JPH03292884A - Cell cultivation - Google Patents

Abstract

PURPOSE:To improve adhesiveness, propagability and extensibility, etc., of cell by keeping a specific crosslinked polymer on the surface of at least a part of porous membrane. CONSTITUTION:(A) A polymerizable monomer (e.g. maleic anhydride) having acid anhydride as a functional group is copolymerized with (B) a monomer component containing >=30wt.% divinylbenzene in a molar ratio of B/A=1/1-2/1 to obtain a crosslinked polymer. Next, 7-150wt.% said crosslinked polymer is kept on the surface of at least a part of porous membrane 2 having 0.01-5mum fractionated particle diameter and 10-90% porosity. Then, a culturing region 6 having a cell-adding port 3 and a region 7 having a medium inlet 4 and an outlet 5 are separated by said porous membrane 2, thus the regions 6 and 7 are filled with a liquid medium and necessary numbers of cell are added through the adding port 3, then the medium is flowed in the region 7, thus the medium in the region 6 is adjusted at a fixed temperature to culture cell.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a cell culture method using a porous hollow fiber membrane suitable for culturing cells.

[Conventional technology]

In recent years, bioactive substances such as monoclonal antibodies, interferon, interleukin, and TPA have been used in medicine.
Its usefulness in the industrial field is becoming clear. Accordingly, there is an increasing need for high-density cell culture methods for producing these useful substances in large quantities and at low cost.

In particular, a method by KNAZEK et al. in which cells are attached to the hollow fiber membrane surface, a medium is circulated through the hollow part of the hollow fiber, and nutrients are supplied to the cells through the hollow fiber membrane surface (Japanese Patent Laid-Open No. 49-415
No. 79) is attracting attention as a method for efficiently culturing cells.

The advantage of this hollow system method is that by securing a large surface area within a limited space, it can provide a wide adhesion surface for adherent cells. Through the hollow fibers, various nutrients and oxygen necessary for cell survival and proliferation are supplied, and waste products generated by cell metabolism are removed by diffusion or by accompanying the flow of liquid caused by the pressure difference between the inside and outside of the hollow fibers. It is very easy to do this. As a result, high-density culture of cells becomes possible, and product concentration and productivity can be increased.

Conventionally, there have been examples of using hollow fibers made of cellulose acetate (Japanese Patent Publication No. 54-6634), hollow fibers made of polysulfone (Japanese Patent Application Laid-open No. 130678/1984), etc. for hollow fiber cell culture.

Additionally, a method using a porous membrane made of polyolefin such as polyethylene or polypropylene (Japanese Unexamined Patent Publication No. 63-17
No. 685) has been proposed.

[Problem to be solved by the invention]

However, the porous membranes that have been used for cell culture using conventional porous membranes are often not always sufficient in terms of cell adhesion, proliferation, and survival, and improvements in these areas have been desired. .

For example, polysulfone hollow fibers have superior properties in cell attachment, proliferation, and extensibility compared to cellulose-based hollow fibers. However, compared to the polystyrene dates that are currently widely used,
Cell attachment, proliferation, and survival are not satisfactory.

Furthermore, according to the research conducted by the present inventors, even in porous membranes made of polyolefin, cell adhesion and proliferation were not necessarily sufficient.

On the other hand, to prevent contamination of the culture system with other microorganisms,
Sterilization of the culture vessel is required.

Sterilization can be carried out, for example, by γ-ray irradiation, gas sterilization using a sterilizing gas such as ethylene oxide gas, steam sterilization, or the like.

However, in culture vessels using conventional resinous porous membranes, it has been difficult to perform simple and inexpensive sterilization without impairing membrane performance.

For example, in the method using gamma ray irradiation, the gamma ray irradiation equipment is expensive. In addition, with gas sterilization methods, it is difficult to remove gas components that adhere to porous surfaces after sterilization by aeration, so it is necessary to wash and remove gas components that remain attached to the membrane surface using a medium or water. There is. However, it takes a long time to remove the residual gas to the extent that it does not affect the culture performed after sterilization.

Furthermore, the steam sterilization method itself has the advantage of being simpler and cheaper than the other methods mentioned above, but since the porous membrane is exposed to high temperature conditions, it has problems with heat resistance. Difficult to apply to sterilization process. For example, when a porous membrane made of polyethylene, polypropylene, etc. is subjected to steam sterilization, the porous membrane will shrink significantly and its membrane performance will be significantly reduced.

The purpose of the present invention is to cultivate good cell culture at low running costs by using a porous membrane that has excellent properties necessary for good cell culture, such as cell adhesion, proliferation, and spreadability, and is also applicable to steam sterilization. The object of the present invention is to provide a method that enables culture.

[Means to solve the problem]

The cell culture method of the present invention is a method of culturing cells in a culture region separated from other regions by a porous membrane, in which an acid anhydride is added as a functional group on the surface of at least a portion of the porous membrane. It is characterized by retaining a crosslinked polymer obtained from a composition containing a polymerizable monomer and divinylbenzene.

The porous membrane used in the present invention is a porous membrane made of a polyolefin material such as polyethylene, polypropylene, poly4-methylpentene-1, etc.; polysulfone; polyvinyl alcohol; polymethyl methacrylate; cellulose acetate; It can be obtained by maintaining the union.

For example, a crosslinked polymer can be retained on the surface of a porous membrane made of a polyolefin material by the method described in Japanese Patent Application No. 63-221584.

The pore size of the porous membrane used in the present invention is large enough to ensure a sufficient amount of movement of substances such as culture medium, gases such as oxygen, products, and waste materials, and to prevent cells from leaking. It may be selected as appropriate depending on the culture form. Further, the shape of the hole is not particularly limited.

The pore size of the porous membrane is, for example, a fractional particle size of 0.0.
It is preferably about 1 to 5 μm, and 0.1 to 2 μm
It is more preferable that the amount is within a certain range.

When the porous membrane is made porous by a stretching method, the fractional particle diameter is expressed as the average diameter of the slits between the fibrils of the pores surrounded by the knots and microfibrils.

Furthermore, the porosity may be within a range that allows sufficient flow of the culture medium etc. and maintains sufficient strength for practical use, specifically 1.
It may be 0 to 90%, preferably 40 to 80%. The film thickness may be within a range that can maintain a practically sufficient strength, but it is preferably about 20 to 200 μm.

As the porous membrane used in the present invention, any form such as a hollow fiber, a flat membrane, or a tubular membrane can be used. In particular, hollow fibers produced by the drawing method have a pore structure in which slit-like microspaces (pores) formed by microfibrils and knots communicate with each other three-dimensionally on the membrane surface of the hollow fiber. Because it has a high porosity, when culturing adherent cells using this material, the cells attached to the surface are substantially fully absorbed through the communicating pore structure. It is preferable because nutrients, oxygen, etc. can be uniformly supplied, cell products, metabolic products, etc. can be easily removed, and there is little performance deterioration due to clogging. The manufacturing method is disclosed in Japanese Patent Publication No. 56-52123 and Japanese Patent Application Publication No. 5
This can be carried out according to the method described in Japanese Patent Publication No. 7-42919.

The crosslinked polymer held on at least a portion of the surface of the porous membrane is made from a composition containing a monomer component (A) consisting of a monomer having an acid anhydride structure as a functional group and a monomer component (B) containing divinylbenzene. can get.

As the monomer component (A), for example, maleic anhydride, itaconic anhydride, himic anhydride, etc. can be used.

The molar composition ratio of the monomer component (A) and the monomer component (B) in the composition for forming a crosslinked polymer is B/A=1/1.
It may be about 2/1 to 2/1, preferably 1/1 to 1.5/1.

Incidentally, as the fluorine component (B), divinylbenzene alone, a mixture of divinylbenzene and styrene, a mixture of divinylbenzene and ethylvinylbenzene, a mixture of divinylbenzene, ethylvinylbenzene and styrene, etc. can be used.

When a mixture is used, the proportion of divinylbenzene in the mixture may be 30% by weight or more.

In addition, in the crosslinked polymer formed, it is preferable that the polymerizable child units are substantially uniformly distributed at the molecular level without forming a block copolymer.

In the present invention, "the surface of at least a portion of the porous membrane" refers to a portion or all of the pore surface and the outer surface.

That is, it is sufficient that the crosslinked polymer is substantially retained, and it is not necessarily necessary that the crosslinked polymer be retained on all surfaces, but it is sufficient that the crosslinked polymer is retained at least on the outer surface on the side to which cells are attached. The amount of crosslinked polymer retained on the surface depends on the porosity and pore diameter of the porous membrane, but is approximately 7 to 150% by weight, preferably 5 to 150% by weight based on the weight of the porous membrane.
It is 120%, more preferably 10 to 100%.

Furthermore, "retained" means that the crosslinked polymer is bonded or adhered to the surface of the pores of the porous membrane to such an extent that it does not easily detach during storage or use.

The porous membrane used in the present invention has a crosslinked structure obtained by holding on the surface of the crosslinked polymer obtained using at least the monomer component (A) and the monomer component (B) as described above. The heat resistance of the porous membrane is improved, and the temperature of 120 to 13
Steam sterilization at 0°C is possible, and it also has better cell adhesion than, for example, polyolefin-based porous membranes or porous membranes with a cross-linked polymer made of divinylbenzene retained on the surface. Proliferation and spreadability are obtained.

In addition, in the present invention, the adhesiveness of cells is increased due to the following:
This is thought to be due to the presence of a crosslinked structure containing a polar acid anhydride.

Specific aspects of the culture of the present invention include the type of cells to be cultured,
It is selected as appropriate depending on the type of substance to be transferred through the porous membrane.

According to the present invention, for example, African green monkey kidney cells (V
ero cells), epithelial cells (940C3), mouse fibroblasts (3T3), Chinese hamster ovary cells (CHO), Chinese hamster lung fibroblasts (V-79), cervical cancer cells (
Hela), human fetal lung-derived normal diploid cells (Wl-3
8) of various cells such as adherent cells such as lymphocytes, myeloma cells, white blood cells, etc., and floating cells such as hybrid cells (hybridomas) obtained by cell fusion of these cells with other cells. Efficient culture can be performed.

In addition, in the porous membrane used in the present invention, good cell adhesion and spreadability can be obtained when adherent cells are used.

The medium used in the present invention may be selected depending on the cells to be cultured, and includes, for example, inorganic salts, amino acids, sugars, fatty acids, vitamins, coenzymes, nucleobases, hormones,
A liquid medium in which components selected from albumin, transferrin, various other cell growth factors, components in serum, etc. are dissolved in water is used.

Specific embodiments of the method of the present invention include, for example, the following.

1) As shown in FIG. 1, a culture vessel is prepared in which a culture region 6 having a cell addition port 3 and a region 7 having a culture medium population 4 and an outlet 5 are partitioned with a porous membrane 2, and the regions 6 and 7 are separated by a porous membrane 2. With the inside filled with a liquid medium, a required number of cells are added from the cell addition port 3, and the culture medium in the culture area 6 is adjusted to a predetermined temperature to perform culture. At that time, the medium is allowed to flow in from the medium outlet 4, and the culture solution is allowed to flow out from the medium outlet 5 to circulate within the region 7. The culture medium flowing out from the culture medium outlet 5 may be returned to the culture medium population 4 and circulated within the region 7. Substances such as nutrients and oxygen necessary for the growth and maintenance of cells in the medium are supplied from within region 7 to region 6 via porous membrane 2 and utilized for cell proliferation. In addition, products, waste products, etc. generated in the region 6 as the cells proliferate are discharged into the region 7 through the membrane wall, allowing efficient culture.

2) As shown in FIG. 2, a culture vessel is prepared in which a culture region 6 having a cell addition port 3 and a medium outlet 5 and a region having a medium population 4 are partitioned by a porous membrane 2, and a liquid is placed in the region 6.7. With the medium filled, the required number of cells is added through the cell addition port 3, and the temperature of the medium in the culture area is adjusted to a predetermined temperature to perform culture. At that time, the medium flows into the region 7 from the medium population 4 and flows out from the medium outlet 5. The medium outlet 5 may be provided with a filter or the like to remove outflow of cells, if necessary. In addition, the culture medium flowing from the culture medium outlet 5 is
The medium may be returned to the culture medium 4 and circulated within the incubator 1.

When using a hollow fiber membrane as the porous membrane, for example, a region communicating with the hollow part of the hollow fiber membrane and a region in contact with the outer wall of the hollow fiber membrane 6 are formed, and these two regions are separated from each other as shown in FIGS. It is used as area 6.7 shown in the figure.

An example of a case where a hollow fiber membrane is used and the area in contact with the outer wall of the hollow fiber membrane is used as a culture area is shown below.

In Figure 3, both ends of the hollow fiber membrane 9 are fixed with botting material,
End conduit 10 communicating with the space connected to the hollow part of the hollow fiber
．． 11 can be used for supplying nutrients and oxygen and removing waste products.

In addition, the side conduits 12 and 13 are the inlet for cells at the start of culture, and when culture is continued for a long time and the cell concentration increases, it is necessary to drain the medium containing waste products and dead cells from here. can.

FIG. 4 shows a cell culture device incorporating the cell culture device shown in FIG.

Above, we have described the case where two areas partitioned by a porous membrane are used, but two or more areas, the culture area and the area partitioned by the porous membrane, are provided, and culture medium components and gases such as oxygen are supplied separately. Alternatively, the area may be divided into an area for supplying the culture solution and an area for removing products and wastes.

Furthermore, two or more culture areas may be provided.

〔Example〕

The present invention will be explained below using examples, but the present invention is not limited to these examples.

In addition, in the examples, porous membranes in which slit-like spaces (pores) formed by microfibrils obtained by a stretching method and knots are three-dimensionally connected were used as porous membranes. The pore diameter of the porous membrane was expressed by the average width and length of the slit-like spaces.

The amount of cross-linked polymer retained is determined by the dissolution fractionation method in which the porous membrane is dissolved under tetralin reflux and expressed as weight % of the porous membrane.
It was displayed in

Example 1 Polyethylene porous hollow fiber (slit-like pore width 0.8
μm, length 2.2 μm, porosity 70%, film thickness 55 μm,
Styrene (s) with a concentration shown in Table 1
t) After dipping for 10 seconds in an acetone solution containing divinylbenzene (DVB), maleic anhydride as a polymerizable monomer, and 0.2% by weight of benzoyl peroxide, the sample was air-dried at room temperature for 30 minutes to volatilize the acetone. Then, it was heated in a nitrogen atmosphere at 60° C. for 20 minutes to polymerize the monomers and hold the crosslinked polymer on the pore surface. Table 1 shows the amount of polymer retained in the porous membrane.

The 150 porous hollow fibers thus obtained were bundled together to form a polycarbonate cylindrical container (inner diameter 8 mm x effective length 150 mm).
mm) and fixed both ends of each hollow fiber with urethane resin so as to keep both ends open, thereby creating a cell culture vessel 8 as shown in FIG. 3.

After steam sterilizing this cell culture device at 120℃ for 30 minutes, 70%
After moistening with ethanol and thoroughly washing with sterile distilled water, the cell culture device was connected to a culture medium reservoir [16] using a silicone tube 14 that had been sterilized separately by steam, thereby producing a cell culture device as shown in FIG.

This medium storage tank 16 contains Han' containing 10% tallow serum.
sF-12 medium 21 was placed in the flask, and air containing 5% CO2 was supplied through a filter 17.

Next, Chinese hamster ovary-derived CHO-Kl (ATCCCCCL-
61) Transfer the cells (5 x 105 cells) to the side conduit 12 in Figure 3.
After sealing, culturing was started.

For culturing, the cells are uniformly dispersed in the incubator by rotating the entire incubator, and after standing still for 3 hours, the device umbrella is
Place it in a constant temperature bath at 7℃, and pump 2.5℃ of culture medium from pump 15.
The experiment was performed while circulating at mIL/hr. On the 3rd day, the medium was stored [15] After replacing the medium with a new medium, the flow rate was increased to 5 m
After replacing the medium with fresh medium once more on the 6th day, the flow rate was increased to 25 m it/hr and culture was continued.

On the 8th day after the start of culture, the culture vessel was filled with Dulbecco's PBS (
-), trypsin 0.25%, EDTAo, 0
Cells were collected in Dulbecco's PBS (-) containing 2%, and the number of cells was measured using a cell counter. In addition, the recovered cells were stained with 0.025% Trivan Blue to determine the cell survival rate and the number of living cells was calculated. As a result, the number of living cells in the culture vessel was 5.2×10′.

Example 2 Polyethylene porous hollow fibers similar to those used in Example 1 were mixed with divinylbenzene (DVB) at the concentrations shown in Table 1, maleic anhydride as a polymerizable monomer, and 0.2% by weight of benzoyl peroxide. After immersing the fibers in an acetone solution for 10 seconds, a porous hollow fiber having a crosslinked polymer retained on its surface was obtained in the same manner as in Example 1.

A culture device was prepared using the obtained hollow fiber in the same manner as in Example 1, and one cell was cultured in CHO- under the same conditions. The number of cells on the 8th day after the start of culture was measured in the same manner as in Example 1, and was found to be 5.4 x 107 cells.

Table 1 Comparative Example 1 150 polyethylene hollow fibers similar to those used in Example 1 (without holding a crosslinked polymer) were bundled and filled into a polycarbonate cylindrical container (8φ x 150mm), and urethane A cell culture vessel was prepared using resin as a potting material and fixed at both ends.

The cell culture vessel was sterilized with ethylene oxide gas, made hydrophilic with 70% ethanol, and then thoroughly washed with sterile distilled water.

Using this cell culture device, a cell culture device having the configuration shown in FIG. 4 was prepared in the same manner as in Example 1, and CHO-Kl was
As a result of culturing the cells, the number of viable cells after 8 days was 1.7.
There were 107 pieces.

Example 3 Using the culture apparatus prepared in Example 1, culture of CHO-Kl cells was started under the same conditions as in Example 1, and then the culture was continued for 1 time.
Stop after 7 hours, remove the hollow fiber from the incubator, and incubate with PB.
After washing with S (-) buffer, the hollow fibers were stained with Kimza staining, and the proliferation morphology of cells attached to the outer wall surface of the hollow fibers was observed using a stereomicroscope. It was confirmed that it was attached.

Comparative Example 2 Using the culture device prepared in Comparative Example 1, CHO was grown under the same conditions.
- Culture of Kl cells was started. After 17 hours, the hollow fiber was taken out and stained with Giemsa in the same manner as in Example 3, and the growth morphology of the cells was observed. Although the cells were attached, the degree of extension was small compared to Example 3. The number of attached cells was also small.

〔Effect of the invention〕

As described above, in the cell culture method of the present invention, a crosslinked polymer obtained from a composition containing divinylbenzene and a polymerizable monomer having an acid anhydride as a functional group on the surface of the porous membrane is used as the porous membrane. Because it uses a membrane that maintains coalescence, steam sterilization before culturing is possible, and compared to membranes made of other materials, it has improved cell proliferation, adhesion and spreadability when using adherent cells. Therefore, this method has the characteristic that good cell culture can be performed.

[Brief explanation of drawings]

1 to 3 are schematic sectional views showing an example of a cell culture device that can be used in the present invention, and FIG. 4 is a diagram showing an example of a cell culture device using the cell culture device shown in FIG. 3. It is. In the figure, 1, 8: cell culture vessel, 2: porous membrane (plain clothes), 3: cell addition port, 4: medium inlet, 5: medium outlet, 6
: Culture area, 7: Area, 9: Porous hollow fiber membrane, 10, 1
1: End conduit, 12° 13: Side conduit, 14: Silicone tube, 15: Tube pump, 16: Medium storage tank, 1
7°18: Insert the filter.

Claims (1)

[Scope of Claims] 1) A method for culturing cells in a culture region separated from other regions by a porous membrane, wherein an acid anhydride as a functional group is provided on the surface of at least a portion of the porous membrane. A cell culture method characterized in that a crosslinked polymer obtained from a composition containing a polymerizable monomer and divinylbenzene is retained. 2) The cell culture method according to claim 1, wherein the porous membrane has a hollow fiber shape.