JPS63283576A - Substrate for cell culture - Google Patents

Abstract

PURPOSE:To improve adhesiveness to cells, extension of cells and to improve multiplication and activity maintenance, by coating a high polymer substrate with a film of a polymerized organosilicon compound by plasma treatment and using the coated high polymer substrate as a substrate for cell culture. CONSTITUTION:A pair of facing electrodes connected to a high-frequency electric source are set in a reactor consisting of a bell-jar and a high polymer substrate is supported between the electrodes. Then, the bell-jar is sufficiently evacuated and a gaseous organosilicon compound is introduced into the bell-jar. Plasma is generated between the electrodes to form a plasma polymerized film of the organosilicon compound on the surface of the high polymer substrate. A porous high polymer material is preferable as the high polymer substrate and tubular or hollow shape is preferable as the shape for high-density culture. The prepared substrate for cell culture is useful for production system of hormone, etc., by cultivation of animal cell.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a substrate for cell culture. More specifically, the present invention relates to a cell culture substrate used for culturing animal cells.

<Prior art and problems to be solved by the invention> In recent years,
BACKGROUND OF THE INVENTION Research is actively being carried out to cultivate biological cells and produce useful physiologically active substances, such as vaccines, hormones, and interferons, through the metabolic activities of the cells.

In such methods, adherent animal cells have conventionally been cultured using glass or plastic petri dishes, test tubes, culture bottles, and the like.

Recently, attempts have been made to use microcarriers and hollow fibers as culture substrates to achieve higher-density culture and longer-term culture. In order to allow adherent animal cells to adhere and proliferate on a culture substrate, it is necessary to have good adhesion between the cells and the surface of the substrate, as well as the morphology and arrangement of the adhered cells.
It is necessary to have a form that is effective for cell expansion and proliferation.

However, although the polymer materials conventionally used as substrates for cell culture have excellent shapeability and durability, they are unsuitable in terms of adhesive properties, etc., and cannot be used for high-density and long-term cell culture. However, none of them have been able to achieve sufficient results.

In order to improve these problems, cell culture substrates made of polymeric materials that are surface-treated to make them hydrophilic, such as tissue culture materials in which polymeric materials are treated with ozone (Japanese Patent Laid-Open No. 52-412
91), tissue culture carrier particles treated with low-temperature plasma (see JP-A-57-22891), and a plasma polymerized film formed using a mixed gas of a hydrocarbon compound containing a double bond and oxygen. cell culture bed (Unexamined Japanese Patent Publication No. 6
1-52281) etc. have been proposed.

However, although all of the cell culture substrates obtained by the above methods have hydrophilic surfaces, they do not have sufficient adhesion with cells, and do not have sufficient cell spreadability and proliferation, allowing cells to be grown at high density and for long periods of time. The problem is that it cannot be carried out over a period of time.

Purpose This invention was made in view of the above-mentioned problems, and is a cell culture product that has excellent adhesion with cells, as well as cell spreading, proliferation, and maintenance of activity, and enables high-density, long-term culture. The purpose is to provide a base material.

Means and Effects for Solving the Problems> The cell culture substrate of the present invention, which has been made to achieve the above object, has a polymer substrate whose surface is coated with a plasma polymerized film of an organosilicon compound. It is characterized by the fact that

As shown in the above structure, this invention consists of a polymer base material whose surface is coated with a plasma polymerized film of an organosilicon compound, and the plasma polymerized film of an organosilicon compound has heat resistance, chemical resistance, and wear resistance. It is known as a membrane with excellent properties, and its surface tends to be hydrophobic. Conventionally, the development of cell culture substrates through surface modification has been carried out with the aim of improving compatibility with cells by making the surface of the substrate hydrophilic. We have also discovered that a plasma-polymerized membrane made of an organosilicon compound with a strong hydrophobic tendency has excellent adhesion to cells, and also allows the adhered cells to spread and proliferate well. This invention was made based on the above knowledge, and a material in which the surface of a polymer base material is coated with a plasma-polymerized film of an organosilicon compound can improve adhesion and proliferation with cells, and can also be used at high temperatures. It is an excellent cell culture substrate that is resistant to sterilization and stable against pH changes.

Note that the polymer base material is preferably a porous material. Further, it is preferable that the base material is a hollow fiber. When the polymeric material is a porous material, substance metabolism is facilitated through the pores of the porous material, and cells can be cultured for a long period of time.

In particular, when the base material made of the polymeric material is a hollow fiber, cells can be grown and multiplied at high density on the hollow fiber by perfusing a culture solution or the like into the hollow portion or outside of the hollow fiber. can.

The present invention will be explained in detail below.

As the polymer base material, any material can be used as long as it has formability and mechanical strength, such as polyethylene, polypropylene, chlorinated polyethylene, olefin polymers such as ionomers, polytetrafluoroethylene, Fluorine resins such as polyvinylidene fluoride, styrene resins such as polystyrene, acrylic resins such as polymethyl methacrylate, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetal, polyacrylonitrile, polyvinyl chloride, polyvinylidene chloride, polycarbonate, polyarylate , polyester resins such as polyphenylene oxide, polyethylene terephthalate, and polybutylene terephthalate, various polymers or copolymers such as epoxy resins, polyamides, polyimides, polysulfones, cellulose resins, silicone resins, and polyurethanes, or blends thereof. .

The above-mentioned polymeric base material can be formed into various forms, such as molded products such as petri dishes and flasks, as well as forms such as films, tubes, hollow fibers, fibers, and fine particles. Among these forms, for long-term cell culture, porous polymeric materials with pores that facilitate material metabolism are preferable, and for high-density culture, tubes and hollow fibers are preferable. suitable. Particularly preferred are hollow fibers made of porous polymeric materials that are easy to metabolize and can be cultured at high density for a long period of time. When using this hollow fiber, the culture solution is
Cells can be grown and proliferated on the hollow fiber by perfusing the hollow portion or the outside of the hollow fiber and sending carbon dioxide gas, air, etc. into the hollow portion of the hollow fiber as necessary. Note that the hollow fibers can be of various sizes, and for example, those having an inner diameter of about 50 to 1000 μm are used.

Further, when the cell culture substrate of the present invention is used as a bead carrier in a microcarrier method, the polymer substrate used has a particle size of about 100 to 300 μm.

Organosilicon compounds that are raw materials for the plasma-polymerized film of organosilicon compounds formed on the surface of the polymer material base material include alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, octyl, and dodecyl. ; Alkoxy groups such as methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, octyloxy; Unsaturated hydrocarbon groups such as vinyl, ethynyl, allyl; Aryl groups such as phenyl, naphthyl; benzyl, benzhydryl, phenethyl, etc. Examples include silane compounds, siloxane compounds, and silazane compounds having organic substituents such as aralkyl groups, halogen atoms such as chlorine, bromine, iodine, and fluorine, such as trimethylsilane, tetramethylsilane, triethylsilane,
Tetraethylsilane, octylsilane, dichlorodimethylsilane, dimethoxydimethylsilane, methyltrimethoxysilane, ethoxytrimethylsilane, jetoxysilane, dibutoxysilane, triphenylsilane, dimethylphenylsilane, diethylphenylsilane, trichlorosilane, trimethylvinylsilane, dimethyl Methoxyvinylsilane, dimethylethoxyvinylsilane, ethynyldimethylmethoxysilane, dimethyldivinylsilane,
Methyltrivinylsilane, trimethoxyvinylsilane,
Silane compounds such as tetravinylsilane, allyltrimethylsilane, dibenzylsilane, tetramethyldisiloxane, hexamethyldisiloxane, hexapropyldisiloxane, hexamethylcyclotrisiloxane, octamethylcyclotrisiloxane, hexaphenyldisiloxane, 1.3 Examples include siloxane compounds such as -dimethoxy-1,1,3,3-tetramethyldisiloxane, pentamethylvinyldisiloxane, and pentamethylphenyldisiloxane, and silazane compounds such as hexamethyldisilazane and tetramethyldisilazane. Two or more types of organosilicon compounds may be used in combination.

Plasma polymerization of the above-mentioned organosilicon compounds can be carried out using conventional methods and equipment. For example, a pair of opposing electrodes connected to a high-frequency power source is provided in a reaction vessel constituted by a Pelger, and the electrodes are A polymeric substrate is held in between. Next, after the pressure inside the Pelger is sufficiently reduced, a gaseous organosilicon compound is introduced, and plasma is generated between the electrodes to form a plasma-polymerized film of the organosilicon compound on the surface of the polymer substrate. At this time, along with organosilicon compounds, argon, helium, nitrogen, ammonia, oxygen,
Plasma polymerization may be performed by mixing a carrier gas such as hydrogen. Note that the conditions for the plasma polymerization mentioned above, such as the degree of vacuum in the reaction vessel, the amount of introduced organosilicon compound, etc., are the same as the reaction conditions for normal plasma polymerization.
For example, the degree of vacuum inside the reaction vessel is 1 mm Torr ~
10Torr, the volume of the reaction vessel is 1j as the flow rate of the organosilicon compound! Per I x 10-3m11min~1
Approximately 0 m1/min is appropriate. In addition, the thickness of the plasma polymerized film to be formed is not particularly limited, but is usually 1 to 1000.
The film thickness can be adjusted as appropriate by adjusting the amount of the organosilicon compound introduced, the polymerization time, etc.

The cell culture substrate of the present invention can be used to culture various cells, and the type of cells is not particularly limited, and examples include living body-derived cells, hybridomas, etc. For example, Chinese hamster lung-derived cells v- 79, human uterine cancer-derived cells HeLa, human fetal lung-derived cells MRC-5, human liver-derived cells Chang Livers, human lung-derived eudiploid fibroblasts IRC-90, human lymphoma-derived Namalva cells, and the like.

Furthermore, when culturing animal cells using the cell culture substrate of the present invention, various culture solutions are used depending on the type of cells to be cultured, and conditions such as optimal temperature and pH suitable for cell proliferation are used. Culture is carried out in

<Examples> The present invention will be described in more detail below based on examples.

Example 1 and Comparative Example 1 Polytetrafluoroethylene porous membrane (manufactured by Sumitomo Electric Industries, Ltd., Fluorobor FP-065) was used at 13.56MH
It was installed in a Pelger type plasma device with a high frequency power supply of Plasma polymerization was performed for 3 minutes. The film thus obtained is 10mm
10-layer, 8-chamber slide glass (Lab T
After high-pressure steam sterilization, human liver-derived Chang L1ver cells were cultured. The culture solution is
Using Eagle's MEM culture solution containing fetal bovine serum, 2×10 4 cultured cells were seeded per chamber and cultured in an incubator (in an atmosphere of 5% carbon dioxide gas and 95% air).

After 24 hours, the cells attached to the film were separated and the number of cells was calculated using a hemocytometer.
4, showing excellent adhesion and proliferation.

On the other hand, as Comparative Example 1, a culture test was conducted in the same manner as in Example 1 using a polytetrafluoroethylene porous membrane that was not coated with a plasma polymerized membrane, but the number of cells was 4×10 3 cells.

Example 2 and Comparative Example 2 Polystyrene petri dishes (10 plates) were treated in the same manner as in Example 1 using a mixed gas of tetramethylsilazane and ammonia gas under conditions of a pressure of 0.3 Torr, a discharge output of 50 W, and a treatment time of 5 minutes. Plasma polymerization was performed. After sterilizing these petri dishes with ethylene oxide, separately cultured Chinese hamster lung-derived V-79 cells (separated by trypsin-EDTA) were placed in each petri dish, and Eagle M containing 10% tallow serum was added.
The cells were cultured in an incubator (in an atmosphere of 5% carbon dioxide gas and 9.5% air) using EM culture solution. 2 hours later
Each petri dish was removed from the incubator, the cells adhered to the surface of the petri dish were released, and the number of cells was calculated using a hemocytometer.

Then, the ratio of the number of adherent cells to the number of prepared cells (adhesion rate) was determined. The results are shown in the table below.

On the other hand, as Comparative Example 2, polystyrene petri dishes (10 pieces) not coated with the plasma polymerized film were used as they were.
A culture test was conducted in the same manner as in Example 2 above, and the cell adhesion rate was determined. The results are also shown in the table below.

(The following is a blank space) As is clear from the above table, the petri dish of Comparative Example 2 has a low adhesion rate to cells, whereas the petri dish of Example 2 shows a high adhesion rate and has excellent adhesion to cells.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, the surface of the polymer substrate is coated with a plasma polymerized film of an organosilicon compound. Since it has excellent adhesion to cells and can increase proliferation, it has the unique effect of enabling high-density and long-term cell culture. Therefore, the cell culture substrate of the present invention can be used in a system for producing useful products such as hormones by culturing animal cells, and can also form an artificial pancreas by, for example, attaching and culturing insulin-producing cells to the surface of the substrate. It can be used to construct artificial organs.

Patent applicant: Sumitomo Electric Industries, Ltd. (3 others)

Claims (1)

[Claims] 1. With plasma polymerized membrane of organosilicon compound It is characterized by being made of a coated polymeric base material. Substrate for cell culture. 2. The above where the polymer base material is a porous material For cell culture according to claim 1 Base material. 3. The above patent in which the polymer base material is a hollow fiber Either claim 1 or 2 The cell culture substrate described in Crab.