JPS62215386A - Culture of adhering animal cell and equipment therefor - Google Patents

Abstract

PURPOSE:Adhering animal cells are allowed to adhere to a porous substance impregnated with the culture solution and cultured to enable the high-density cultivation of adhering animal cells in a large amount. CONSTITUTION:The culture solution in the tank 3 is fed through pipe 23 to a wetted-wall column 2 to absorb the gas fed from gas bombs 4 such as air bomb 41 and carbon dioxide bomb 42 and sent through the circulation pipe 21 into the cultivation tank 11. Then, a porous substance such as polyurethane foam 110 which has pores of 10mum-5mm average diameter, 0.02-0.1 apparent specific gravity and 10-50 times foaming degree is placed on the perforated plate 11 in the tank 1 and impregnated with the cultivation solution. The remaining culture solution is passed through the circulation path 12 to the trap 20 equipped with a laminar flower 201 and allowed to flow down on the walls in the column 2 and recycled. Then, adhering animal cells such as kidney cells of African green monkey are allowed to adhere to the substance 110 about 7.5X10<6>cells/cm<3>.

Description

[Detailed Description of the Invention] U's Turn, Now■ (Industrial Application Field) The present invention relates to a high-density culture method for adherent animal cells in which adherent animal cells are cultured using a porous substance as an adherent object, and a method for culturing adherent animal cells at a high density. Regarding a culture device.

(Conventional technology) Animal cells are roughly divided into two cell groups based on their culture form.

One type is floating cells, typified by blood cells, and the other type is adherent cells, such as fibroblasts and epithelial cells, which require a surface to adhere to. Since planktonic cells grow while suspended in a medium, they can be easily grown by a method similar to the culture of microorganisms. Adherent cells, on the other hand.

Since it requires an attachment surface (object to be attached) that serves as a scaffold for proliferation, it is difficult to culture in large quantities and at high density.

In recent years, microcarrier culture methods, holofiber culture methods, ceramic porous tube culture methods, microcapsule culture methods, and the like have been proposed as methods for culturing adherent animal cells in relatively large quantities and at high density. Among these, the microcarrier culture method is preferably used because it does not require complicated equipment and is relatively easy to operate. The microcarrier culture method uses objects with a diameter of 100 to 3
This method uses polymer fine particles (microcarriers) of about 00 μm. Microcarrier materials include polyacrylamide and dextran.

Examples include gelatin and polystyrene. For example, a surface-charged microcarrier (Cytodex 2; manufactured by Pharmacia) by introducing a bridge H and l- is preferably used. The culture density in the microcarrier culture method is 9. For example, the above Cytodex 2
When culturing Vero cells (described later) using 3 μg per 1 mJ of culture solution, approximately 2.0 × 10 h to 4
．． It has been reported that OX 10' pieces/IIIl (microcarrier solid content) [Microcarrier Cell Culture Principles and Methods, Pharmacia Fine Chemicals (Micro
carrier cell culture pudding
ciples & nethods, Pharmaci
a Fine Chemicals, ) ) *However, with this microcarrier culture method, it is still not possible to achieve sufficiently high-density and large-scale culture.

There is a desire for the development of a method capable of culturing adherent animal cells at high density and in large quantities for purposes such as obtaining trace amounts of physiologically active substances contained in animal cells.

(Problems to be Solved by the Invention) An object of the present invention is to provide a method for culturing adherent animal cells at high density and in large quantities. The purpose of the present invention is to provide a culture device that can absorb water.

(Means and effects for solving the problem) The method for culturing adherent animal cells of the present invention involves impregnating a porous substance with a medium solution,
Alternatively, it includes immersing a porous material in a medium solution, allowing adherent animal cells to adhere to the porous material, and culturing the adherent animal cells.

This achieves the above objective.

The adherent animal cell culture device of the present invention includes a culture tank in which adherent animal cells that grow attached to a porous substance are accommodated on a perforated plate together with the porous substance, and a culture tank is connected to the culture tank via a medium circulation path. and a wet wall tower connected to the tower.

It has a culture medium supply tank connected to the wet wall tower via a culture medium supply path and a gas supply means connected to the gas supply path, and the medium liquid and the gas are supplied to the wet wall tower. The above object is achieved by bringing the culture medium into contact with the culture medium and supplying the gas-supplied culture medium to the culture tank through the culture medium circulation path.

The porous substance used in the present invention only needs to be a substance that is not metabolized by the animal cells to be cultured and has a water-absorbing ability. For example, natural products such as synthetic resin foams and sponges can be used. As the synthetic resin foam, for example, a foam such as polyvinyl alcohol or polyurethane is used. especially.

A polyurethane foam having a polyoxyethylene structure is preferably used because it has excellent hydrophilicity and water retention ability.

Such polyurethane is obtained by reacting a polyol having a polyoxyethylene structure with a polyisocyanate. Examples of polyols having a polyoxyethylene structure include 9, for example, polyethylene glycol; a mixture of polyethylene glycol and polypropylene glycol;
Examples include polyethylene glycol-polypropylene glycol, which is a copolymer of ethylene oxide and propylene oxide. Basic polyols can also be used.

The basic polyols mentioned here include ethylenediamine,
diethylenetriamine, methylamine, butylamine,
piperazine, ethanolamine, propatoolamine,
It is obtained by adding ethylene oxide to an amine such as N-methyljetanolamine in the form of polyoxyethylene.

As the polyol having a polyoxyethylene structure, polyethylene glycol having a molecular weight of about 400 to 1,000 is preferably used. When the polypropylene component is added to polyethylene glycol, its content is about 6
Preferably it is less than 5 parts by weight. If it is in excess, the hydrophilicity of the resulting foam will decrease.

Examples of the above polyisocyanates include aromatic,
Various polyisocyanates such as aliphatic and polyether are used. For example, tolylene diisocyanate,
Diphenylmethane diisocyanate, diphenyl diisocyanate, naphthalene diisocyanate, xylene diisocyanate butane diisocyanate, triphenylmethane-4-4'4'-) diisocyanate, and the like.

A polyol without a polyoxyethylene structure can be used alone or in combination with the polyol having a polyoxyethylene structure. Examples of such polyols include glycerin, trimethylolethane,
Trimethylolpropane, 1,2,6-hexandriol, pentaerythritol, sorbitol, sucrose.

α-methyl glucoside is mentioned. Natural polymers such as cellulose, carboxymethylcellulose, and hydroxymethylcellulose and derivatives thereof can also be used as polyols.

During the reaction between polyols and polyisocyanates.

When a bebutide such as collagen, albumin, or gelatin is allowed to coexist in the reaction system, a polyurethane in which a peptide matrix is formed within one molecule can be obtained. When a polyurethane foam with a peptide matrix formed within its molecules is used as a porous material, the moisture content of the porous material is high (
This results in good wetting between the porous material and the culture medium. As a result, the affinity between adherent animal cells and porous materials increases.

The cells proliferate more rapidly.

As the foam, both semi-open foam and open foam can be used. It is preferable to use open foam because the easier the contact with the oxygen-containing medium, the better.

The average pore diameter of the porous material is 10 μm to 5 mm, preferably 100 μm to 1 mm. If the pore size is too small, it will be difficult for cells to grow inside the porous material.

If it is too large, the internal surface area of the porous material becomes small. The apparent specific gravity of such porous materials is 0.02 to 0.1
If it is a 9 foam, the foaming ratio is about 10 to 50.
It's double.

Methods for culturing adherent animal cells using the porous material of the present invention include static culture methods and packed bed culture methods.

In order to carry out the culture method of the present invention, it is necessary to provide a culture tank in which adherent animal cells that grow attached to a porous material are housed on a perforated plate together with the porous material, and a culture medium circulation path in the culture tank. An apparatus is used which is equipped with a wetted wall column which is connected to the cell via a wetted wall column and which supplies gas to the culture medium.

A culture medium supply tank is connected to the wet wall tower.

To perform static culture, place 2, for example, 10 cells on a perforated plate in 8 culture tanks.
A porous material containing 1 to 3 pieces of sodium bicarbonate, preferably 1 to 3 pieces of baking soda, is placed, and a culture medium is supplied to impregnate the porous material. Inoculate (seeding) cells.

The amount of the medium is not particularly limited as long as it is at least the amount that the porous substance can absorb. Since the culture is static, the culture medium is not circulated, but it is preferable to replace the culture medium once every 1 to 3 days.

When cultured at a temperature and atmosphere necessary for cell growth, the animal cells rapidly proliferate using the porous material surface as a scaffold. The larger the volume of the medium relative to the porous material, the higher the growth rate of the animal cells, and the higher the saturation density of growth of the animal cells in the porous material. For example, Vero cells described below are cultured using polyurethane foam (PUF 23■).

Medium 2IIll) and 7.5X10' pieces/d! A high saturation density of (PUP in medium solution) can be obtained.

To carry out the packed bed culture method, porous substances are stacked and packed in multiple layers on a perforated plate in a culture tank, and a gas-containing medium solution from a wet wall column is supplied thereto at a growth temperature.

Inoculate desired animal cells. Then, the gas-containing culture medium from the wet wall tower is continuously supplied through the culture medium circulation path. According to such a packed bed culture method.

Since the animal cells are supplied with a medium containing sufficient oxygen and other necessary gases, high-density culture is achieved. For example, when Vero cells are cultured using 5.8 g of PUP while sufficiently supplying air containing CO1 into the medium, the cell number doubling time in the logarithmic growth phase is 71 hours, and 2
On the fourth day, the cells proliferated 75 times and a high density culture of 1.7×10' cells/d was achieved.

Using the above device, a continuous culture method can be achieved by continuously supplying the culture medium and continuously removing the obtained culture products such as physiologically active substances from the system. As a means for taking out the culture product, for example, the culture tank may be provided with a medium solution outlet with pulp. It is also possible to use an ultrafiltration membrane or the like that selectively takes out low-molecular substances.

Agitation culture can be achieved by stirring slowly enough to prevent adherent animal cells from detaching from the surface of the porous material. In this case, the degree of contact between the cells and the medium containing a large amount of oxygen increases, so the growth rate of the cells becomes faster, and the culture products effectively diffuse into the medium, increasing the yield of the product. will improve. A stirring means is appropriately provided within the culture tank.

(Experiment example) Preliminary experimental examples for implementing the present invention are shown below.

The cells used here are Vero cells (their origin and characteristics are shown below).

Vero cells: Adherent cells derived from the kidneys of African amputial monkeys. The morphology is fibroblast-like. It is an established cell line.

Proliferate infinitely. The spherical cells have a diameter of approximately 13.5 μi.

Used for the production of viruses such as SV40. When cultured using the bottom of the Petri dish as the attachment surface, the cells will double in about 18 hours.

Na) Ices on DME synthetic medium (manufactured by Nippon Pharmaceutical Co., Ltd.) on a large plate.
HEPIES.

Add penicillin G and streptomycin.

Final concentration of NaHCOs 12.5mM, HEPES
After adjusting the concentration to 5mM, penicillin G10'U/l, and streptomycin O, Lg/12, fetal bovine serum (FBS) was further added to prepare a medium solution.

Pt1F with a peptide matrix formed within the molecule
(expansion ratio: 25 times) was applied to a mixer and ground into pieces of 311 or less. After removing impurities and washing with distilled water, sterilization was performed in an autoclave. This was immersed in the above medium solution, and the attached glycoproteins in the serum were sufficiently adsorbed onto the PUF surface. Put this PUP and the above medium into a Petri dish in a clean bench.

Vero cells were seeded into petri dishes. 5 for this
The cells were incubated at 37° C. with air containing % COW. After several hours, it was confirmed by a microscope that Vero cells had attached and spread to the bottom of the Petri dish and the PUF/surface 6. It was also confirmed that cells proliferated over time. It was finally confirmed that Vero cells had grown to saturation on the PUF surface. In the latter half of the stationary phase, the cells became round and aggregated to form large cell clusters. When you enter the annihilator.

The cells on the bottom of the Petri dish were detached, and the Vero cells on the PUF surface also formed a significantly large aggregate.

Disaster Burning 1 The same culture medium as in Experimental Example 1 was used, and PUP treated in the same manner and with the same attached glycoprotein adsorbed was used.

This PUF and culture medium were placed in a Vetri dish with a diameter of 5311, and air containing 5% COt was added at 31/win.
The ink was incubated at 37°C for 30 minutes while feeding at a rate of . PUF 40■ treated in this way and culture medium 4f
Three sets of Vetri dish medium containing f11 were prepared.

104 cells/m j for these three sets of PUPs in the medium! ,
V at a rate of 10' pieces 7 ml and 106 pieces/la1
ero cells were seeded respectively. Here, 104 pieces/l
ll1 is 10 per r'UF1cd containing medium solution.
' indicates that cells were seeded. This was cultured for 38 hours according to Experimental Example 1, and the number of cells was counted. Seeding density and P
The relationship with the number of cells per UP Ig is shown in FIG. From Figure 1.

It was confirmed that the rate of cells adhering to PUP was almost constant regardless of the seeding density.

(Example) The present invention will be described below with reference to Examples.

The same medium solution and the same PuF as in Experimental Example 1 were used on surface spots. A petri dish medium containing 23 mg of PIF and 2 mf of medium was prepared, and Vero cells were seeded on the put surface at a rate of 1.04 x 10'' cells/g (PUF) (7). Containing 5% Cow. 3 j the air!
After incubating at 37°C for 23 hours while supplying the cells at a rate of 100 μl/min, sample the cells carefully to prevent them from detaching from the PUF surface, and count the Vero cells attached to the PIIF using the multiplication counting method. did. The number of cells was approximately 4.5 x 106, and it was observed that the number of cells was almost saturated on the PUP surface. This is almost the same number of cells when culturing was carried out using the bottom surface of a Vetri dish of the same size as the attachment surface. The PUF used was 23
■ Therefore, the cell density is 1.95 x 10' cells/g
(PUF). The bulk volume of PUP in the medium is approximately 2
Since it is 6cJ/g, what is this? , 5 x 10' pieces/
- corresponds to (PUF in the medium). This is 2 to 3 times better than the microcarrier culture method using Cytodex 2 shown in the prior art section. In this way, high-density culture can be effectively achieved by the method of the present invention.

Chiasel Spot 1 The same medium solution and the same PIIF as in Experimental Example 1 were used. pup
A Vetrizoisossu medium containing 2raβ medium in 90+++g, a Vetrizoisossu medium containing 6ml of the medium in PUF 90■, and a Vetrizoisossu medium containing only 5ml of the medium were prepared. 8 x 10', 8 x 10' and 2 x 10' Vero cells were seeded therein, respectively, and cultured according to Experimental Example 1. FIG. 2 shows the relationship between culture time and cell number. P
The doubling time (D, T,) in the logarithmic growth phase during the above culture using UF was measured. Separately, add the amount of culture medium to 41
I11, and the PUFs to be added are θ■, 50■, 70■,
Experiments were conducted with 190 ml of each sample, and R and T during the logarithmic growth phase were measured. Percentage of PUF in the culture medium (
g/1) and T, is shown in Figure 3.

From Figures 2 and 3, it can be seen that the larger the volume of medium is for pup, the faster the cell growth rate (, D, T, is smaller). This is thought to be due to inhibition of material exchange of cells growing on the PUF surface.When cultured in a Petri dish without PUF, the proliferation rate is high, but the bottom surface of the Petri dish is saturated in a short time, resulting in a high density. cannot be cultivated.

Emperor ±1 The culture device includes a culture tank 1, as shown in FIG.

It has a wet wall tower 2, a culture medium supply tank 3, and a gas supply means 4. The culture tank 1 houses a perforated plate 11 therein, and a PUF 110 is placed on the perforated plate 11.

A wetted wall tower 2 is connected to the culture tank 1 via a culture medium circulation path 12. This wet wall tower 2 has a culture medium supply channel 23.
The medium liquid supply tank 3 is connected to the gas supply means 4 via the gas supply path 24 .

In the wet wall tower 2, the returned medium (or culture liquid) from the culture tank 1 flows in from the medium liquid inlet 20, and while flowing down along the inner wall surface of the tower, a gas supply means (for example, an air cylinder 41 and a COt gas cylinder) is used. 4) and absorbs the gas. The culture medium that has absorbed fresh gas passes through the culture medium circulation path 21 and returns to the culture tank 1.
supplied to In wet wall tower 2, an effective gas supply system functions as described above, and useful gas is supplied to culture tank 1.
will be supplied again. The culture medium inlet 20 of the wet wall tower 2 has a liquid reservoir part 200 along the wall surface of the wet wall tower, and the culture medium returned from the culture tank 1 enters this liquid reservoir part 200 and gradually overflows from the tower wall. The structure is such that it flows into the tower. In order to maintain a more uniform flow rate from the liquid distillation section 200 into the tower, a laminar flow creation means 2 such as beads such as glass beads or a perforated plate is used.
01 may be placed within this liquid reservoir 200. A condenser 202 and a filter 203 are provided above the wet wall tower 2. A filter 240 is also provided in the gas supply path 24 as required. In the culture medium circulation path and the culture medium supply path, liquid supply means 60 and 61 such as roller pumps are appropriately arranged.

For example, a culture solution outlet with a valve is provided in the culture tank 1 so that the above device can be used for continuous culture in which products such as physiologically active substances contained in the culture medium can be continuously taken out of the system.
00 is set. Together with or in place of this outlet 100, it is also possible to arrange an ultrafiltration membrane for selectively removing low-molecular substances in the culture medium circulation path or other appropriate location.

The above device can be used for culture with stirring.

It is also possible to arrange the stirring means within the culture tank 1, for example.

After the medium liquid from the medium liquid supply tank 3 was supplied to each part of the above apparatus via the medium liquid supply path 23 in the following manner, the valve 101 of the medium liquid supply path 23 was closed. And culture tank 1
The culture medium was circulated between the and wet wall tower 2 through the culture medium circulation paths 12 and 21 at a speed of 10On+17ml for 1 hour.

PUP 5.8g in culture tank 1 and culture medium 2001I
200 nIl of culture medium in wet wall tower 2; and culture medium circulation path 12.21 and roller bob 60
Add a total of 100 ml of culture medium to PUF 1.
10 cells were seeded with 3.4 x 10' Vero cells, and 2
After standing for an hour, 50 nj! Filled bed culture was started by circulating the medium solution at 1/111n. FIG. 5 shows the relationship between the culture time and the number of cells per 1ffi PUF unit.

In FIG. 5, the arrows indicate opening the valve 100 of the culture tank 1 to recover the culture medium in the culture tank 1, and also releasing the culture medium from the culture medium supply tank 3 by opening the valve 101 of the culture medium supply path 23 to recover the medium in the culture tank 1. Indicates that it was supplied to each part of the device according to the instructions.

From FIG. 5, it can be seen that the number of cells after 150 hours of culture was 3×10′ cells, 7 g, indicating that high-density culture was performed.

(Effects of the Invention) According to the present invention, adherent animal cells can be effectively cultured using a porous substance as an adherent object. Even higher density culture can be achieved than conventional methods such as microcarrier culture and holofiber culture. According to the present invention, cells can be effectively grown using either the static culture method or the packed bed culture method. In particular, when a packed bed culture method is adopted, it is easy to scale up the culture, and high-density and large-scale culture is possible. Porous materials such as polyurethane foams are also inexpensive, allowing large quantities of adherent animal cells to be cultured inexpensively. The present invention can be suitably used for research on viruses using adherent animal cells, and for the production and research of physiologically active substances produced by adherent animal cells. In addition, the porous material such as pup allows cells to be easily peeled off after culturing, and is reusable.

4. Figure 1 is a graph showing the relationship between the seeding density and the number of cells per unit volume of porous material when adherent animal cells are statically cultured by the method of the present invention. The figure is a graph comparing changes in cell number with culture time when adherent animal cells were statically cultured using the method of the present invention and the conventional method, respectively.
Figure 3 shows the weight of porous material per unit medium during static culture according to the method of the present invention. , the fourth graph showing the relationship between
The figure is a schematic diagram showing one embodiment of the culture apparatus of the present invention, and FIG. 5 is a graph showing changes in the number of animal cells with culture time when packed bed culture is performed using the apparatus of FIG. 4.

1... Culture tank, 2... Wet wall tower, 3... Culture medium supply tank.

4... Gas supply means, 11... Perforated plate, 12.2
1... Culture medium circulation path, 20... Culture liquid inlet, 23
...Medium solution supply path.

110...PUF, 200...Liquid distillation section.

Arubi t4Ivl Figure 3 昂Z4藺

Claims (1)

[Claims] 1. A porous substance is impregnated with a medium solution, or a porous substance is immersed in a medium solution, adherent animal cells are attached to the porous substance, and the adherent animal cells are cultured. A method for culturing adherent animal cells comprising: 2. The culture method according to claim 1, wherein the porous material is a polyurethane foam. 3. The culture method according to claim 2, wherein a peptide matrix is formed within the polyurethane molecules. 4. The culture method according to claim 3, wherein the peptide forming the peptide matrix is collagen, albumin, or gelatin. 5. The culture method according to claim 2 or 3, wherein the polyurethane foam has a foaming ratio of 10 to 20 times. 6. The adherent animal cells are African green monkey kidney cells (Vero cells), normal bat lung cells (TblLu)
, epithelial cells (940c3), mouse fibroblasts (3T3)
), human striated muscle sarcoma cells (RD), human striated muscle sarcoma cells (A204), primary chick embryonic fibroblasts, Chinese hamster ovary cells (CHO), infant hamster kidney cells (BHK), Chinese hamster ovary cells ( CHO), primary chick embryo fibroblasts, primary fetal cheek cells, human diploid cells (W1-38.MRC-5), monkey kidney cells (Vero, LLC-MK2, CV-1), primary monkey kidney cells, human Embryonic lung cells (MRC-5), rabbit kidney cells (
RK-13), infant hamster kidney cells (BHK), pig kidney cells (IBR-52), dog kidney cells, infant hamster kidney cells (BHK), cat lung fibroblast-like cells, or fish cells. The culture method described in Section 1. 7. The culture method according to claim 1, which is a static culture method, a packed bed culture method, a continuous culture method, or a stirring culture method. 8. A culture tank that accommodates adherent animal cells that grow attached to a porous material on a perforated plate together with the porous material, and a wet wall tower connected to the culture tank via a medium circulation path; It has a culture medium supply tank connected to the wet wall tower via a culture medium supply path and a gas supply means connected to the gas supply path, and the medium liquid and the gas are supplied to the wet wall tower. A culture device for adherent animal cells, in which a culture medium supplied with gas is supplied to a culture tank via the culture medium circulation path. 9. The culture device according to claim 8, wherein the porous material is a polyurethane foam. 10. The culture device according to claim 9, wherein a peptide matrix is formed within the polyurethane molecules. 11. The culture device according to claim 10, wherein the peptide forming the peptide matrix is collagen, albumin, or gelatin. 12. The foaming ratio of the polyurethane foam is 10 to 20.
The culture device according to claim 9 or 10, which is twice as large. 13. The adherent animal cells are African green monkey kidney cells (Vero cells), normal bat lung cells (TblLu
), epithelial cells (940c3), mouse fibroblasts (3T
3), human striated muscle sarcoma cells (RD), human striated muscle sarcoma cells (A204), primary chick embryo fibroblasts,
Chinese hamster ovary cells (CHO), infant hamster kidney cells (BHK), Chinese hamster ovary cells (CHO), primary chick embryo fibroblasts, primary fetal cheek cells, human diploid cells (W1-38.MRC-5) , monkey kidney cells (Vero, LLC-MK2, CV-1), primary monkey kidney cells, human embryonic lung cells (MRC-5), rabbit kidney cells (RK-13), infant hamster kidney cells (BHK),
11. The culture device according to claim 10, which is a pig kidney cell (IBR-52), a dog kidney cell, a baby hamster kidney cell (BHK), a cat lung fibroblast-like cell, or a fish cell. 14. The culture apparatus according to claim 8, wherein the culture tank has means for recovering physiologically active substances in the culture solution. 15. The culture device according to claim 14, wherein the physiologically active substance recovery means is an ultrafiltration membrane. 16. The culture device according to claim 8, wherein the physiologically active substance recovery means is a culture solution outlet with a valve provided in the culture tank. 17. The culture apparatus according to claim 8, wherein the culture tank is provided with a culture solution stirring means. 18. The culture apparatus according to claim 8, wherein the culture medium inlet of the wetted wall tower has a liquid reservoir along the wall surface of the wetted wall tower. 19. The culture device according to claim 18, which has a laminar flow creating means for making the liquid flow into a laminar flow in the liquid reservoir. 20. The culture device according to claim 19, wherein the laminar flow creating means is a bead-like object.