest diameter.

Example 1 Viscosity 2 at 5°C prepared from purified linter as raw material
Poise, cellulose copper ammonium solution with cellulose concentration 2%, copper concentration 1.2%, and ammonia concentration 4% was sprayed with nitrogen gas using a two-fluid nozzle, and silicone oil (SH200, 1 .5cs, Toray Industries, Inc.) was received in 10 liters with stirring. Thereafter, the temperature of the silicone oil was raised to -20°C, stirring was continued for 20 minutes, and 10 liters of 40% sulfuric acid cooled to -35°C was added. After adding sulfuric acid, stirring was continued for 8 hours, the temperature was raised to 20° C., silicone oil was removed, and the remainder was taken out and washed with water, yielding a large amount of cellulose spherical particles. Next, only particles with a particle diameter of 180 to 210 μm were taken out using a classification sieve, and after introducing some positive charges through surface modification, freeze-drying was performed to obtain large-pore porous microcarriers. Separately, the surfaces and cross sections of the particles were observed and photographed using a scanning electron microscope, and the pore diameter and film thickness were measured. Both the surface and interior of the particles are composed of vacuoles with a diameter of 20 to 40 μm.
The thickness of the septum between vacuoles was 1-2 μm.

Example 2 1. Culture tank As a culture tank for microcarriers, a 500 ml spinner flask (Bellco) was equipped with a temperature sensor (outer diameter 3φ, ABLE), a pH electrode (DPAS/325 type, Ingold), and a dissolved oxygen electrode (10AN type,
ABLE), porous Teflon tube (inner diameter 3mm,
Outer diameter: 4 mm, length: 1 m, coiled, ABLE Co., Ltd.), culture medium supply/discharge nozzle, microcarrier precipitation tube (precipitation tube with constant temperature jacket, ABLE Co., Ltd.), sample collection port (inner diameter: 3
φ, ABLE Inc.) was assembled and used. pH is CO2 gas or NaHCO3 solution,
Dissolved oxygen can be added to the cell culture controller (System Fermentor MODEL) by supplying air or pure oxygen through a porous Teflon tube.
AIS-12, ABLE Inc.) was used for control.

2. The microcarrier microcarrier was obtained by preparing 2g of the large-pore porous microcarrier of Example 1 according to a conventional method (Pharmacia
, Microcarrier cell culture
re principles and methods
) After swelling, it was sterilized and used.

3. The cells used were M-CSF producing genetically modified CHO cells.

4. Adhesion of cells to adhesive microcarriers was carried out by the Ficoll method (Kobayashi Tissue Culture 15 (1989)).One 500 ml spinner flask culture tank contained 4
% Ficoll (Pharmacia), 5% fetal bovine serum (Biocell), 150μg/ml proline (Wako Pure Chemical Industries) containing DME medium (Flow) 250ml
The large-pore porous microcarrier of Example 1 and M-
CSF-producing genetically modified CHO cells (1 x 108 cells)
was mixed in and adhered while stirring at 10 rpm on a magnetic stirrer.

5. After the culture adhesion was completed, an additional 250 ml of DME medium was added, the stirring speed was increased to 25 rpm, and culture was carried out (37°C, pH 7.
4. Mixed ventilation of sterile air and pure oxygen was started. Culture 2
Culture was performed while continuously replacing the medium from day one. Collect the culture solution containing microcarriers from the first day of culture,
Nuclear staining method (Sanfor) was used to quantify the number of cells on microcarriers.
d, K 11 773 J. Nat. Cancer)
In addition, M-CSF activity in the culture solution was determined by mouse bone marrow cell colony formation method [J. Biol. Med. , 44,
287-300 (1966)]. The course of the 20-day culture is shown in Figure 1.

Furthermore, sections were prepared to observe whether the accurate number of cells proliferating inside the large-pore microcarrier could be counted by nuclear staining. The microcarriers on day 18 of culture before and after cell counts were counted using nuclear staining were washed twice with PBS. The microcarriers were placed in 2% glutaraldehyde and left at 4°C for 3 hours to fix the cells. Next, the resin was washed with water to remove excess glutaraldehyde, and the resin was gradually replaced with a water-soluble methacrylic resin. After 100% resin replacement, the resin was solidified at 60° C. for 12 hours. After creating a section with a thickness of 1 μm using an ultramicrotome, only the cells were stained by toluidine blue staining. When 100 sections prepared in this way were observed using an optical microscope, it was found that approximately 40% of the cells before counting remained inside the large-pore microcarriers after counting using the nuclear staining method. understood. Therefore, it is clear that the cell numbers shown in Figure 1 are much lower than the actual numbers because the nuclear staining method cannot accurately count the number of cells that have proliferated inside the large-pore microcarriers. Ta.

Comparative Example 1 The same culture vessels and cells as in Example 2 were used. The microcarrier was prepared by adding 2 g of Cytodex I (Pharmacia) in a conventional manner (Pharmacia).
, Microcarrier cell culture
re principles and methods
) After swelling, it was sterilized and used. Cell adhesion to the microcarriers was performed by the Ficoll method [Kobayashi Tissue Culture 15 (1989)].

[0043] In the microcarrier culture tank, 8% Fi
coll, Cytodex I in 250 ml of DME medium containing 5% fetal bovine serum, 150 μg/ml proline.
and M-CSF producing genetically modified CHO cells (1×
108 pieces) and stirred on a magnetic stirrer.
Adhesion was carried out while stirring at 0 rpm.

After completion of adhesion, add DME medium for an additional 250 m
1 was added, the stirring speed was increased to 25 rpm, and culture was started. From the second day of culture, culture was carried out while continuously replacing the medium. From the first day of culture, the culture medium containing microcarriers was collected, and the number of cells on the microcarriers was determined using nuclear staining method (
Sanford, K 11 773 J. Nat. C
Ancer), and the M-CSF activity in the culture medium was determined using the mouse bone marrow cell colony formation method [J. Biol. M
ed. , 44, 287-300 (1966)].

[0045] The course of the 20-day culture is shown in Figure 2. From the results of Example 2 and Comparative Example 1 and FIGS. 1 and 2, the following can be understood. Example 2 has a cell density at least 3 times higher than Comparative Example 1. Furthermore, the M-CSF productivity was 8 times higher, and it was clear that the large-pore porous microcarrier of Example 1 was superior in both cases.

Example 3 1. Culture tank As a culture tank for microcarriers, a 500 ml spinner flask manufactured by Bellco was equipped with a temperature sensor and a pH sensor.
Electrode, dissolved oxygen electrode, porous Teflon tube (inner diameter 3
mm, outer diameter 4 mm, length 1 m, coiled, ABLE)
A device equipped with a medium supply/discharge nozzle, a microcarrier sedimentation tube, and a sample collection port was assembled and used. pH
was CO2 gas or NaHCO3 solution, and dissolved oxygen was controlled by a cell culture controller by supplying air or pure oxygen through a porous Teflon tube.

2. Microcarriers Microcarriers were obtained by adding 2g of the porous microcarriers of Example 1 according to a conventional method (Pharmacia,
Microcarrier cell culture
principles and methods) After swelling, it was sterilized and used.

3. The cells used were interferon-producing genetically modified CHO cells.

4. Adhesion of cells to the adhesive microcarriers was performed by the Ficoll method [Kobayashi Tissue Culture 15 (1989)]. The 500ml spinner flask culture tank contains 4% F.
The porous microcarrier of Example 1 and interferon-producing recombinant CHO cells (1 x 10 8 cells) were mixed into 250 ml of DME medium (Flow Inc.) containing icoll, 5% fetal bovine serum, and 150 μg/ml proline, and magnetically incubated. Adhesion was carried out while stirring at 10 rpm on a stirrer.

5. After completion of culture adhesion, an additional 250 ml of DME medium was added, the stirring speed was increased to 25 rpm, and culture was started. Culture 2
From day 1 onwards, culture was carried out while continuously replacing the medium. From the first day of culture, the culture medium containing microcarriers was collected, and the number of cells on the microcarriers was determined using nuclear staining method (Sanf).
ord, K 11 773 J. Nat. Cance
r). The course of the culture is shown in Figure 3. After 16 days of culture, the cell density reached 1 x 107 cells/ml, making it possible to obtain about 50 times the inoculated cell density.

[Brief explanation of the drawing]

FIG. 1 shows the course of the cell culture of the present invention and its product activity.

FIG. 2 shows a progress chart of cell culture and product activity in a comparative example.

FIG. 3 shows a progress chart of cell culture in the present invention.

Claims (2)

[Claims] Claim 1: It has a large number of vacuoles separated by a membrane and having a diameter of more than 2 μm, and the vacuoles form a continuous pore structure that communicates with each other through openings in the membrane that separate adjacent vacuoles. A genetic combination characterized by forming a suspension in a cell culture medium using a microcarrier, inoculating the suspension with genetically modified CHO cells to obtain a cell culture medium, and maintaining this under cell growth conditions. Method for culturing recombinant CHO cells. [Claim 2] It has a large number of vacuoles separated by a membrane and having a diameter of more than 2 μm, and the vacuoles form a continuous pore structure that communicates with each other through openings in the membrane that separate adjacent vacuoles. Using microcarriers, a suspension is formed in a cell culture medium, this is inoculated with genetically modified CHO cells to obtain a cell culture medium, this is maintained under cell growth conditions, and the cells are grown from the culture medium. A method for producing a cell growth product of genetically modified CHO cells, which comprises collecting the product.