JPH05308953A - Cell culture membrane - Google Patents

Abstract

PURPOSE:To obtain a porous cell culture membrane capable of readily culturing a large amount of high-density attachment cells demanding substrate function in culture. CONSTITUTION:The surface of a porous synthetic substrate is provided with a basic functional group having >=4.0pKa and a hydrophilic coating layer substantially covering the substrate to give a cell culture membrane.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a porous cell culture membrane suitable for cell culture and a cell incubator using the same.

[0002]

2. Description of the Related Art With the recent advances in genetic engineering, the need for industrial large-scale culture of animal cells is rapidly increasing. Various methods such as a tank culture method and a roller bottle method are used as a large-scale culture method for animal cells. Among these, especially Nazek (RA Knazek, S
Science, 178: 65, 1972) et al., reported cell culture method using hollow fibers (hereinafter referred to as hollow fiber culture method) as an excellent large-scale culture method. ..

The hollow fiber culture method can secure a large amount of a water-permeable membrane surface excellent in substance exchange in a limited volume, and thus can very effectively supply nutrients and enzymes and remove products and waste products. Of. In the culture of adherent cells, the hollow fiber membranes used here need to play a role as a substrate as a scaffold for cell proliferation as well as substance exchange.
Therefore, the hollow fiber membrane for hollow fiber culture has a high liquid permeability capable of removing a waste product by supplying a sufficient amount of nutrients to a large number and high density of grown cells, and also a place for cell growth. Excellent substrate function is required.

Hollow fiber membranes that have been used in the hollow fiber culture method so far include polysulfone hollow fibers, cellulose hollow fibers, and hollow fibers having a hydrophilic coating layer on the surface of a polyolefin porous synthetic support. It has been known. However, these hollow fibers have a low substrate function as a place for cell adhesion / proliferation, and there is a demand for hollow fibers having excellent cell adhesion / proliferation properties and high substrate functions.

By the way, it is known that the crosslinked dextran beads for tank culture have a substrate function enhanced by retaining a functional group having a positive charge such as a tertiary or quaternary amino group (JP-A-53). -628
89). According to these studies, the charge capacity suitable as a substrate is 0.01 or more and 0.45 meq / ml or less (the swelling rate is 10 as in the case of Sephadex G-50).
Calculated as ml / g).

A polyolefin hollow fiber membrane having a hydrophilic coating layer on its surface has a high liquid permeability, and therefore exhibits extremely high growth performance in floating cells that do not require a substrate for growth. Unlike a hollow fiber membrane whose surface is hydrophobic, it has advantages such as easy moistening of the hollow fiber prior to culturing. Regarding the function as a substrate for cell growth, it is known that some cells such as cervical cancer cells exhibit excellent functions. However, although its function as a substrate is superior to that of other hollow fiber materials, it did not show an excellent substrate function for any of various cells currently cultured. Especially, high density culture of Chinese hamster cells (CHO cells), which are widely used in genetic engineering, could not be performed.

[0007]

The present inventors have completed the present invention as a result of researches for enhancing the function of a hollow fiber such as polyolefin having a hydrophilic coating layer. That is,
A hydrophilic coating layer on the surface of the porous hydrophobic support and a basic functional group having a pKa of 4.0 or more, 0.5 or more and 50 μeq / m
A cell culture hollow fiber membrane characterized by having 1 or less.

The substrate function of the hollow fiber referred to in the present invention can be shown also as the adhesion of the cultured cells to the beads or the extensibility of the cultured cells on the beads, which is referred to as the crosslinked dextran beads. The adhesion-dependent cells are three-dimensional,
In addition, the function of high-density culture is important. The present inventors started culturing Chinese hamster-derived CHO cells by a known hollow fiber culturing method at a cell density of 1 × 10 ⁷ cells / ml per growth space, and cultivated for 14 days. Later when 1 × 10 ⁸ cells / ml can be reached,
The substrate function was considered good.

The porous synthetic support referred to in the present invention is a hollow fiber-like porous membrane made of a synthetic polymer material, which is known in the art such as a wet phase inversion method, a melt phase separation method, a melt spinning drawing and opening method. Obtained by the method. After forming the crystalline polymer into a hollow fiber shape in this, the crystalline lamella is cleaved by cold drawing, and the pore size is expanded by hot drawing to obtain a porous structure composed of stacked lamellae and microfibrils. The stretch-opening method is preferable because it does not use a solvent or other additives in the synthetic polymer material and thus does not have a problem of residual solvent.

Examples of synthetic polymer materials used for the porous synthetic support include polyolefins such as polyethylene and polypropylene, acrylic polymer materials such as polymethylmethacrylate, polystyrene, polysulfone, and Teflon. Of these, polyolefin is particularly preferable because the desired pore size can be easily obtained by the stretch opening method.

The basic functional group on the surface of the present porous synthetic support means a functional group which exhibits a positive charge in a solution, and a nitrogen-containing functional group is preferable. These include amines, amine derivatives and the like, and examples thereof include tertiary and quaternary amino groups, but those having a pKa of 4.0 or more are preferred. Particularly preferably, it is represented by the following formula (a).

[0012]

[Chemical 1]

There is no particular limitation on the substituents R1, R2 and R3, and any substituent can be provided, but any substituent is connected to the main chain of polymerization by a covalent bond. .. For example, it may be a hydrocarbon substituent such as hydrogen, a methyl group, an ethyl group, a propyl group, a phenyl group, and a benzyl group, or a substituent containing an atom of a heteronuclide such as methylol and ethylol, resulting in a base. It is sufficient that the pKa of the sexual functional group is 4.0 or more. R1,
Two or more R2 and R3 may be cyclic, and examples thereof include pyridine, imidazole, piperidine, pyrrole, and pyrimidine.

The basic functional group does not necessarily have to be present on the side chain of the polymer chain constituting the support or the hydrophilic coating layer, and may form the main chain. However, it is easier in terms of production method to introduce a basic functional group as a side chain into the hydrophilic coating layer. Specific examples of the basic functional group are represented by monomer units: allylamine, diallylamine, N,
N-dimethylallylamine, N, N-diallylpepyrazine, N, N-diallylaniline, N, N-diallylmelamine, aminostyrene, N, N-dimethylaminostyrene, N, N-diethylaminostyrene, vinylbenzylamine, vinyl Phenethylamine, N, N-dimethylvinylphenethylamine, N, N-diethylvinylphenethylamine, N-propylvinylphenethylamine, vinylpyridine, 2-methyl-5-vinylpyridine, 2-
Ethyl-5-vinyl pyridine, 2-vinyl quinoline, 2
-Vinylimidazole, 4-vinylimidazole, vinylpyrazoline, vinylpyrazine, 4-vinylpyrimidine, vinylamine, vinylcarbazole, ethyleneimine, N-phenylethyleneimine, N, N-diethyl-
There are N-vinylphenethylamine and the like, and oligomers or polymers thereof may be used. Of these, the reason is not clear, but especially dimethylamino group, diethylamino group,
Particularly preferable results have been obtained with a product having a trimethylamino group or a triethylamino group.

The above-mentioned basic functional group is a method in which it is introduced into the support or the hydrophilic coating layer by a grafting method such as radiation or electron beam, and a nucleophilic reactive group having an active hydrogen in the support or the hydrophilic coating layer. A method in which an active group capable of substitutional and / or addition reaction or an active group chemically introduced into a support or a hydrophilic coating layer is chemically covalently bonded, or a polymerizable functional group having a basic functional group is introduced. It is obtained by coating a copolymer containing a compound and a hydrophilic polymerizable compound. Of these, the method of grafting a basic functional group onto the hydrophilic coating layer and the method of chemically covalently bonding with an active group introduced into the hydrophilic coating layer are probably the basic functional groups outwardly facing the hydrophilic coating layer. Is more preferable because The amount of the basic functional group to be introduced is preferably 0.5 or more and 50 μeq / ml or less per membrane volume including the pore volume of the hollow fiber membrane when measured by the neutral salt decomposition method. Further, a particularly preferable range is a range of 1 or more and 10 μeq / ml or less.

The hydrophilic coating layer in the present invention means that the surface of the support has a coating layer made of a hydrophilic polymer in order to improve the affinity with an aqueous solution. The cell culture membrane has a risk that the permeability of the culture solution originally possessed by the cell culture membrane is not exhibited due to the presence of air bubbles in the pores of the membrane,
It is necessary to avoid the risk that the protein in the culture solution will be nonspecifically adsorbed and not sufficiently supplied to cells. The hydrophilicity of the hydrophilic coating layer may be such that these dangers can be avoided, and if a particularly preferable range of hydrophilicity is shown, air bubbles on the solid surface of a sheet or film in water are shown. The contact angle at 25 ° C. is 110 degrees or less.

The hydrophilic coating layer is preferably a polymer in order to prevent peeling during culture. As specific examples of the hydrophilic coating layer, if the polymer unit is exemplified by the name as a monomer, hydroxystyrene, hydroxymethylstyrene,
Block copolymerization of vinyl alcohol, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, vinylamine, diethylaminoethylstyrene, diethylaminoethyl methacrylate, methoxytriethylene glycol methacrylate, dimethylaminoethyl (meth) acrylate segmented polyurethane, segmented polyester, etc. Polymers, graft copolymers such as monomers having polyethylene oxide chains and other polymerized monomers, ethylene-vinyl alcohol, polyester,
Examples thereof include polyethylene glycol. In particular, it is preferable that the polymer has a hydroxyl group. There is no particular limitation on the bonding mode of the hydroxyl group in the polymer. Of these, ethylene-vinyl alcohol, polyethylene glycol, and methoxytriethylene glycol methacrylate are preferable from the viewpoint of hydrophilicity, and particularly ethylene-vinyl alcohol is a functional group that easily introduces an active group when a basic functional group is introduced. It is more preferable because the hydrophilicity is less likely to decrease due to peeling when introducing a group.

The hydrophilic coating layer may be a homopolymer of the above-mentioned polymer units, or may be a copolymer of two or more units, such as a linear polymer, a graft polymer or a crosslinked polymer. There is no particular relation to the polymerization form of.

The hydrophilic coating layer can be obtained by dipping in a liquid in which the compound forming the coating layer is dissolved or by spraying the liquid, or by a grafting method using radiation or electron beam. There is a method of covalently bonding to the surface of the porous synthetic support or a method of covalently bonding via a functional group on the surface of the porous synthetic support by a chemical method. Among them, the coating method is particularly advantageous because it can be easily carried out industrially. The coating method referred to here may be a method in which a polymerizable compound is also allowed to coexist in the compound forming the coating layer and crosslinking is performed after coating. The amount of the coating layer thus obtained, that is, the coating rate, is preferably 0.1% or more and 20% or less when expressed by the ratio of the weight of the coating layer to the dry weight of the cell culture membrane. Particularly preferably 2
% Or more and 15% or less.

The hollow fiber membrane composed of a porous synthetic support having a basic functional group and a hydrophilic coating layer on the surface, as referred to in the present invention, has a maximum pore size determined by the bubble point method of 0.01 μm or more and 10 μm or less Preferably 0.0
5 μm or more and 1 μm or less, and / or the average pore size determined by mercury porosimetry is 0.001 μm or more and 1 μm
Refers to the following: The volume ratio of the pores in the film structure, that is, the porosity is preferably 10% or more and 90% or less, and particularly preferably 40% or more and 80% or less. Furthermore, the permeability of distilled water from the inside of the hollow fiber in the wet state to the outside,
That water permeability is at 0.05L / hr · m ² · mmHg or more, preferably 0.5L / hr · m ² · mmHg or more, more preferably 2L / hr · m ² · mmHg or more. At this time, the upper limit of the water permeability does not have to be particularly limited, but when it has a particularly high water permeability, it may cause a decrease in the film strength. Therefore, 30 L / hr · m ² · m.
It is preferably mHg or less for handling. The hydrophilicity of the surface of the hollow fiber membrane of the present invention is the ratio (%) of the permeability of distilled water of the hollow fiber in the dry state, that is, the hydrophilicity, when the water permeability of the hollow fiber in the wet state is 100%. It is indicated by the degree and is preferably 5% or more.

The incubator filled with hollow fibers as referred to in the present invention means a space in which at least one or more cells grow and a space in which at least one or more culture fluid flows, due to the hollow fiber membrane in the incubator. And each of the spaces has one or more space contents withdrawal and / or intake ports. Among the incubators filled with these hollow fibers, those having a ratio of the outer volume of the hollow fibers to the total inner volume of the incubator of 20% or more and 80% or less are preferable, and 30% or more and 70% or less are given in a more preferable range. Especially excellent.

[0022]

EFFECT OF THE INVENTION On the surface of the porous synthetic support of the present invention,
A hollow fiber membrane for cell culture characterized by having a basic functional group and a hydrophilic coating layer that substantially coats the support, and a culture vessel filled with the hollow fiber membrane have a substrate function in culture. The required adherent cells can be easily mass-cultured in high density.

[0023]

【Example】

Control Example High-density polyethylene (density 0.968, MI value 5.5,
The product name Hi-Zex 2208J) was spun at a spinneret temperature of 150 ° C. using a circular double spinneret, and the obtained hollow fiber was stretched and opened to obtain a support membrane having a hollow fiber-like polyethylene porous structure. This support membrane was immersed in a solution in which an ethylene / vinyl alcohol copolymer having an ethylene content of 29 mol% was dissolved in a 75% by volume aqueous ethanol solution at 1.0% by weight to obtain a hydrophilic coating layer of the ethylene / vinyl alcohol copolymer. A polyethylene hollow fiber having This polyethylene hollow fiber
Inner diameter 330 μm, film thickness 50 μm, average pore size by bubble point method 0.20 μm, water permeability 12 L / hr · m ²
The amount of the coating layer was 3.2 × 10 ⁻³ g / m ² , which was determined by mmHg, the degree of hydrophilicity was 83%, and the weight change before and after the immersion treatment.

[0024]

Example 1 A polyethylene hollow fiber (10 cm, 800 fibers) having a hydrophilic coating layer obtained in the control example was mixed with 5 volumes of dimethyl sulfoxide and 4 volumes of epichlorohydrin.
In 100 ml of a mixed solution of 1 volume of N sodium hydroxide aqueous solution, reaction was carried out at 40 ° C. for 2 hours to introduce an epoxy group. Then, in a 10% aqueous solution of diethylamine, the mixture was reacted at 40 ° C. for 4 hours to introduce a positive charge. The charge capacity that could be introduced at this time could be measured as 16.5 μeq / ml by the neutral salt decomposition method.
The water permeability was 11.8 L / hr · m ² · mmHg and the degree of hydrophilicity was 73%.

[0025]

Example 2 A polyethylene hollow fiber (10 cm, 800 fibers) having a hydrophilic coating layer obtained in the control example was immersed in a 10% ethanol aqueous solution containing 5% dimethylaminoethyl methacrylate, and 25 kGy of γ-ray was applied. Irradiated and covalently bonded by the graft method. The charge capacity of the obtained hollow fiber membrane was 8.5 μeq / ml by the neutral salt decomposition method. The water permeability was 11.8 L / hr · m ² · mmHg and the degree of hydrophilicity was 78%.

Culture Example Each of the hollow fiber membranes obtained in the control example, the example 1 and the example 2 had an inner diameter of 8 with a cell inlet / outlet port on the side surface.
An incubator was prepared by immobilizing a potting agent with a urethane adhesive in a polycarbonate container having a size of 10 mm and a length of 10 cm. Culture was performed by connecting a culture vessel as shown in FIG. 1 and using CHO cells, the number of added cells was 1 × 10 ⁷ cells / m 2.
The procedure was the same as that of the ordinary hollow fiber culture method in Example 1. At this time, as the culture medium, an Eagle MEM culture medium (manufactured by Dainippon Pharmaceutical Co., Ltd.) to which fetal bovine serum was added at a concentration of 10% was used. The culture results are shown in FIG. The cell density of each incubator 14 days after the start of culture was 3.2 × 10 ⁷ cells / ml in the control example.
In contrast, in Examples 1 and 2, each was 1.1.
× and 10 ⁸ cells /Ml,1.8×10 ⁸ cells / ml, both also at 1 × 10 ⁸ cells / ml or more showed excellent substrate function.

[Brief description of drawings]

FIG. 1 is an example of a culture system using a cell culture device.

FIG. 2 is a growth curve obtained in the culture example, in which A is a control example and B is a control example.
Shows the results obtained in Example 1 and C in the cell incubator filled with the cell culture membrane obtained in Example 2.

[Explanation of symbols]

 1 Polycarbonate container 2 Cell culture membrane 3 Cell inlet / outlet port 4 Culture fluid reservoir 5 Ironing pump 6 Ironing tube 7 Silicon tube

Claims (1)

[Claims] 1. A pKa of 4. on the surface of a porous synthetic support.
A cell culture membrane having 0 or more basic functional groups and a hydrophilic coating layer that substantially coats the support.