JPH04278081A - Method for culturing cell - Google Patents

Abstract

PURPOSE:To remarkably improve yield of a cell growth product with excellent cell adhesion and proliferating properties by carrying out culture of various cells using a porous micro-carrier forming a specific open-cell structure. CONSTITUTION:A micro-carrier forming an open-cell structure in which many vacuoles, separated with a membrane and having >=2mum diameter and the vacuoles are mutually communicated with openings of a membrane separating the adjacent vacuoles is used to form a suspension in a cell culture medium. A cell such as yeast or microorganism is then inoculated into the aforementioned suspension to form a cell culture solution, which is subsequently kept under good growth conditions for the cell. Thereby, high-density cell culture can be carried out to remarkably improve the yield of a cell growth product which is the objective substance of production.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel method for culturing cells. More specifically, the present invention relates to a method for culturing cells at high density using a porous microcarrier having a continuous pore structure.

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. Since it is possible to maintain a high cell density per cell, it is currently widely used in culturing adhesion-dependent cells.

[0003] Van et al. was the first to report that adhesion-dependent cells could be cultured on suspended beads by stirring.
This is Wezel. (A.L. van Wezel
,”Growth of Cell strains
and primary Cell on Micro
carriersin homogeneous cul
ture”, Nature, 216, 64 (1967)
) He is a member of Pharmacia Fine Chemical
Diethylaminoethyl (D
DEAE) substituted dextran beads (DEAE-Seph
We demonstrated that adhesion-dependent cells, such as human diploid fibroblasts, can be cultured in large quantities in a homogeneous suspension microcarrier system by using adex A-50) at a concentration of 1 mg/ml. However, DEAE-Sepha
It has been found that when dex A-50 is used at concentrations above 1-2 mg/ml, toxicity appears and cell production does not increase proportionately.

[0004] After that, D. W. Levine,
D. I. C. Wang, W. G. Tilly discovered that if the charge capacity of microcarriers is controlled within the range of 0.1 to 4.5 meq/g, a wide variety of attachment-dependent cells can be grown favorably. (Tokuko Sho 57-3595
3, Special Publication No. 56-14270, Special Publication No. 57-5514)
This improved microcarrier is manufactured by Pharma
Cyto by cia Fine Chemicals
It is manufactured industrially under the name dex I. Chemically, this microcarrier has a diethylaminoethyl group covalently attached to the dextran chain, resulting in a
．． Manufactured from a cross-linked dextran matrix with a charge capacity of 5 meq/g. With the commercialization of this improved microcarrier, microcarrier culture methods have greatly advanced, and it has now been shown that more than 100 cell types can be cultured on microcarriers.

The microcarrier culture method has been applied to the production of interferon, various viruses, monoclonal antibodies, embryonic cancer antigens, and the like. (Cell culture technology; edited by Saburo Fukui and Yukio Sugino, Kodansha Scientific (1985)) Clark and Hirtenst
ein has reported that they have optimized the culture method for cell growth using a microcarrier system and obtained a yield of 3 x 104 units of INF-β per 106 human fibroblasts. (J.M.Clark and M.D.
Hirtenstein, J., INF Res. ,1.
391 (1981)) Microcarriers themselves have been further improved, with the aim of cultivating cells at high density by increasing the culture surface area/
Microcarriers consisting of a porous structure with a large pore diameter have been developed to increase the volume and allow cells to enter and proliferate inside the microcarrier beads.

Kjell Nilsson et al.
A gelatin microcarrier with a porous structure was developed by granulating an aqueous gelatin solution containing minute benzene droplets, removing the benzene, and crosslinking the gelatin solution. Using this porous gelatin microcarrier, BHK cells and Ve
As a result of agitation culture of ro cells, it was possible to obtain approximately twice the number of cells in both cases compared to gelatin microcarriers without pores. (Kjell Nilsson, Fere
nc Buzsaky and Klaus Mosb
sch, Biotechnology vol. 4,1
1 (1986)) It is clear that microcarriers composed of large-pore porous structures are useful for improving cell culture density, but Kjell Nilsson
These methods have not yet been able to sufficiently increase the culture surface area/volume.

[0007]

[Problems to be Solved by the Invention] Microcarriers made of porous structures with large pores improve cell culture density because they increase the culture surface area per microcarrier volume, but if the opening diameter is the same, As long as the strength is not compromised, the thinner the wall separating the openings, the larger the culture surface area and the effective internal culture volume of the microcarrier. Ultimately, porous microcarriers with a membrane-separated structure are desirable, and the emergence of such porous microcarriers that enable higher-density cell culture has been awaited.

[0008] In order to solve the conventional problems, the present inventor has created a large number of vacuoles with a diameter larger than about 2 μm separated by a membrane, and the vacuoles are formed by openings in the membrane separating adjacent vacuoles. We have developed a microcarrier characterized by a continuous pore structure in which some parts communicate with each other, and have previously filed a patent application. (Unexamined Japanese Patent Publication No. 64-43530, Unexamined Japanese Patent Publication No. 2-2077
850, JP-A-2-208331) When various cells were cultured using this microcarrier, surprisingly, the cell density increased several times compared to the conventional microcarrier culture method. It was discovered that the target substance, a cell growth product, could be obtained with nearly 10 times the efficiency.

[0009] Accordingly, an object of the present invention is to solve the conventional problems and provide a method for culturing cells at high density using the porous microcarrier previously proposed by the present inventor.

0010

[Means for Solving the Problems] The method for culturing cells provided by the present invention includes a method for culturing cells using microcarriers. Microcarriers (hereinafter referred to as microcarriers) have 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. forming a suspension in a cell culture medium using large pore microcarriers; b. inoculating the suspension with cells to form a cell culture; c. This can be achieved by maintaining the cell culture medium under cell growth conditions.

The present invention will be explained in detail below. First, the average particle diameter of the large-pore porous microcarrier used in the present invention is preferably 100 μm to 1000 μm. If it is smaller than 100 μm, the number of cells retained per microcarrier will be small and separation from the medium will be difficult. If it 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.

[0012] Furthermore, the average porosity of the large-pore porous microcarrier is preferably 80% to 99%. More preferably 9
It is 0% to 98%. If the average porosity is less than 80%, there will be insufficient space for holding cells, making it unsuitable 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, the polymeric substance
If it accounts for 0% or more, the specific gravity of the microcarriers becomes too large, making it difficult to perform suspension culture with gentle stirring. On the other hand, when the average porosity exceeds 99%, the strength of the large-pore porous microcarrier decreases, and in long-term agitated suspension culture,
There is a risk of deformation and damage.

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

[0014] There are no particular restrictions on the thickness or structure of the vacuole diaphragm, other than the opening that connects the vacuoles to each other, but the size of the opening is It is preferable that the size is not too small compared to the vacuole diameter, and the size is 1 μm or more or 1/1 of the vacuole diameter to allow cell entry.
Approximately 30 is desirable. If the opening is too large, the strength of the particle structure will be insufficient, leading to destruction during use, which is undesirable.
It is desirable that it be below the level of

[0015] 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 solution. The thickness of the membrane separating the vacuoles of the large-pore porous microcarrier used in the present invention is not particularly limited, but may be 1/4 or less of the vacuole diameter or 5 μm.
m or less, preferably 3 μm or less, more preferably 1 μm
It is as follows.

The membrane constituting the large-pore porous microcarrier used in the present invention is substantially made of a polymeric substance. Here, the polymer is preferably a rubber, a polysaccharide, a protein, a water-soluble synthetic polymer, or an organic solvent-soluble synthetic polymer, but is not particularly limited, and inorganic polymers are also included in 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, and xanthan gum. , natural animal compounds such as gelatin,
Starch-based semi-synthetic polymer compounds such as casein, casein potassium salt, caseinate sodium salt, or chondroitin sulfate sodium salt, collagen, eslatin, fibroin, chitosan, chitin, hyaluronic acid, etc., such as carboxymethyl starch, methylhydroxypropyl starch, Dextrin, dextran, etc., alginic acid semi-synthetic polymer compounds such as alginate propylene glycol ester or alginates, etc. Synthetic polymer compounds such as polyvinyl alcohol,
Polyamides, such as polyvinylpyrrolidone, polyvinylethyl ether, carboxyvinyl polymers, polyacrylic acid, polyacrylamide, polystyrene, polyvinyl chloride, polyvinyl acetate, polyacrylonitrile, etc., or copolymers of their respective monomers with other vinyl monomers, etc. Examples include condensation polymers and epoxy reactants such as polyester and polyaramid, and addition polymers such as polyurethane. Derivatives, salts, crosslinked products, and blends of the polymeric substances listed above are also included, and the polymers are not particularly limited, but among these, cellulose and those derived from cellulose derivatives are particularly preferred.

[0017] The average molecular weight of the polymer forming the large-pore porous microcarrier is not particularly limited. In addition, metals, low molecular weight inorganic substances, low molecular weight organic substances,
Even if gas is mixed, it is allowed as long as it 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.
Special objects include cylindrical, cylindrical, cubic, rectangular,
It can also have a saddle shape or the like.

[0018] The large-pore porous microcarrier used in the present invention can be produced 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 or reducing its dissolution ability. Manufactured by a method that causes the loss of 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.
Small droplets with a uniform diameter can be easily produced, and the particle size can be controlled as desired. The diameter and shape of the vacuole are basically determined by the size and shape of the solvent crystals that form 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.

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

[0020] In principle, microcarrier culture is a very flexible technique and the culture can be carried out in virtually any cell culture vessel. Microcarriers are suitable for use in microcarriers such as Petri dishes, plastic bags, tubes, hollow fibers, spiral films, Jensen spiral films, gyrogens, columns, multiwell trays, multiplates, culture bottles and roller bottles.
et al. , Trend in Biotech. ，
1,105 (1983)) can be used to increase the surface area of conventional containers. However, when used in stirred 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.

[0021] A container for microcarrier culture is selected depending on the purpose of culture and the amount of culture. Laboratory scale microcarrier cultures are typically less than 5 liters and a variety of vessels are used. Large scale microcarrier cultures range from about 5 liters to several thousand liters and require specially designed vessels that can monitor parameters such as gas pressure and pH.

[0022] As described above, in normal culture, the amount of microcarriers used may range from several to several hundred microcarriers/ml. An example of a microcarrier culture tank used for agitation suspension culture is Bellco 500.
Examples include ml spinner flasks. This spinner flask can be used by assembling a device with a temperature sensor, pH electrode, dissolved oxygen electrode, porous Teflon tube, culture medium supply/discharge nozzle, microcarrier sedimentation tube, sample collection port, etc. as necessary. . At the same time, a cell culture controller (System Fermentor)
When using MODEL AIS-12, ASLE, etc., pH can be controlled with CO2 gas or NaHCO3 solution, and dissolved oxygen can be controlled by supplying air or pure oxygen through a porous Teflon tube.

[0023] As cells used for culture, bacteria, yeast, plant cells, etc. can be used, but animal cells mainly including fish, insects, etc. can also be used. Since large-pore porous microcarriers can hold these cells inside the microcarriers, they can be used not only for attachment-dependent cells but also as carriers for floating cells such as hybridomas. When culturing, the cells themselves may be the purpose of production, and viruses and the like for vaccine production may also be the cell growth products that are the purpose of production. In that case, an appropriate cell may be selected as a host. Depending on the purpose, it is also a preferable embodiment to use genetically modified cells into which a gene for producing the cell growth product to be produced is introduced.

[0024] Cultivation is usually carried out by the following method, but the method is not particularly limited. The microcarriers were placed in a suitable glass bottle in Dulbecco's phosphate buffer solution (hereinafter referred to as PB) with calcium and magnesium ions removed.
(S) and stir gently from time to time for several minutes. However, in the case of the large-pore porous microcarrier of the present invention, PB
There is no problem even if calcium ions or magnesium ions are added to S. Gently remove the supernatant and wash the microcarriers with fresh PBS with gentle agitation. Throw away the PBS again and add new PBS at a rate of 1 for 1 g of microcarriers.
Add 00ml and autoclave using pure water (
Sterilize the microcarriers (120°C, 20 minutes). Depending on the surface modification treatment method, the
In some cases, it may be better to sterilize with 0% ethanol. In this case, after washing three times with 70% ethanol in the same way as the PBS washing described above, leave it at room temperature overnight, and then wash it with PBS three more times.
Wash with S. Prior to use, the sterilized microcarriers are allowed to settle and the supernatant is removed, leaving a serum-free solution of approximately 3
Add 100 ml of the medium heated to 7°C to 1 g of microcarriers and wash. (Pharmacia, Mic
rocarrier cell culturepri
There are various methods for adhering cells to microcarriers (ciples and methods).
It is important to bond as uniformly as possible using methods such as 89)).

[0025] The concentration of the cells to be prepared is, for example, 1×
107 to 5 x 108 cells/ml, and the cell concentration at the time of adhesion is, for example,
It may be used at a concentration of about 1×10 5 to 1×10 6 cells/ml. In the agitated 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, such as 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.

[0026] The product may be collected and purified by conventional methods by appropriately selecting and combining filtration, 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 that case. While optimizing culture conditions, etc., check the amount of cell growth products in the replaced culture solution, and collect when the desired amount has been produced. The desired production amount varies depending on the purpose.

[0027]

[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. After 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 and enlarged to an appropriate magnification using a scanning electron microscope, and the diameter of the pores was observed and measured. The aperture diameter and film thickness inside the microcarrier were determined by freezing the microcarrier in liquid nitrogen as described above, cutting it at the same temperature, drying it in a vacuum, and then observing and measuring it with a scanning electron microscope. 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.

[0028]

[Example 1] Using purified linter as a raw material, the viscosity at 5°C was 2 poise, the cellulose concentration was 2%, and the copper concentration was 1.
Using a two-fluid nozzle, spray 500 cc of cellulose copper ammonium solution with 2% ammonia concentration and 4% ammonia concentration with nitrogen gas, -
Silicone oil (SH200, 1.5
CS, 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 to raise the temperature 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 a slight positive charge 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 were composed of vacuoles with a diameter of 20 to 40 μm, and the thickness of the membrane between the vacuoles was 1 to 2 μm.

[0029]

[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: 3 m).
mφ, ABLE), pH electrode (DPAS/325 type,
Ingold), dissolved oxygen electrode (10AN type, ABL
Company E), porous Teflon tube (inner diameter 3 mm, outer diameter 4
mm, length 1m, coiled, ABLE), culture medium supply/discharge nozzle, microcarrier precipitation tube (temperature jacketed precipitation tube, ABLE), sample collection port (inner diameter 3mmφ,
A device equipped with a BLE Inc.) was assembled and used. pH was measured with CO2 gas or NaHCO3 solution, and dissolved oxygen was measured with a cell culture controller (Sy) by supplying air or pure oxygen through a porous Teflon tube.
stem Fermentor MODEL AIS-
12, ABLE Inc.).

2. The microcarrier microcarrier was prepared by adding 2g of the large-pore porous microcarrier of Example 1 according to a conventional method (Pharmacia, M
icrocarrier cell culture
principles and methods) After swelling, it was sterilized and used. 3. Cells are Vero cells (derived from African green monkey kidney)
and HeLa cells (derived from human cervical cancer) were used.

4. Cell adhesion to the adhesive microcarriers was carried out by the Ficoll method (Kobayashi Tissue Culture 15 (1989)). On the other hand, in the 500 ml spinner flask culture tank, 4% Fic
oll (Pharmacia), 5% bovine serum (Flow)
Example 1 large-pore microcarriers and Vero cells (
1 x 108 pieces) were mixed in and adhered while stirring at 10 rpm on a magnetic stirrer. the other 5
00ml spinner plastic culture tank contains 4% Fico.
ll (Pharmacia), 10% bovine serum (Flow)
The large-pore porous microcarrier of Example 1 and HeLa cells (1×10 8 cells) were mixed into 250 ml of MEM Earl's medium (Flow), and allowed to adhere while stirring at 10 rpm on a magnetic stirrer.

5. After completion of culture adhesion, an additional 250 ml of Ficoll-free medium was added to each culture tank, the stirring speed was increased to 25 rpm, and culture was started (37° C., pH 7.4, mixed aeration of sterile air and pure oxygen). From the second day of culture, culture was carried out while continuously replacing the medium. The culture medium containing microcarriers was collected from the first day of culture, and the number of cells on the microcarriers was determined using nuclear staining method (Sanford, K 11 773 J).
．． Nat. Cancer).

[0033] After 16 days of culture, the cell density of Vero cells reached 2 x 107 cells/ml, making it possible to obtain cells approximately 100 times the inoculated cell density. Also,
The HeLa cell density reached 5 x 107 cells/ml, making it possible to obtain about 250 times the inoculated cell density. Furthermore, sections were prepared to observe whether the nuclear staining method could accurately count the number of cells proliferating inside the large-pore microcarriers. The microcarriers on day 16 of culture were washed twice with PBS before and after counting cells using a nuclear staining method. 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 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 can be seen that there are considerably more cells inside the large-pore microcarrier than the number of cells counted by the nuclear staining method.

[0034]

[Comparative Example 1] The same culture tank and cells as in Example 2 were used. The microcarrier is Cytodex I (
Pharmacia) 2g according to the usual method (Pharmac)
ia, Microcarrier cell cult
ure principles and methods
s) After swelling, it was sterilized and used.

[0035] Cell adhesion to microcarriers is Fic
This was carried out by the oll method (Kobayashi Tissue Culture 15 (1989)). In one 500 ml spinner flask culture tank, 4% Ficoll (Pharmacia), 5%
MEM Earl medium (Flow) containing bovine serum (Flow)
Cytodex I and Vero in 250ml
Cells (1 x 108 cells) were mixed and allowed to adhere while stirring at 10 rpm on a magnetic stirrer. The other 500 ml spinner flask culture tank contains 4% F.
icll (Pharmacia), 10% bovine serum (Flow
Cytodex I and Hela cells (1 x 108
) on a magnetic stirrer for 10 rpm.
It was adhered while stirring at m.

After completion of adhesion, add another 250 ml of Ficoll-free medium to each culture tank and increase the stirring speed to 25 ml.
The rpm was raised to start culturing (37° C., pH 7.4, mixed aeration of sterile air and pure oxygen). From the second day of culture, culture was carried out while continuously replacing the medium. From the first day of culture, the culture solution containing microcarriers was collected, and the number of cells on the microcarriers was determined using each staining method (Sanford, K 11).
773J. Nat. Cancer).

[0037] After 16 days of culture, the cell density of Vero cells reached 5 x 106 cells/ml, approximately 25 times the inoculated cell density. In addition, the HeLa cell density was 6 × 10
6 cells/ml, which was approximately 30 times the inoculated cell density. The results of Example 2 and Comparative Example 1 reveal the following. Example 2 has a cell density of at least Ver.
It was 4 times higher in O cells and 8 times higher in HeLa cells. In all cases, it was clear that the large-pore porous microcarrier of Example 1 was superior.

[0038]

[Example 3] 1. Culture tank Bellco 50 as a culture tank for microcarriers
Temperature sensor, pH electrode, 0ml spinner flask
Dissolved oxygen electrode, porous Teflon tube (inner diameter 3mm,
A device having an outer diameter of 4 mm, a length of 1 m, coiled shape (Able Inc.), equipped with a medium supply/discharge nozzle, a microcarrier sedimentation tube, and a sample collection port was assembled and used. pH is CO
2 gas or NaHCO3 solution, dissolved oxygen was controlled with a cell culture controller by supplying air or pure oxygen through porous Teflon tubing.

2. Microcarriers Microcarriers were obtained by adding 2g of the porous microcarriers of Example 1 according to a conventional method (Pharmacia, Micr
oarrier cell culture pudding
After swelling, it was sterilized and used. 3. Erythropoietin-producing genetically modified BHK cells (derived from Syrian hamster kidney) were used as cells.

4. Cell adhesion to the adhesive microcarriers was performed by the Ficoll method (Kobayashi Tissue Culture 15 (1989)). 5
00ml spinner flask culture tank contains 4% Fico
Example 1 in 250 ml of BME medium (Flow) containing ll, 10% bovine serum, 10% trypto-zurinate broth.
Porous microcarriers and erythropoietin-producing genetically modified BHK cells (1×10 8 ) were mixed and allowed to adhere while stirring on a magnetic stirrer at 10 rpm.

5. After completion of culture adhesion, add another 250 ml of Ficoll-free medium to the culture tank, increase the stirring speed to 25 rpm, and culture (37°C, pH 7.4, mixed aeration of sterile air and pure oxygen).
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.
．． Cancer).

[0042] After 16 days of culture, the cell density of BHK cells reached 2 x 107 cells/ml, making it possible to obtain cells approximately 100 times the inoculated cell density.

[0043]

Example 4 In Example 2 and Comparative Example 1, 10 ml of the homogeneous suspension medium was collected from each of the 500 ml spinner flasks in which Vero cells were cultured on the 16th day of culture. Remove the medium from 10 ml of the collected sample, and
Washed twice with BS. Then 1% bovine serum (Flow)
and ME with sufficient amount of herpes simplex virus type 1 added.
Infection of the cells with herpes simplex virus type 1 was caused by injecting 100 ml of M Earl's medium (Flow) into each cell.

Virus was harvested by low centrifugation 24 hours after infection. Virus quantification is performed using plaque
e) method, plaque-forming units (P
FU/ml). In the system using the large-pore porous microcarrier of Example 2, 73×109 PFU/ml
and 9. in the system using Cytodex I of Comparative Example 1.
1 x 109 PFU/ml, and the virus production capacity is approximately 8 times higher when using large-pore microcarriers.
It was clear that it was superior.

[0045]

[Effects of the invention] The method for culturing cells of the present invention has a diameter of 2 μm.
Because larger pore size porous microcarriers are used,
It has good cell adhesion and proliferation, and since cells are cultured inside the microcarrier, the shearing force caused by agitation does not damage the cells. In addition, by effectively utilizing the internal porous surface as a cell adhesion surface, the cell culture concentration can be dramatically increased, and at the same time, the yield of the cell growth product that is the production objective can be greatly improved. In short, according to the method of the present invention, the drawbacks of conventional cell culture methods can be overcome, and the cell culture concentration and yield of cell growth products can be significantly improved.

Claims (1)

[Claims] Claim 1: A method for culturing cells using microcarriers, comprising: a. Using a microcarrier that has a large number of vacuoles separated by a membrane and having a diameter of more than 2 μm, the vacuoles forming a continuous pore structure that communicates with each other through openings in the membrane that separate adjacent vacuoles. , form a suspension in cell culture medium, b
．． inoculating the suspension with cells to form a cell culture; c.
A method for culturing cells, which comprises maintaining the cell culture medium under cell growth conditions.