JPH02234670A - Substrate for cell culture - 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] [Industrial application field] The present invention relates to a cell culture substrate.

[Conventional technology]

Useful live-embedded active substances, such as interferon and plasminodan activator, which are difficult to produce by organic synthesis or microbial fermentation, can be produced by culturing adherent cells such as fibroblasts. The development of culture technology has become active.

When culturing adherent cells on a small scale, a flat method using glass or plastic flasks, petri dishes, etc. is adopted, and when producing the above useful substances, production efficiency is In order to improve this, three-dimensional methods are being used using microcarriers, hollow fibers, etc.

In either case, the technical point is how to improve the adhesion and compostability of cells during culture, and various efforts have been made in the past.

Among these, JP-A-58-7188/I proposes a method of coating the surface of glass or plastic molded into a flat, spherical, or hollow fiber shape with an adhesive protein such as collagen or gelatin. Furthermore, in JP-A No. 61-274, adhesive proteins such as collagen and gelatin are molded into membranes, spheres, hollow fibers, etc., and the molded products are used as substrates for subculture. A method has been proposed.

4 West 7 However, the adhesive protein mentioned above is a natural high-t! -It is currently extremely expensive because it uses protein raw materials and has a high purity obtained through a complicated purification process.

Furthermore, when the adhesive protein is irradiated with gamma rays, it becomes low molecular weight and loses its adhesive properties, so a substrate for cell culture using this adhesive protein has the disadvantage that it cannot be sterilized with gamma rays.

[Problem to be solved by the invention]

Under these circumstances, the present invention provides inexpensive and γ
The purpose of this invention is to provide a carrier for animal cell culture that can be subjected to various sterilization operations including radiation sterilization.

The present inventors focused on the fact that the main component of eggshell membranes is protein that is insoluble in fresh water, and that animal cells adhere well to the surface of eggshell membranes, and as a result of intensive research, completed the present invention. This is what I did.

[Failure to solve the problem]

The present invention relates to a substrate for cell culture, and is characterized in that it uses eggshell membrane as a carrier.

In the present invention, the eggshell membrane refers to a membrane attached to the inside of the eggshell of a reptile egg such as a bird egg.

In addition, the carrier refers to a member on which cells are supported and grow when culturing adherent animal cells such as fibroblasts and hybridomas.

Furthermore, the cell culture substrate refers to containers, microcarriers, films, hollow fibers, etc. for culturing cells.

To obtain the cell culture substrate of the present invention, first, the eggshell membrane adhering to the eggshell is separated manually or mechanically. If the eggshell membrane is attached to the eggshell membrane, it is difficult to process it into a carrier, so it is desirable to remove the eggshell by treating the obtained eggshell membrane with an acid.

Next, there are various methods for using the eggshell membrane thus obtained as a carrier, but one example is to dry the eggshell membrane and then crush it into powder, preferably having a particle size of 100 μm to 400 μm. Then, it can be used as it is as a microcarrier (a type of carrier). In addition, eggshell membranes are finely powdered (
If this fine powder is supported on the surface of small spheres made of plastic such as polystyrene by a known method, a microcarrier whose surface is coated with eggshell membrane can be obtained.

As another example, an aqueous solution of soluble eggshell membranes obtained by treating eggshell membranes with acids, alkalis, organic solvents, oxidizing agents, reducing agents, etc. is applied to a container such as a flask or a petri dish, and then air-dried. In this way, a cell culture container coated with eggshell membrane can be obtained. However, microcarriers or films coated with eggshell membranes can be obtained by immersing small spheres or films made of glass, plastic, etc. in the aqueous solution of soluble eggshell membranes and then air-drying them.

Furthermore, to give another example, eggshell membranes are reduced and dissolved with β-mercazotoethanol/thioglycolic acid to obtain an aqueous solution of soluble eggshell membranes, and this aqueous solution is applied to a glass plate.
After drying by heating, a water-insoluble film can be obtained by peeling it from the glass plate.

[Effect]

It exhibits the same cell adhesion and proliferation effect as conventional cell culture substrates.

Although we have not investigated the principle in depth, it is speculated that the eggshell membrane has a strong affinity for adherent cells.

〔Example〕

Example 1. After cracking chicken eggs with shells and removing the egg liquid, the obtained eggshells with eggshell membranes were placed in clear water, and the eggshells were removed manually to obtain eggshell membranes.

Next, the obtained eggshell membrane was immersed in a 1st volume hydrochloric acid aqueous solution for 1 hour to dissolve minute eggshells attached to the eggshell membrane, and then heated to 80°C.
The sample was immersed in hot water for 3 hours, and then washed with water.

Next, this eggshell membrane was dried in the sun, and then pulverized in a screen mill.Then, the pulverized product was sieved to obtain an eggshell membrane powder with an average particle size of 200 μm.

Then, the obtained eggshell membrane powder was directly used as a cell culture substrate.

Example 2. After cracking chicken eggs with shells and removing the egg liquid, the resulting eggshells with eggshell membranes were placed in clean water, and the eggshells were completely removed manually to obtain eggshell membranes.

Next, the obtained eggshell membrane was 0. After being immersed in a 1% caustic soda aqueous solution for 2 hours, it was washed with water.

Next, this eggshell membrane was dried in the sun, and then ground in an immersion mill to obtain eggshell membrane powder with an average particle size of 300 μm.

The obtained eggshell membrane powder was then used as a substrate for culturing 1-year-old cells.

Example 3 After cracking chicken eggs and removing the liquid, the resulting eggshells were crushed.

Next, the crushed eggshell membrane example was placed in clear water and stirred, and the eggshell membrane that separated from the eggshell and floated to the surface was collected.

Next, the obtained eggshell membranes were immersed in a 1st volume aqueous hydrochloric acid solution for 1 hour, washed with water, and then dried in the sun.

Next, after coarsely pulverizing this eggshell membrane in an umber mill,
Geno 1 Mill (■ Seinon Enterprises, product name r Co-
JET sysTEMo! JJ) Kte fine grinding,
A fine powder of eggshell membranes with an average particle size of 12 μm was obtained.

Next, a particle size of 200 μm was prepared separately from this eggshell membrane fine powder.
After mixing with m spheres made of polystyrene to form an ordered mixture state, the microcarriers were treated by impact method in a high-speed air stream, and the surface of the small polystyrene spheres was stably supported by the eggshell membrane. was gotten.

Example 4.

Dried eggshell membrane obtained by the same method as Example 3]. 0 0
g to 1-200 ml of 2N aqueous sodium hydroxide solution. 1 and 800 ml of absolute ethanol. was added and treated at 40° C. for 5 hours with stirring to solubilize the eggshell membranes.

Next, this liquid is separated by F through a cloth filter, the F liquid is desalted, and then freeze-dried to form a powder of soluble eggshell membranes 53.
I got g. Then, 90μ of the obtained soluble eggshell membrane powder
g was dissolved in 2 ml of clean water, and then sterilized by heating at 121° C. for 15 minutes.

Next, this aqueous solution was added to a polystyrene Petri dish (diameter 35 mm) prepared separately and air-dried aseptically, resulting in a cell culture Petri dish whose surface was covered with eggshell membrane of 10/1 g/Cr/L2. Obtained.

Example 5, 100 g of dried eggshell membranes obtained by the same method as Example 3,
After adding 500 ml of 1M aqueous 2-mercaptoethanol solution and adjusting the pH to 9.5, the mixture was treated at 60° C. for 4 hours to solubilize the eggshell membranes.

Next, this solution was centrifuged (6000 rpm).
After removing insoluble matter (20 minutes), dialysis was performed to obtain a solution with a protein content of 8.

Next, the solution obtained in the above method was applied to a separately prepared polyethylene sheet (surface treated with plasma), and when air-dried, the cell culture showed that the surface was strongly coated with 5 μI/crn2 eggshell membrane. A sheet was obtained.

[Test example]

Test Example 1 After dry heat sterilizing 2 g of the eggshell membrane powder obtained in Example 1 at 160°C for 2 hours, the flask was charged with Dulbesoco's modified Eagle's medium (10 g).
50 ml of fetal bovine serum (containing bovine fetal serum) was added and stirred for 30 minutes to swell the eggshell membrane powder.

Next, human dermis-derived fibroblasts were added to the flask at 4×1.
Add 50 ml of dispersed medium (same composition as the Dulbesoco modified Eagle medium) and incubate at 50 r.p.
After stirring at m for 2 minutes, the mixture was allowed to stand for 1 hour. After that, 50 r.p. every hour. p. Repeat the stirring operation for 2 minutes at 50 r.m six times, then stir at 50 r.m. p. Stir continuously at m,
37 Product name r KOKEN CELLGEN 1 -
The same test as the test group was conducted using 2J of microcarriers (particle size: 200 μm) prepared by a conventional method (control group).

When used as the number of living cells after 2 days, 4 days, and 6 days, it has a cell proliferation effect comparable to that of conventional microcarriers.

Table 1 The numbers in the table indicate the number of living cells per ml of culture solution.

Test example 2. 5. Human dermis-derived fibroblasts were placed in the Vetri dish obtained in Example 4. Add 2 ml of Dulphecco's modified Eagle's medium 40 (containing fetal bovine serum) in which OX104/ml was isolated, and mix at 37°C.
The cells were cultured for 7 days (test group).

As a control, a polystyrene vetri dish with an untreated surface (
Collagen 4f#-θ prepared according to Example 4 using control group 1) and commercially available purified colladan (manufactured by Koken, trade name "KOKEN CELLGEN 1-PCJ")
(10μ#/cm2) The same test as above was performed using a Petri dish (control group 2).

Then, when the number of living cells in the whole culture dish was measured after 2 days, 4 days, and 7 days, the results shown in Table 2 were obtained.

As is clear from Table 2, the eggshell membrane-coated vitriol dish has a cell growth effect comparable to that of the conventional collage-coated vitriol dish.

After sterilizing 2 g of the microcarriers obtained in Test Example 3 and Example 3 with gamma rays, the microcarriers were placed in a spinner flask.

Next, add 2x10 human dermis-derived fibroblasts to this flask.
Dulbesoko's modified Eagle's medium (10
Add 100 ml of fetal bovine serum and incubate for 30 r. p
．． The cells were cultured at 35° C. for 6 days with stirring.

Then, the number of living cells in the entire flask was measured after 2 days, 4 days, and 6 days, and the results shown in Table 3 were obtained.

As is clear from Table 3, the microcarriers carrying eggshell membranes do not deteriorate in cell adhesion or proliferation even when irradiated with gamma rays, and have a good proliferation effect.

table Note that the numbers in the table indicate the number of living cells per ml of culture medium.

〔Effect of the invention〕

As described above, since the cell culture substrate of the present invention is made of eggshell membrane as a carrier, the cell culture substrate of the present invention is processed into a cylindrical shape without special purification, etc. If vascular endothelial cells are cultured, it can be applied to a hybrid artificial blood vessel, and if processed into an organ shape and cultured with hepatic parenchymal cells on the inner surface, it can be applied to a hybrid artificial liver. .

A silicone or polyvinyl alcohol sheet supported with eggshell membrane can be used as a wound dressing.

It exhibits a culture effect comparable to that of cell culture substrates.

Furthermore, even when sterilized with gamma rays, eggshell membranes do not decompose, so the base material can be reliably sterilized within a few hours.

Claims (1)

[Claims] A cell culture substrate characterized by using eggshell membrane as a carrier.