JPH02504221A - Cell culture methods, culture products and culture products - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cell culture methods, culture products and culture products The present invention provides a method and culture for culturing mammalian cells, particularly epithelial cells, on a culture substrate. , particularly regarding the use of the culture as a wound dressing.

Most normal mammalian cells derived from solid organs are anchorage dependent. Sunawa In other words, these cells cannot substantially proliferate in suspension culture, but in a growth medium containing the cells. It can grow on culture substrates that are in contact with the ground.

Epithelial cells are anchorage dependent. When cells are cultured with a culture substrate surface present, this It attaches to surfaces and proliferates in layered colonies, eventually reaching full growth. This stage At the bottom, the culture enters steady state, with dividing cells proliferating in the basal layer, and cells at the top growing in the culture layer. It overflows from the nutrient substrate surface.

That is, differentiation occurs and, to some extent, the culture becomes similar to the intact skin. behave. This type of cell culture is used to test skin growth, differentiate skin cells, and control this differentiation. It is useful not only for treatment (but also for implantation into the skin as a permanent wound repair). It is also useful for obtaining usable sheet materials. Rolbrook and Ilen nings considers the in vitro growth of epidermal cells (J, Invest, 　Dermeng aLd.

81; 115-245.1983).

The ability to proliferate with anchorage-dependent cell power depends not only on the culture substrate surface (but also on the components of the growth medium). and culture conditions. The properties of the culture substrate that allows cells to attach and grow are currently unknown. Currently, it is not completely understood. Cultures are usually grown in solid containers, e.g. glass or regular containers. Usually, a bedding dish or flask made of a harder transparent plastic material such as polystyrene is used. Generally, the cells of the mammalian animal are sufficiently attached to such a surface, and Proliferate.

In many cases, cells are cultured and then some or all of the cells, sometimes the grown layer, are removed. It is desirable to move it to another location without destroying it. This changes the nature of the sample must be examined by examination or by microscopic examination in the case of multiple may be necessary. A layer of epithelial cells and fibroblasts protects burns and other wounds. It may be necessary to apply skin grafts to treat the condition.

Usually used as a means to determine the factors necessary for cell surface adsorption and cell attachment. , studies have been published investigating the properties of the substrate surface required for cell attachment. for example , Applied Biochetis Lry and Biotechnol In Ogy 8.115-126 (1983), Yoshii et al. The cell culture of tumor cells and liver intracellular cell lines is described. Various acrylic Cast a solution of sulfuric acid ester monomer to form a film on the inner surface of the flask and polymerize it. Cultures are carried out in polymer-coated flasks by initiating. Polymer is Since it swells a little, the maximum water content of the gel swollen with pure water is 25%. culture Once the vessel surface is coated with polymer, it is difficult to remove the adherent cells from the culture vessel.

Other methods for culturing fixation-dependent cells include various types of collagen, fibronectin, and Laminin, a molecule present in the extracellular stroma of cells in the normal surrounding environment Cover the hard culture surface with J, Ce1lular Physiology83 ．． 379-379 (1973), Macieira-Coelha, (Amino acids) A polymer-based bovine serum solution coated with charged materials such as histones and heparin. Fibroblasts are cultured in containers coated with rubumin. Either of these methods also promotes cell growth, but still suffers from the drawbacks mentioned above regarding rigid container surfaces. It is.

Experimental Ce1l Re5earch 143, p. 15-25 (1983), Paris et al. Culture of endothelial cells and fibroblasts on the sheet surface of methacrylate) (HHMA) It describes what to do. Preparation of the polymers may in some cases include e.g. This is done in the presence of proteins such as collagen and elastin. This IIEM eight ho Moherimer has low swelling property and absorbs 0.9% physiological saline. A gel with a maximum content of 38% can be formed.

Ce1l (1977) 11. Green announced on Pp, 405-416. Paper = Ters + 1nal Differentiation or Cu1t ured Human Epiderval Ce11g” Keratinocytes from the foreskin are cultured to form stratified full-scale colonies. Cultivation The nutrients are used for testing differentiation. Experimental Cell Re5e Jepsen et al. (1980) 125, pp. 141-152. “Fine 5structure or Sub Cultivated 5Lratiried 5quasous Epitheliu+s” Rat tongue epithelium was cultured and primary explants were grown on the surface of a plastic culture flask. The tissue has been subcultured to produce long-term sustainable cultures. Journal or InvestigaLive Dermatology 19g7.88:31 4-319, Arenholt-Bindslev et al. This article describes an experiment in which cells were cultured and subcultured in hard plastic flasks. ing.

Feed cells are often used to promote epithelial cell growth and differentiation. example For example, in the above-mentioned document, Green describes the supply of lethally irradiated 3T3 cells. It is used as. Ce1l (1975) j2, pp, 331-344 5erial cultivation announced by Rheinwald et al. r　5trainsor　human　epider+sal　kera tinocytes: the formationor keraLin ising colonies from single cells”, The use of fibroblasts as feed cells has been described.

There are various disclosures regarding the use of cultured epithelial cells as skin grafts. J Oural or Trauma (1986) 26, pp, 955-96 In 2, Madden et al. Describes the use of the epidermis) as a graft for 11th or 111th degree burns. are doing. The consequence of burn injury is immunosuppression that prevents early rejection of the allograft. , these allografts will eventually be rejected unless drugs are used as immunosuppressants. receive. Additionally, using someone else's tissue is especially dangerous for virus infections such as HIV. It goes without saying that it is desirable to avoid this, as there is a high risk of infection.

Therefore, it is preferable to use autologous cultured epithelium. Journal or 1ledicine (August 16, 1984), pp , 448-451, Gallico et al. describes its use as a skin graft. Also, Lancet (1986)! , PP, 1123-1124, Cuono et al. A whole skin allograft is used as the first step in the explantation procedure, and then the epidermal layer of the allograft is removed. describes the replacement of skin allografts by autologous epidermal cultures. There is. This method was successful. This is because the immunoreactivity of the dermis is lower than that of the epidermis. It is et al. However, the risk of virus infection remains high.

Using hard plastic or glass surfaces as culture substrate surfaces for epidermal cell culture One of the problems involved is that the medium is supplied from above the surface, so that the basal cell medium does not reach the surface. The degree of access to the site will be lower. This slows down the growth rate. why Then, the number of cells that can divide is y! This is because they are basal cells. cell sheet skin graft Another drawback when trying to use the sheet as a The point is that it must be removed from the surface of the culture substrate and handled without support; This is inconvenient and may also damage the seat.

To facilitate handling, support the sheet after removing it from the culture substrate surface. It is known that For example, Gallico et al. (cited above) It states that it is to be fastened to the case with a chestnut. Also, Cuono et al. A supporting material "Terrace" is provided on the culture medium. child Although these methods solve some of the problems in handling sheets, they still As well as having disadvantages associated with hard surface growth, the sheet is removed from the substrate surface. There is a need. Removal from hard surfaces is disclosed in U.S. Pat. No. 4,304,866. It can be easily done by applying enzymes such as dispase, which has been .

In addition, epithelial cell growth was performed on collagen gel. In this case, the epithelial cells The collagen along with the layer was used directly as an epidermal substitute. For example, Proc, Na1l.

Acad, Sci, USA (1979) 76, I) p, 1274-127 In 6, El et al. described the use of collagen gel as a culture substrate for epithelial cells. It is written as follows. Fullergen lattice should use human fibroblasts as feeding cells to be inoculated. Journal of InvestigaLive Ders+ aLology (1983) 81% pp, Be11 etc. in 2S-IOS The skin equivalent consists of fibroblasts in a collagen matrix and collagen matrices. grown from “plating” fern keratinocytes on This paper describes the reconstruction of raw skin consisting of an epidermal equivalent.

Several studies have shown that at air/liquid interfaces that approximate in-vivo conditions, When growing epithelial cells, the epithelial cells have a higher volume of epithelial cells than cells that are completely submerged in the culture medium. Cells grown on Gengel may have better stratification (differentiation). be.

Although the growth of epithelial cells on collagen gels is excellent, the use of collagen There are some problems with this. Collagen is expensive, especially in isolated form. In the case of powders that are difficult to prepare and require to be dissolved and gelled, it takes time to configure them. It takes. Isolating and constructing gels in the laboratory from natural sources is also time consuming.

Additionally, collagen is still not completely understood. I mean, Because it is a natural substance, its behavior varies from batch to batch, making standardization difficult. be. This is important when applying collagen as a skin graft. very undesirable from this point of view. Furthermore, collagen is difficult to handle in the laboratory. However, one of the problems is that the size of the gel, and therefore its water content, is limited when used as feeding cells. This point depends on the presence of other cells, especially fibroblasts. The size of the gel Although it changes during use, this is when the gel is used as the main part of the skin substitute. This is a particularly big drawback. When the water content is reduced to a certain level, the gel becomes opaque. Therefore, it is not easy to observe the degree of wound healing under the graft made of this gel.

Another problem is that the protein collagen cannot be steam sterilized, so The point is that sterilization of living organisms is only possible with antibiotics and other sterilization methods. Heaven In the case of natural collagen gels, both are favorable for the growth of undesirable microorganisms. It is a good substrate for wounds, and its use on wounds increases the risk of infection. yet another A major drawback is that when the gel is used as a skin substitute with epithelial cell cultures, the epithelial cells The point is that it is necessary to provide a separate barrier directly above the cell culture.

The present invention provides water-insoluble and water-swollen materials that are swollen in an aqueous medium in the presence of the medium. Fixed on the surface of a removable substrate, which is a gel made of a hydrophilic synthetic polymer that exhibits moisture properties. A novel method of culturing dependent mammalian cells is provided. This method is , the polymer absorbs at least its dry weight of 0.9 weight 1% saline. It is characterized by its swelling properties.

The method of the invention can be used in particular for the culture of human cells, especially epithelial cells and fibroblasts. However, the use of polymeric matrices is widespread in mammalian anchorage-dependent cells. It is also advantageous for culturing.

This novel method requires a small amount of the culture process (and in some parts, a substratum above the aqueous medium). The cells on the surface can be exposed to a gaseous medium. Exposure of cells is at the beginning of the culture process. It may be carried out during or during the later stages (or may be carried out throughout the culture process).

Throughout the cultivation process, the synthetic polymer gel is swollen in an aqueous medium and nutrients are transferred to the gel base. Supplies the cells through the cells. Exposure of cells to gaseous medium during the culture process While growing cells in contact with ambient air or other gaseous media, This is done by maintaining the substrate surface continuously feeding the gel with an aqueous medium.

For example, a solid support may be used to support the gel on a substrate surface, or the gel may be It is possible to partially immerse the gel in an aqueous medium by floating it on the surface of the water-based medium. can.

This method exposes the surface layer of the skin to the atmosphere while providing nutrients from beneath the skin. The growth and differentiation of epithelial cells in vitro can be monitored by approximating in vivo conditions. It is useful as a research tool.

Apart from the use of specific substrates, regular nutrients and components that control growth rates It is convenient to use a cell culture medium containing Therefore, for epithelial cells, the medium can contain substances known to promote epithelial cell growth. Ru. These may be growth factors such as epidermal growth factor (EGF), and are usually is derived from feeder cells, such as normal human fibroblasts, 3T3-cells, Used in conjunction with glial cells and other brain cells.

A preferred method involves growing feed cells on the surface of a medium container and allowing the cells to grow. Once a steady state has been reached, epithelial cells are introduced. Swelling synthetic polymer gel grows may be introduced into the supplied cell layer. This allows the cells to be Epithelial cells grow in a physically separated state. Alternatively, swelling The fed cells are grown on the surface of the synthetic polymer gel, and after the growth reaches steady state, the epithelium Cells may also be introduced.

Sometimes we grow epithelial cells on the same surface as the feeding cells, but as mentioned earlier, we grow epithelial cells on different surfaces. Different cell types are grown on different surfaces. When the supply cells are fibroblasts, Gr Fibroblast cells, as described in U.S. Patent No. 4,016,036 to can be treated to prevent them from multiplying. In addition, culturing is a two-step process. When freezing cells between steps, French Patent No. 2589165 describes The cells may be grown by the method described (the second step is according to the invention). Implemented.

The culture medium may include those known to those skilled in the art, such as those described in the above-mentioned documents and publications. Any of the known conventional ingredients can be incorporated. Therefore, the medium may contain other growing crops. substances, serum, nutrients, enzymes, sequestrants, buffers and/or antibiotics. Other conventional materials can be included. In addition, substrates include proteins and other Natural substances may be incorporated or the substrate may be coated with these to affect cell growth. may be given. Examples include proteins such as elastin and collagen. these Depending on the composition, certain types of cells can be isolated, as described by Paris et al. (op. cit.). can choose about growth.

Preferably, the polymer gel used in this method is fully synthetic. This is one Generally, it takes the form of a swollen sheet, and its thickness is, for example, 0.1 to 5 cm, preferably 0°. There are 5 to 2 temples.

In order for the polymer to be sufficiently impregnated with nutrients and water, the polymer must be It has a swelling property that allows it to absorb 0.9% by weight of physiological saline by its own weight. I have to be there. That is, the polymer swells in 0.9% physiological saline. , forming a gel consisting of at least 50% by weight saline. Usually this port Rimmer absorbs 0.9% physiological saline, 60% or more - 1-1 preferably 70-1 A gel consisting of 95% saline can be formed. Polymer chopsticks forming the matrix It is often a polymer that can absorb up to 100 times its dry weight of water. water Since the sexual medium contains dissolved substances, e.g. The swelling property of polymers used in water tends to be low in pure water, but the swelling property is usually 0.9. % is the same in saline. Preferred polymers have a dry weight of 1 to 5 0 times, preferably 1.5 times, more preferably 2 times, or 3 to 20 times It is a polymer that can absorb twice as much, for example about 10 times as much, of an aqueous medium. oxygen and epithelium To improve the permeability of polymers to substances necessary for cell growth, It is better to have as high a swelling property as possible. Regarding preferred gas permeability of polymer matrices The DK value should be at least 20, preferably in the range 20-35. stomach. In addition, gels with high swelling properties have a tendency to separate from the liquid when first introduced into the culture medium. Equilibrium of the liquid phase within the medium is achieved quickly, which is advantageous for cell growth.

The polymer consists of ethylenically unsaturated monomers, preferably acrylic monomers. form from. The most suitable monomers are those in which the alkyl group has 2 to 4 carbon atoms, for example. is a hydroxyalkyl (meth)acrylate. An example of such a monomer is is hydroxyethyl acrylate (HEA), hydroxyethyl methacrylate (HEMA), hydroxypropyl methacrylate, hydroxypropyl acetate There are acrylates and hydroxytrimethylene acrylates. The monomer is preferably The material is composed of HEA and/or HEMA. Such hydroxyalkyl (meth) ) Acrylates are generally either hydrophilic or hydrophobic, resulting in copolymers one or more components selected to impart specific chemical and physical properties to Copolymerizes with nomer. Examples of comonomers include alkyl (meth)acrylates, acrylates, Rukoxyalkyl (meth)acrylate, polyalkylene glycol (meth)acrylate Acrylate, (meth)acrylic acid, (meth)acrylamide, styrene, N-bis Nillactams include N-vinylpyrrolidone. Generally, hydrophilic monomers, For example, when (meth)acrylic acid is blended, the swelling property of the polymer increases.

Preferred polymers contain high amounts of hydroxyalkyl (meth)a It is formed from acrylate and a small amount, ie 50-0%, of comonomer. most preferred The mixture contains 50-100%, preferably 75-99%, more preferably 90-98% HEMA and/or HEA and a small amount of comonomer, preferably a hydrophilic comonomer. It will be.

The polymer is preferably crosslinked. Crosslinking makes the polymer insolubilized and Swellability is controlled and physical strength is imparted to the polymer. Crosslinking is preferably is a covalent bond and generally contains di- or polyfunctional ethylenically unsaturated bonds in the polymerization mixture. The compound is added in a suitable amount, generally less than 2% by weight of the monomer mixture, e.g. 1 to 2%, preferably 0.05 to 1.5%, generally 0.05 to 1%. This is done by blending in the amount required. Examples of this type of crosslinking agent are known and include (meth)a It is a di- or tri-ester of acrylic acid. For example, alkylene carbon Alkylene di(meth)acrylates having 2 to 4 prime atoms, or alkylene di(meth)acrylates in general di- or poly-alkylene glycol (meth)acrylic acid whose carbon group has 2 to 4 carbon atoms; rylate, and alkylene bis(meth)acrylamides, usually methylene bis( There is meth)acrylamide.

Polymerization may be carried out by known methods in the absence of a diluent or in water or the like. Other suitable diluents may also be present. About polymerization of monomer aqueous solution is described in US Pat. No. 2,976,576. Liquid diluent virtually Polymerizations carried out in their absence are described in US Pat. No. 3,520,949.

Polymerizations carried out in the presence of non-aqueous diluents are described in GB-A-2097805. It is. This publication describes an ester formed from boric acid and three or more hydroxyl groups. Polymerization is carried out in the presence of tel. In EP-A-0182659, carvone Acids or anhydrides and polyols, sometimes ester reactants of difunctional carboxylic acids and polymers. Polymerization is carried out in the presence of a water-excluding solvent, including lyol itself or a mixture. . In order to form the polymer used in the present invention, the method described in the above publication is Both can be used. Polymerization can also be carried out in the presence of proteins and other natural compounds can do. The protein may be, for example, collagen or elastin.

Polymerization can be carried out by known means, such as thermal or redox initiators and/or by suitable methods. It may be started by irradiation with radiation containing a radiation-sensitive catalyst. Radiation irradiation is ultraviolet It may be ray irradiation, electron beam irradiation, or radiation irradiation from a radiation source. These radiations Curing agents suitable for use with radiation are known.

Sometimes, the gel surface can be made rough or porous to enhance adhesion to cells; This can be carried out by adding a suitable diluent to the polymerization mixture.

After polymerization, the polymer may be molded into a desired shape, but generally it is molded into the desired shape immediately. Then, it polymerizes into a sheet. Therefore, the polymerization mixture is polymerized in the mold. or, Typically, the polymerization mixture is cast onto a flat surface suitably coated with a protective sheet material, and then the polymerization mixture is do. The thickness of the mixture layer is between 0.1 and 2-9 m, preferably between 0.2 and 1 m, more preferably between It is almost 0.5-.

After polymerization is complete, the polymer is washed to remove low molecular weight impurities consisting of excess unpolymerized material. leave This polymer is used in contact with cells, so excess monomer is removed. It is very important to remove this by cleaning. Cleaning is suitable for this purpose. It can be carried out by any suitable known method. The cleaning method is to use deionized water. several continuous cleaning steps using water of different conductivities or several continuous cleaning steps using water of different conductivity It is fine as long as it is. One suitable method involves exposing the polymer to water of increasing conductivity. Soak.

Polymers can be supplied in polymer-filled packages as described above. This puff cage then contains the components of the cell culture medium. This package is the invention It is suitable for the new method by. The puff cage contains a dry polymer containing the above substances. Preferably, it is swollen with an aqueous solution containing the above components to form a gel. Made of polymer. Therefore, the package contains serum, growth agents, nutrients, enzymes. , a sequestering agent, a buffer and/or an antibiotic.

In addition, gel is a container suitable for direct use as a container for the culture medium in the method of the present invention. It can also be provided as But more conveniently, it is easy to use in the laboratory. , polymer as a sterile sealed package in foil, generally metal foil or plastic foil I will provide a. The sealed puff cage is preferably kept under external pressure to prevent rupture. , sterilize using steam sterilization. External pressure is applied by holding the puff cage inside the mold. However, it is more convenient to use gas pressure. Preferably, auto Use a clave, preferably an autoclave whose pressure can be changed independently each time and sterilize it.

The present invention also provides synthetic hydrophilic polymers that are swollen in an aqueous solution and exhibit water-insolubility and water-swellability. a gel matrix with cultured mammalian cells on its surface. It also provides new products that This new product is manufactured by the new method of the present invention. therefore, the cells are preferably fibroblasts or epithelial cells.

A particularly useful application of the product is, for example, animal or human epidermal grafts. like this In some applications, cells coated with a layer (generally essentially a monolayer) of epithelial cells or fibroblasts are used. The gel is applied to a wound, eg a burn, cell side down. This gel is especially effective against bacteria. It acts as a barrier, and has sufficient oxygen permeability to maintain its healing effect. Demonstrate. For further protection and to keep the gel in place, To keep it moist, it is preferable to cover the bandage with another covering.

Generally, the gel is left in place for several days until the cells engraft into the wound. , remove the gel and cover with another swollen gel of the same type or with a regular wound dressing. . The aqueous solution containing the swollen gel is loaded with nutrients, growth factors, and Antibiotics, analgesics, anti-inflammatory drugs, etc. can be added.

When a product carrying a fibroblast layer is applied to a wound, the fibroblasts are present at the wound site. Acts as a supply cell for existing natural epithelial cells, stimulating them to proliferate and differentiate. induce wound healing. The epithelial cells are stimulated to form a gel. Because it grows under the wound contact side, the gel can later be pulled away from the wound. − I- Because gels are permeable to skin cell nutrients, they are Can spread to cells. Preferably, the fibroblast culture originates from a patient's wound. for this purpose This is particularly useful. This is because fibroblasts are relatively easy to culture in vitro. Also, since the growth of epithelial cells is stimulated in vivo by cultured fibroblasts, This is because there is no need to culture bithelial cells in vitro. This is done in vitro. This is particularly advantageous given the great difficulties in growing skin cells. particularly severe For burns, the wound can be inoculated with epithelial cells or cultured with fibroblasts. At the same time, cultured epithelial cells are used.

Products with cultured epithelial cells could also be applied to wounds by other means.

Such products can be used in any of the methods described in the literature. cultured epithelium or skin consisting of a collagen gel supporting an epithelial cell culture It can be used in place of a cell sheet. If necessary, immediately after applying to the wound. , or later, as disclosed in U.S. Pat. No. 4,016,036. Gels can be separated from adherent cells by using spases.

The gel used in the present invention has numerous advantages over previously used substrates. ing. Also, when compared to a hard surface, it is permeable, which means that nutrients in the medium and its The advantage is that other components are supplied to the basal cells of the medium, thus making growth more efficient. Ru. The cost is much lower than that of collagen, and it is well understood, making it a clinical choice. Extremely advantageous on the floor. Furthermore, it is very easy to physically handle and chemically Since it is well understood, standardization and control are also easier. In addition, composite Therefore, it is an unfavorable substrate for the growth of microbial cells, and this It is advantageous not only in terms of quality but also in the final product and eliminates the risk of bacterial or other infections. Since the swollen gel is relatively permeable to oxygen, bacteria and other Prolongs healing while acting as a barrier against undesirable components of the body.

The gel can also be steam sterilized prior to use, which is highly advantageous. Additionally, the gel is transparent. Since it is bright, it is possible to observe the state of wound healing through this gel.

When the product of the present invention is used as a wound treatment adjuvant, foreign body tissue and collagen, etc. The use of natural substances can be avoided, thus minimizing the risk of infection or immune rejection. Can be done to a minimum.

Another use of the method of the invention is particularly when subculturing is required or for samples of cultured cells. testing of the cells themselves when it is necessary to perform a number of different tests on be.

The present invention will be explained below with reference to Examples.

97.5 parts (7) HEMA (200 ppm hydrobovine nonmonomethyl ether HQME), 1 part HEA (containing 200 ppm HQME) and 0. 75 parts of methacrylic acid to 0.5 parts of ethylene glycol di-methacrylate of the crosslinking monomer. 0.25 parts of curing agent (DARO CUR1173) was added and then cast on a flat surface to a thickness of 0.5-. 200 The mixture was irradiated with UV at a wavelength of 365 nm for approximately 60 minutes at an intensity of Hardened.

After polymerization, the polymer was peeled off from the flat surface and the conductivity was 750μS/cm'17) It was soaked in water for about 12 hours. Next, it was exposed to water with a conductivity of 1550μS/cs''. for about 12 hours, and then for several hours in a double-distilled aqueous solution of 0.9% sodium chloride. The polymer was immersed in. Next, polypropylene with excess impregnating agent sodium chloride solution A pyrene foil container was filled with gel sheet pieces. Heat-seal this bag and then heat it to 121℃. Sterilize in an autoclave for 15-20 minutes at temperature.

Polymers prepared in this manner can absorb 0.9% saline and produce 70% % of the impregnating agent liquid could be formed.

Use as culture substrate The sheet of polymer gel formed as described above was heated at 34°C for 3 days before the experiment. Incubation was carried out in sterile MEM (minimum essential medium). gas atmosphere is air and 5% CO1.

Cut the polymer sheet into 1 square piece and culture flask: 2 (Nunc Jon R) and a small amount of sterile silicone fat to prevent the polymer sheet from floating. It was fixed to the surface of the culture flask using a loft. Three gel pieces were placed in each culture flask. .

a) Rat mouths were prepared according to the method described by Jepsen et al. (cited above). Cultures of tectal epithelial (RPE) cells were created, maintained, and subcultured. main research Cells that had been passaged 20 times were used.

b) derived from the above RPE cell line following the same method as non-tumorigenic RPE cells. The derived transformed tumorigenic cell lines were maintained and subcultured.

c) As described by Arenholdt-Bindslev et al. Cultures of normal hydrocoel epithelial cells were created, maintained, and passaged according to methods described in Cultured.

Immediately after introducing the gel fragments into the culture flask, a total of 5XIO” cells were added. Cell suspension7. 5ml' was placed in each flask.

Each cell was introduced into two flasks. Leave the cells for 4 days to allow them to attach. Ta. After cell attachment, phase-contrast microscopy was performed, and the biopsy material was removed and optically removed on the 6th day. Examined with a microscope. The photo was taken on the 6th day.

After 4 days, all three types of cells were attached to the gel fragment surface. from the next day Cell proliferation was observed on the gel. On day 6, tumorigenic and non-tumorigenic The gel incubated with both rat oral epithelial cells was covered entirely with epithelial cells. It was covered with layers. Human epithelial cells also proliferated, but at a slower rate.

Submission of translation of written amendment (Article 184-8 of the Patent Law) October 20, 1999

Claims (12)

[Claims] 1. In the presence of an aqueous medium, the water-insoluble and water-swellable properties swelled with the medium A gel consisting of a hydrophilic synthetic polymer is immobilized on the surface of a removable substrate. in a method of culturing mammalian cells in which the polymer contains 0.9% by weight physiological saline. Characterized by exhibiting swelling properties that allow it to absorb at least as much salt water as its dry weight. The above method. 2. 70% to 95% by weight when the gel is swollen with 0.9% physiological saline. The method according to claim 1, characterized in that it exhibits swelling properties such that it consists of. 3. Claim 1 or 2, wherein the polymer forming the gel is a fully synthetic polymer. The method described in Section 2. 4. The polymer is preferably made from an ethylenically unsaturated monomer consisting of an acrylic monomer. 4. The method according to claim 1, wherein a mer is formed. 5. Any one of claims 1 to 4, wherein the cells consist of fibroblasts and/or epithelial cells. or the method described in paragraph 1. 6. the cells are epithelial cells and the medium is preferably derived from a feeding cell Claims comprising one or more substances that promote the growth of said epithelial cells consisting of a substance. The method described in Section 5. 7. Claim 6, wherein the supply cells consist of vagina cells, fibroblasts, or 3T3 cells. the method of. 8. The medium contains growth agents, serum, nutrients, enzymes, sequestrants, buffers. and an antibiotic. How to put it on. 9. Claim 1, wherein said fixation-dependent cells are under said aqueous medium throughout the process. The method according to any one of items 1 to 8. 10. Made from synthetic hydrophilic polymers that are water-insoluble and water-swellable, swollen in aqueous solutions. A preparation consisting of a gel matrix with anchorage-dependent mammalian cells grown and cultured. Goods. 11. 11. The product of claim 10, wherein the gel is in the form of a sheet. 12. The product of claim 10 or 11 and/or any of claims 1 to 9 A wound dressing, which is a product produced by the method according to item 1.