JPH0310674A - Porous membrane for cell culture and cell culture device using the same - Google Patents

Abstract

PURPOSE:To obtain the subject porous membrane enabling the culture of cells and the recovery of valuable substances in high density and efficiency over a long period and useful for a hybrid-type artificial liver, etc., by forming a cell non-adhesive region and a cell adhesive region on the cell-culture side surface of a membrane. CONSTITUTION:The objective porous membrane has cell non-adhesive regions 5 and cell adhesive regions 4 on the cell culture surface in a state separated from each other in the form of lattice, stripes or sea-island at an areal ratio (non-adhesive/adhesive) of 0.02-20. The thickness of the membrane is 1-500mum (preferably 10-200mum), the porosity is >=20% (preferably 30-80%) and the pore diameter is preferably 0.05-0.5mum. The region 5 is preferably composed mainly of a polyolefin or a modified polyolefin produced by halogenating a part or total of n atoms of a polyolefin.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to a porous material for cell culture and a cell culture vessel using the same. More specifically, the present invention relates to a porous cell culture membrane for culturing attracting-dependent animal cells at high density for a long period of time, and a cell culture vessel using the same.

``Prior art'' Animal cell culture technology is indispensable as a means of producing biologically active substances such as interferon, lymphoid, various growth hormones, and cell growth factors, biologically derived materials, and therapeutic vaccines. In recent years, high-density culture methods that produce these substances efficiently and in large quantities have attracted attention. In animal cell culture, high-density culture of adhesion-dependent cells, which require adhesion to artificial or natural substrates for survival, proliferation, and production of target substances, requires First of all, you need a good surface.

In addition, in order to maintain cell survival and the ability to produce target substances, it is important to maintain a good environment for cells, and it is important to continuously supply nutrients and enzymes and remove waste products. It's coming.

Regarding the former type of cell adhesion, materials whose surfaces have been subjected to surface treatments such as low-temperature plasma treatment, sputtering treatment, ultraviolet treatment, electron beam treatment, and monomolecular cumulative film coating treatment (Japanese Patent Publication No. 58-32589, Kaisho 63-1989
76, JP-A No. 63-198977, etc.), materials treated with cell adhesion factors and cell growth factors such as collagen, fibronectin, vitronectin, and laminin (e.g., Vel, XXXIII Trans, Am, S
oc, Artif, Organs 1987. p
66, 5cho Early Reviews “Ce
1l Adhesion to Biomateria
ss”) is being researched and developed.

On the other hand, methods using porous membranes are considered to be superior in maintaining the growth environment for cells cultured at high density. In other words, by stacking porous flat membranes or bundling hollow fibers, a large surface area can be secured within a limited volume, and the microscopic pores of the porous membrane can be used to supply substances necessary for life activities and remove foreign substances. A good growth environment can be maintained by continuous diffusion through the membrane or by forcing it by a pressure difference between the membranes. An example of this is JP-A No. 63-17686.
(hollow fiber type) and JP-A-60-210982 (laminated type).

``Problems to be solved by the present invention'' The 1N type cell culture device allows cells to be cultured at a higher density than cell culture methods using petri dishes or microcarriers, but it is difficult to culture adhesion-dependent animal cells. When doing so, the inventors often encountered the following problems. In other words, even though cell culture was carried out using a ``membrane with a surface with excellent cell adhesion, spreading, and proliferation'' in order to culture cells at high density on the membrane, the recovery rate of produced substances was not necessarily high. and the inability to maintain cell function. As a result of intensive research by the present inventors, the cause of this problem is that the membrane pores are blocked by the proliferating cells themselves and extracellular substances (such as collagen) synthesized by the cells, which can lead to the supply of necessary substances and the removal of foreign substances. It was found that this was due to the fact that the collection of useful substances could not be carried out smoothly. The present invention was made in consideration of these conventional problems, and has been developed to improve cell adhesion and proliferation. The object of the present invention is to provide a cell culture membrane that has a membrane surface structure and is excellent in supplying necessary substances, removing foreign substances, and recovering useful substances, and a cell culture vessel using the same.

"Means for Solving Problems" The above object is achieved by a porous membrane for cell culture which is characterized by having a cell non-adhesive region and a cell adhesive region at least on the membrane surface on the cell culture side. Furthermore, the above object is achieved by a cell culture device using the porous membrane for cell culture. The shape of the membrane for cell culture is not particularly limited, and examples thereof include a flat membrane, a hollow fiber, and a tube. Furthermore, the cell culture side surface refers to the surface that actually comes into contact with cells.

The ratio and shape of each area on the cell culture side surface are arbitrary, but
Preferably, the area ratio of the cell non-adhesive region/cell adhesive region is in the range of 0.02 to 20, preferably 0.1 to 1.0, and both are divided into, for example, a grid pattern, a striped pattern, or a seabird pattern. It's better to do it before. Also, 11!2 J'! X is 1 to 50
0 μm, especially 10 to 200 μm, porosity 20% or more, especially 30% to 80%, pore diameter 0.01 to 1.0 μm,
In particular, it is preferably 0.05 to 0.5 μm. When the film thickness exceeds 500 μm, the ability to transfer substances through the film decreases, and when it is less than 5 μm, pinholes and strength problems occur. Regarding the porosity, if it is less than 20%, the mass transfer ability will decrease and membrane clogging will easily occur, causing problems in cell growth and recovery of useful substances.

The porous membrane for cell culture of the present invention is characterized in that its main components are polyolefin and polyolefin in which some or all of the hydrogen has been halogenated. For example:
It is a polymer (including homo or copolymer) composed of ethylene, propylene, vinylidene chloride, ethylene chloride, vinylidene fluoride, propylene hexafluoride, Te1-rafluoroethylene, methylpentene, butadiene, etc. These polyolefins do not have functional groups in their molecules, so when designing and synthesizing the molecular structure of the cell-adhesive and non-cell-adhesive regions on the membrane surface, polyolefins have less molecular influence. has advantages.

In addition, untreated polyolefin, in which polar groups have not been introduced to the surface by low-temperature plasma treatment or ozone treatment, has poor cell adhesion and spreading properties, and can be used in the non-cell adhesive region. In that case, it is preferable that the cell non-adhesive region mainly consists of polyolefin and polyolefin in which some or all of the hydrogens are halogenated.

Furthermore, the cell non-adhesive region of the present invention can also have a hydrogel-like surface. A hydrogel-like surface is obtained by insolubilizing a highly water-absorbing polymer or a water-soluble polymer and immobilizing it on the membrane surface, and a hydrogel structure is formed on the membrane surface. Specific methods for forming the hydrogel structure include masking the cell-adhesive region of the porous membrane and then grafting, for example, acrylamide derivatives or (meth)acrylic acid esters to the non-adhesive region; There are methods such as applying polyvinyl ethers and bonding them with covalent bonds, or performing crosslinking and insolubilization treatment to make the coating film insolubilized. Examples of the components constituting the hydrogel include acrylamide, dimethylacrylamide, monomethylacrylamide, ethyl acrylamide,
Polymers containing acrylamide derivatives such as isopropylacrylamide, polyethers such as polyethylene glycol, polyvinyl ethers such as polyvinyl methyl ether, and polymers containing acrylic esters and methacrylic esters as constituent components to form water-absorbing gels. Can be mentioned.

The molecular structure of the cell-adhesive region is not particularly limited as long as cell adhesion and proliferation are superior to that of the non-cell-adhesive region, and the surface may be designed by a known method. - Examples include methods of immobilizing cell-adhesive proteins, peptides, or glycoproteins on the surface, or introducing weakly cationic polar groups. From the viewpoint of practicality, it is preferable to use a membrane for cell culture in which the cell adhesion region contains a compound having a nitrogen-containing heterocycle in the molecule. A nitrogen-containing heterocycle is a compound that has nitrogen in the heterocycle, such as pyridine, pyrimidine, piperidine, pyrrolidone, indole, purine, thiazole, etc. , 1-vinylpyrazole, 5-vinylpyrazoline, 1-vinylimidazoline, 2-vinylpyridine, 4-vinylpyridine, 2-vinyl-4-methylpyridine, 5-bromo-3-vinylpyridine, 3-vinylpiperidine, 4 -vinylpiperidine, 1-vinyl-2-piperidone, vinylpyrrolidone, etc.

"Function" When cells are seeded on the porous membrane for cell culture of the present invention, the cells selectively adhere to the cell adhesion surface and spread and proliferate. Therefore, on the non-cell-adhesive surface, the membrane is less likely to be blocked by cells or intercellular substances synthesized by the cells. As a result, the cell culture membrane of the present invention exhibits excellent performance in supplying necessary substances, removing foreign substances, and recovering useful substances, and cells are cultured in a cell culture vessel using the cell culture membrane of the present invention. If this is done, cell function will be maintained over a long period of time, and a high recovery rate of produced substances will be obtained. Furthermore, by separately forming a cell adhesive region and a cell non-adhesive region in a desired shape on the membrane surface, efficient cell metabolism is achieved.

When a porous membrane for cell culture is made of polyolefin, unlike cellulose-based membranes, it is resistant to swelling in aqueous solvents and enzymatic decomposition, resulting in a porous cell culture membrane with excellent dimensional stability and strength. Furthermore, since polyolefin itself has poor cell adhesion and proliferation, areas that are not subjected to surface treatment can be used as cell non-adhesion areas. Furthermore, since it is a hydrophobic membrane, it can also be used as a cell culture membrane that also has gas exchange ability, or as a cell culture membrane on the gas exchange side.

When the cell non-adhesive region is a hydrogel-like surface, adsorption of not only cells but also proteins, physiologically active substances, etc. to the membrane and clogging of the membrane are suppressed, and useful substances are efficiently supplied and recovered. becomes.

If the cell adhesion region is composed of a compound having a nitrogen-containing heterocycle in its molecule, it will be easier to handle than natural substances such as collagen.

When a compound is used that does not undergo denaturation even during autoclave sterilization and has an aromatic heterocycle in its molecule, the gamma ray resistance improves and gamma ray sterilization becomes possible.

The cell culture device of the present invention fully enjoys the advantages of the above porous membrane for cell culture.

Example 1 A polypropylene sheet (F, OP 60) in which slits with a thickness of 3 m are punched out in stripes at 3 m intervals on a polypropylene sheet (FOP60: Nisho Kagaku Kogyo) with a wall thickness of 50 μm.
and low temperature plasma (Ar, 0.1 torr)
After irradiation for 5 seconds, 4-pyridylethylene gas was supplied and surface graft polymerization was carried out at a temperature of 288 °C for 3 minutes. After peeling off the overlapping polypropylene sheet, the membrane was washed with a solvent (Disolve, manufactured by M Glass Co., Ltd.) for 2 days, dried, and used as a sample.

The sample thus obtained was punched out to a diameter of 29 m, and 5 x 104 liver parenchymal cells separated from an 85-week-old male by the collagenase perfusion method were seeded.
DE containing 10% fetal bovine serum at 37°C in an air atmosphere
(Kyokuto Seiyaku) culture medium for 4 days and observation of cell growth using a phase contrast microscope revealed that cell adhesion regions and cell non-adhesion regions were separated into stripes at approximately 3+n intervals. In this example, the cell adhesion area is the area where a pyridine group, which is a nitrogen-containing heterocycle, is introduced into the surface, and the cell non-adhesion area is an area where the pyridine group is not introduced, which is masked by the stacked polypropylene sheets. there were.

Example 2 The entire surface of a polypropylene sheet (FOP60: Nisho Kagaku Kogyo) with a wall thickness of 50 μm was subjected to surface treatment with 4-pyridylethylene in the same manner as in Example 1, and then one φ5 m hole was formed.
Polypropylene sheets (FOP60) punched out in a polka dot pattern at 0 am intervals were stacked, and low temperature plasma (Ar, 0.1 torr) was irradiated again for 10 seconds, and N,
N-dimethylacrylamide gas was supplied and surface craft polymerization was carried out at a temperature of 288° C. and 0.8 torr for 3 minutes. After peeling off the overlapping polypropylene sheet, the membrane was washed with a solvent (Disolve, manufactured by Asahi Glass Co., Ltd.) for 2 days, dried, and used as a sample.

The sample thus obtained was punched out into a φ29 volume, 5 x 10' liver parenchymal cells separated from an 85-week-old male by the collagenase perfusion method were seeded, and 5% carbon dioxide gas was added to the 95
D containing 10% beef tallow serum at 37°C under an air atmosphere.
As a result of culturing in E (Kyokuto Pharmaceutical) medium for 4 days and observing the growth status of the cells using a phase contrast microscope, it was found that approximately φ51II11
The non-cell adhesion area and the surrounding cell adhesion area were separated into islands. In this example, the cell adhesion region is a region in which a pyridine group, which is a nitrogen-containing heterocycle, is introduced into the surface, and the cell non-adhesion region is a region in which a pyridine group, which is a nitrogen-containing heterocycle, is introduced into the surface.
It was a hydrogel-like surface formed by grafted chains of dimethylacrylamide). Comparative Examples 1-3 Polypropylene sheet (FOP60) and full surface Example 2
Hepatic parenchymal cells were cultured using a polypropylene sheet with a hydrogel-like surface formed by graft chains of poly(N,N-dimethylacrylamide) similar to the above, but the cells did not adhere.

In addition, in the sample in which 4-pyridylethylene gas was supplied as in Example 1 and surface graft polymerization was performed on the entire surface of the polypropylene sheet at a temperature of 288°C for 3 minutes, hepatocytes adhered and proliferated, but no cell non-adhesion area was formed. It wasn't done.

Example 3 400 parts by weight of liquid paraffin (number average molecular weight 324) and 0.3 parts by weight of crystal nucleation per 100 parts by weight of polypropylene mixture with melt flow index of 30 and 0.3 (mixing weight ratio 100:40) 1 as an agent
, 3.2,4-bis(p-ethylbenzylidene) sorbitol was melt-kneaded and pelletized using a twin-screw extruder. The pellets were heated to 150 to 200°C using the extruder mentioned above.
The material was melted and extruded into the air through a T-die with slits of 0 and 6 ++n, introduced into a cooling solidification liquid by the rotation of a cooling liquid phase guide roller placed directly below the T-die, cooled and solidified, and then wound up.

The wound film was cut to a certain length, fixed in both the vertical and horizontal directions, and immersed in 1,1,2-trichloro-1,2,2-trifluoroethane for 10 minutes for a total of 4 times to extract liquid paraffin. Then, heat treatment was performed in air at 135° C. for 2 minutes to obtain a polypropylene porous membrane having a pore diameter of 0.45 μm, a film thickness of 120 μm, and a porosity of 62%. The porous membrane thus obtained was coated with 3 n as in Example 1.
*Polypropylene sheets with striped slits of 3 mm thick are punched out at intervals, and are heated with low-temperature plasma (Ar, 0 mm).
．． 1 torr) for 15 seconds, 4-vinylpyridine gas was supplied and surface graft polymerization was carried out at a temperature of 288° C. for 3 minutes. Next, one layer of polypropylene sheets was peeled off, washed with a solvent (Hashishi Disolve, manufactured by Asahi Glass Co., Ltd.) for two days, and dried. The portion grafted with 4-vinylpyridine is the cell adhesion region 4, and the untreated polypropylene surface is the cell non-adhesion region 5. Using the porous cell culture membrane 1 thus produced, high-density membrane culture of hepatocytes was attempted using a mini module (effective membrane area of about 282) as shown in FIG. That is, the membrane of this example was used as the first membrane 1, and the pore size was 0.05 μm as the second membrane 2.
The second chamber 1 is made of polypropylene gas exchange membrane.
Hepatic parenchymal cells 4' were sealed in chamber 2, and a liquid medium (DE medium containing io% tallow serum) was poured into the third chamber 13 from 3A to 3B, and the liquid medium was collected from chambers 2A and 2B. A mixed gas of IA→IB, 5% carbon dioxide gas, and 95% air was flowed into the first chamber 11. Even after one week had passed since the start of culture, the cells were growing well at the cell adhesion site, and the medium flowed smoothly without clogging the membrane under conditions of TMP (transmembrane pressure difference) of approximately 20 mn+Hg. there were.

Comparative Example 4 An experiment similar to Example 3 was conducted using a polypropylene porous membrane into which 4-pyridylethylene graft chains were introduced over the entire surface, and as a result, clogging of the membrane occurred on the 5th day of culture, resulting in 2B
, 2A is collected at TMP (transmembrane pressure difference) of 20
It became impossible with mmHg.

Example 4 Polyvinylidene fluoride powder (manufactured by Mitsubishi Yui L Co., Ltd., ky
nar K 301) 18 parts by weight, acetone 73.8
A solution prepared by dissolving 8.2 parts by weight of polyethylene terephthalate and 8.2 parts by weight of dimethylformamide was cast onto a polyethylene terephthalate film to obtain a polyvinylidene polyhydride film. The membrane was then immersed in a 1.1.2-trichlorotrifluoroethane bath for 5 minutes and dried to a thickness of 125 μm.
A polyvinylidene fluoride porous membrane with an average pore diameter of 0.45 μm was obtained. Surface graft polymerization was performed on the membrane in the same manner as in Example 3. Then, as in Example 1, the thickness was 3 n at 3 mm intervals.
Polypropylene sheets with wn slits punched out in a striped pattern were stacked and irradiated with low-temperature plasma (Ar, 0.1 torr) for 15 seconds, then 1-vinyl-2-imidasyring gas was supplied and surface graft polymerization was carried out at a temperature of 288 °C for 3 minutes. I did it. Next, the overlapping polypropylene sheets were peeled off, and the solvent (Freon 113-methanol azeotrope) was removed.
After washing and drying for 2 days, the same hepatocyte culture test as in Example 3 was conducted.

Even after one week had passed since the start of culture, the cells were growing well at the cell adhesion site and the TMP (transmembrane pressure difference) was approximately 20 mmHg.
Under these conditions, the membrane did not become clogged and the medium flowed smoothly.

"Effects of the Invention" As is clear from the above explanation, since the porous membrane for cell culture of the present invention has a cell non-adhesive region on the membrane surface,
Cells adhering to the membrane and substances produced by the cells will no longer block the pores of the membrane and impair its function, thereby preventing problems in the supply and recovery of necessary substances. Therefore, the cell culture device using the porous membrane for cell culture of the present invention enables efficient cell culture and recovery of useful substances at high density over a long period of time, and enables the production of various physiologically active substances by cells. It will be effective for systems, biology, and medical 4° research, and as a hybrid type artificial liver and artificial pancreas in which polymers and cells coexist.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing an example of a cell culture vessel equipped with the porous membrane for cell culture of the present invention. 1... First membrane (porous S for cell culture of the present invention) 2.
...Second membrane 4...Cell adhesion region 4'...Cell 5...Cell non-adhesion region 11...First chamber 12...Second chamber 13...Third chamber

Claims (1)

[Scope of Claims] 1. A porous membrane for cell culture, characterized in that it has a cell non-adhesive region and a cell adhesive region at least on the membrane surface on the cell culture side. 2. The area ratio of the cell non-adhesive region/cell adhesive region on the cell culture surface is 0.02 to 20, the two are separated into a grid, stripe, or sea island shape, and the film thickness is 1 to 500 μm.
2. The porous membrane for cell culture according to claim 1, which has a porosity of 20% or more. 3. A cell culture device using the porous membrane for cell culture according to any one of claims 1 to 2.