JPS6379588A - Base material for cell culture and production thereof - Google Patents

Abstract

PURPOSE:To obtain a base material for cell culture, suitable as a wound covering material, having high adaptability to organisms free from immunogenicity, by making a collagen-containing base material support a cytostatic factor derived from blood platelet. CONSTITUTION:A solution of collagen of various animal sources is blended with an equilibrium solution (e.g. Hanks medium), cultivated at about 37 deg.C for 10-15min, refibrillated and gelatinized. The gelatinous material is mixed with a solution containing a blood platelet component such as PRP (platelet-rich plasma), etc., cultivated for 30-60min, the platelet is bonded to and aggregated on the collagen fibrins. The released cytostatic factor is supported on the gelatinous material, washed with a phosphoric acid buffer solution to give the aimed base material. The collagen solution is lyophilized and made into a film to give a collagen layer, which is immersed in 0.05-2% solution of a crosslinking agent such as glutaraldehyde, etc., at 25-30 deg.C for 5-24hr, crosslinked and the crosslinked collagen layer can be blended with a solution containing a blood platelet component to give a product.

Description

Detailed Description of the Invention [5] Background of the Invention (Field of the Invention) The present invention relates to a cell culture substrate and a method for producing the same. Specifically, the present invention aims to provide a cell culture substrate that strongly supports cell proliferation and a method for producing the same.

(Prior Art) In recent years, in the fields of medicine and biology, somatic cells have been studied in vivo or in vitro.
There is interest in culturing in vitro.

For example, in cases of skin tissue loss, especially extensive skin loss, due to diseases or wounds such as burns, skin harvest and abrasion wounds, traumatic skin loss wounds, infection in the affected area and excessive loss of body fluids. Since there is a risk of immediate life-threatening danger, prompt tissue repair is desired. Currently, the best treatment for such affected areas is to remove skin from the person's normal area and perform autografting, but autografting is not always applicable, and for example This becomes extremely difficult when the defect is spread over a wide area, and even if it is applicable, it is impossible to transplant all the defects at once, and it is often necessary to repeat the transplant many times over a long period of time. To this end, it is desirable to develop wound dressings that can temporarily or permanently cover the affected area in place of autografts, prevent bacterial infection and loss of body fluids, and promote tissue repair by proliferating tissue cells. It is rare.

Items to cover such wounds include gauze, etc.)
Erasable cotton has been used for a long time, but these have low antibacterial properties, deteriorate due to exudate, need to be replaced frequently, and are rarely effective in promoting tissue repair. . In addition, as alternatives to these, coating membranes made of artificial materials such as silicone gauze, silicone rubber membranes, and synthetic fiber sheets such as nylon and Teflon with velour-like surface markings have also been developed.
These have problems in terms of biocompatibility, and also have various problems in terms of adhesion to the affected area, water vapor permeability, cracking, etc. Furthermore, allogeneic skin grafts and xenografts such as dried pig skin, amniotic membrane, fibrin membranes from xenobiotic animals, and collagen membranes are also known. Although these biologically derived coating membranes are excellent in terms of biocompatibility, most of them are immunogenic (antigenic) and have the potential for bacterial infection and deterioration due to exudates. Many of them are large.

Among these, allogeneic skin grafting has excellent clinical effects, but
It is difficult to obtain as it requires a kidney donor, and amniotic membrane has low antigenicity, so comparatively good results can be expected, but this has also been difficult to obtain.

Collagen is relatively easy to obtain and has often been used not only as a wound covering membrane but also as a base material for cell culture systems, and has been treated with trypsin, pepsin, and papain. It has also been widely studied to reduce collagen to a substance that exhibits little to no oxidation, and to inhibit collagenase degradation by fibrillizing or crosslinking collagen. However, on the other hand, it is also known that collagen suppresses the spread and proliferation of cells in a concentration-dependent manner, and collagen membranes have not been sufficient as substrates for cell culture.

Furthermore, in recent years, a method of transplanting a material similar to the skin, which can be called a cultured transplantation method or a cultured skin method, has been carried out. This is aimed at severely ill patients, by collecting the remaining normal skin of the patient and culturing the isolated cells in a test tube.
After proliferating several tens to hundreds of times the original cell size, the covering,
It is transplanted together with a membrane into a patient's wound site to form epidermis and promote healing. As an example of clinical application of this method, Galico et al.
New England Journal of Medicine, Vol. 311, No. 7, No. 448-
451 pages (1984) [New, Eng, J.
, Med, , Vol. 311. NO, 7pp44
8-451 (1984)]). In this clinical case, gauze is used as a dressing, which gives it a classic feel.

TI, OBJECTS OF THE INVENTION Accordingly, the present invention provides a novel cell culture substrate and its)
′! The purpose is to provide a manufacturing method. Another object of the present invention is to provide a cell culture tool that strongly supports cell proliferation and a method for manufacturing the same. The present invention also has high biocompatibility and is non-immunogenic, and when applied as a wound dressing, promotes cell proliferation, promotes early granulation layer formation or epidermis formation, and promotes healing. The purpose of the present invention is to provide a cell culture substrate and a method for producing the same.

The above-mentioned advantages can be achieved by a cell @ cultivation substrate characterized by having a collagen-containing substrate support platelet-derived cell growth factors.

The present invention also provides a substrate for cell culture in which collagen is made into oriented fibers. The present invention also provides a cell culture substrate in which collagen is crosslinked. The present invention further provides a substrate containing collagen, which is a substrate for cell culture, which is obtained by coating an air-permeable support with collagen.

The present invention also provides a cell culture clogging material in which the collagen-containing base material consists essentially of collagen. The present invention also provides a water-based substrate for cell jf3f3, in which the collagen-containing substrate is attenuated with two breathable support layers. Broadly speaking, the present invention provides a cell culture substrate in which the collagen-containing substrate has a gel-like, film-like, woven fabric-like, non-woven fabric-like, porous, or sponge-like form. . The present invention also relates to a cell culture substrate in which the support layer is made of polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer, polyvinyl chloride, polyester or fluororesin. This indicates the material.

In the present invention, the base material containing collagen has a pore size of 1 to 10
This shows a cell culture substrate having pores of 0 μm. In the present invention, the membrane pores of the substrate for cell culture are roughly 1.
This figure shows a cell PJ culture substrate having a size of about 0 μm to 1 Q mm. The present invention also provides a cell culture substrate that is applied as a wound dressing.

The above method also includes adding a balanced salt solution to the collagen solution, then incubating it at about 37°C to gel it, and then adding a fresh platelet component-containing solution to this gel and incubating it. This is achieved by a method for manufacturing a cell culture substrate characterized by:

The present invention also provides a method for producing a cell culture substrate in which the balanced salt solution is Hank's solution.

The present invention further provides a method for producing a cell culture substrate in which the platelet component-containing solution is platelet-containing plasma. The present invention also provides a method for producing a substrate for cell culture in which a homogenization treatment is performed after adding an equilibrium warm solution to a collagen solution, followed by incubation at about 37°C.

The above method further includes freezing and drying the collagen solution, subjecting the obtained collagen layer to crosslinking treatment, and then impregnating the crosslinked collagen layer with a fresh platelet component-containing solution and incubating it. This is achieved by a method for manufacturing a cell culture substrate characterized by:

The present invention also provides a method for producing a cell culture substrate, wherein the crosslinking treatment uses a treatment agent selected from the group consisting of glutaraldehyde, formaldehyde and glyoxal. The present invention further provides a method for producing a cell culture substrate in which a collagen layer is formed on a support layer. The present invention also provides a method for producing a substrate for cell culture in which the support layer has air permeability. The present invention further provides a support layer for cell culture which is made of polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer, polyvinyl chloride, polyester or fluororesin. This shows a method for manufacturing the base material.

The present invention also provides a method for producing a substrate for cell culture, in which the fresh platelet component-containing solution impregnated into the collagen layer is applied as a mixture with the collagen solution. The present invention generally provides a method for producing a cell culture substrate, in which the collagen solution mixed with the fresh platelet component-containing solution is incubated at 37° C. with a balanced salt solution added in advance. .

II. DETAILED DESCRIPTION OF THE INVENTION Collagen is a major protein component of the connective systems of vertebrates and other vertebrates, and as mentioned above, it is a highly biocompatible and well-purified protein. l,t, Shows almost no immunogenicity. It is also known that collagen causes platelet aggregation when it comes into contact with platelets.

On the other hand, it is known that platelets contain various cell growth factors. This means that, in general, when cells are cultured in vitro, normal cells do not grow in a normal Pi nutrient solution alone, and it is necessary to add an appropriate amount of serum to the culture solution, and also to This is supported by the fact that when comparing the effects of serum and plasma on cells, cells proliferate well in serum, but plasma has very little proliferation-promoting effect.

It is also believed that these cell growth factors are released into serum during platelet aggregation. These platelet-derived cell growth factors are difficult to isolate and identify because they exist in very small amounts in platelets and have properties such as being easily sucked onto glass. There are also very few such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEPF), and epidermal growth factor (EGF).

However, when the present inventors exposed platelets to collagen and caused platelet aggregation on the substrate surface, surprisingly, the above platelet-derived cell growth factors were easily absorbed by the substrate. It has been revealed that when various cells are cultured using a substrate treated in this way, cell proliferation is promoted even without the use of serum.

Hereinafter, the present invention will be described in more detail based on embodiments.

The cell culture substrate of the present invention is characterized in that the substrate containing collagen supports cell growth factors derived from platelets.

Collagen used in the present invention may be derived from various animal sources, such as collagen derived from bovine skin, bovine quince, bovine bone, azolocollagen (ΔC), succinylated collagen (SAC), etc.
), methylated collagen (MAC), and collagen adsorbed with a substance containing a large amount of guanidyl groups such as protamine.

By adjusting the ionic strength and DH':9 of collagen molecules, it can be used as fibers with orientation.
Furthermore, it is also possible to crosslink collagen. This is advantageous in that, for example, when the cell culture substrate of the present invention is used as a wound dressing, decomposition by enzymes such as collagenase can be suppressed. Oriented fibers can be obtained, for example, by mixing a collagen solution with a balanced salt solution, such as Hank's solution, and incubating at about 37°C.

For crosslinking, crosslinking agents such as aldehydes such as glutaraldehyde, formaldehyde and glyoxal, diisocyanates such as hexamethylene diisocyanate and diphenylmethane isocyanate, carbodiimides and azides, preferably aldehydes, particularly preferably glutaraldehyde, are used. It can be done using

The collagen-containing base material according to the present invention may be composed of a collagen layer substantially formed only of collagen as described above, or may be composed of a support coated with collagen. When a cell culture substrate is used as a wound dressing, it should be made by lining a breathable support layer with a collagen-containing substrate to prevent body fluid leakage and bacterial infection. preferable. Such a collagen-containing base material has a gel-like, film-like, woven fabric-like, non-woven fabric-like, porous, or sponge-like form. Examples of the material for the support include nylon, polypropylene, polyethylene, and polyester. Further, as a support layer lining such a collagen-containing base material, polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer,
Desirable materials include polyvinyl chloride, polyester, and fluororesin. In addition, the base material containing collagen has a pore size of 1 to 1
It is desirable to have pores of 1001-I so that cultured cells can grow in the pores and migrate through the pores. The cell IR valley base material of the present invention having such a configuration has membrane pores of about 10 μm to 1 Q mm.

In the cell culture substrate of the present invention, a platelet-derived cell growth factor is supported on the collagen-containing substrate. The cell growth factors supported are those released when platelets come into contact with a collagen-containing substrate and aggregate.
Although the details of the supported cell growth factors are not fully clear, the obtained cell culture substrate supports the proliferation of many normal cells such as epidermal cells, fibroblasts, and vascular endothelial cells. Met.

In the cell culture substrate of the present invention, if necessary, Na, Ca, MQ, P, C! It is also possible to add or impregnate basic inorganic components such as Q, carbon sources, nitrogen sources, amino acids, vitamins, hormones, etc. For example, in the known composition of synthetic media such as Dulbecco's modified MEM medium and MCDB-104 medium. It can be added or soaked according to the standard.

The cell culture substrate of the present invention is stored aseptically until use. For example, the cell culture substrate of the present invention can be aseptically 1'!
The cell culture substrate is sealed in a sealed container made of a non-toxic material such as polyurethane or glass that has been sterilized in advance, or the cell culture substrate is sealed in the sealed container and then sterilized, preferably. are stored by sterilizing them with gamma rays.

The cell culture clogging material of the present invention is obtained by causing platelet aggregation in a collagen-containing base material and supporting the cell growth factors released at this time, and is necessarily obtained by, for example, the following: It can be manufactured satisfactorily by the two manufacturing methods shown below.

In the first method, a balanced salt solution, such as Hank's solution, is added to the collagen solution. Next,
After homogenizing this mixture as necessary, approximately 37°C
The mixture is incubated for 5 to 100 minutes, preferably 10 to 15 minutes, to refibrillate the collagen and roughly gel the mixture. Thereafter, a fresh platelet component-containing solution is added to this gel and incubated for 5 to 100 minutes, preferably 30 to 60 minutes, to cause the platelets to react with collagen and cohere and aggregate on collagen fibers. Fresh platelet component-containing solutions include platelet-containing plasma such as platelet-rich blood Wu (PPP) obtained by centrifugation from anticoagulated whole blood of the same type as cultured cells, or platelet-containing plasma further gel-filtered. For example, platelets obtained by suspending platelets in an appropriate buffer can be used. In this way, after the cell growth factors released by platelet aggregation are supported on the gel, the gel is thoroughly washed with an appropriate washing solution, such as a phosphate buffer, to produce a product. get.

In the second manufacturing method, the collagen solution is first freeze-dried to form a film. The freeze-drying conditions are as follows:
For example, a collagen solution may be immersed in liquid nitrogen to rapidly freeze it, and then degassed to 10" Torr in 1 to 3 minutes while maintaining the temperature at about -10'C to -30'C. It will be done.

The collagen layer thus formed is then subjected to a crosslinking treatment. The cross-linking treatment involves adding the collagen layer to a 0.05-2% solution of a cross-linking agent at a temperature of 25-30°C for 5-24 hours.
This can be done by a time soak treatment. Examples of crosslinking agents include aldehydes such as glutaraldehyde, formaldehyde, and glyogyzal; diincyanates such as hexamethylene diisocyanate and diphenylmethane isocyanate; crosslinking agents such as carbodiimides and heptadides; preferably aldehydes; particularly preferably glutaraldehyde. be. After the cross-linking process is completed, the collagen is thoroughly washed to remove unreacted cross-linking agent. The crosslinked collagen layer thus obtained is formed on the support layer, if necessary. The support layer is preferably one that is breathable; for example, wood containing collagen can be used in the form of a film, a woven fabric, a non-woven fabric, a porous award certificate,
Or it has a spongy form.

Examples of the material used include polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer, polyvinyl chloride, polyester, and fluororesin. For example, when attempting to form a cross-linked collagen layer on a polyurethane film, a urethane solution is applied onto the plug, and this immersion is performed in the following manner: 1. 'Place the crosslinked collagen layer on top of the first layer,
It can be obtained by allowing it to stand at room temperature for an appropriate period of time and then crosslinking it by heating.

A fresh platelet component-containing solution is then added to the crosslinked collagen layer and incubated for 5 to 100 minutes, preferably 30 to 60 minutes, to allow the platelets to interact with the collagen and make the platelets stick to the crosslinked collagen layer. , coagulate. As the fresh platelet component-containing solution, a solution similar to that shown in the first production method is used. Moreover, this solution containing platelet components may be applied as a mixture with a collagen solution. When applied as a mixture with a collagen solution in this manner, the platelet aggregation effect is promoted. Furthermore, it is preferable that this collagen solution is incubated in advance at 37° C. with a balanced salt solution, since platelet aggregation is further promoted. It is also possible to promote platelet aggregation by adding other substances that promote platelet aggregation, such as 80P, thrombin, calcium, etc. In this way, the cell proliferation M released by platelet aggregation
After the factor is supported on the cross-linked collagen layer, the product is obtained by thoroughly washing the cross-linked collagen layer with an appropriate washing solution, for example, a phosphate buffer solution.

The cell bottle base material of the present invention, which can be assembled in this way, can be used in various types of cell culture systems, in vivo or in vitro, and can be used to treat skin defects due to burns, etc.
When this occurs, 01 wound dressing is applied to the affected area in order to cause proliferation of epithelial cells and fibroblasts in the affected area.

(Example) EXAMPLES The present invention will be explained in detail below with reference to Examples.

Example 1 Collagen solution (manufactured by Takasen, 0.3%, for tissue culture)
Double concentrated Hank's M and distilled water were mixed in a ratio (volume) of 3:1:5 at 4°C.

This mixture was poured into a 12-well plate (manufactured by Terumo Corporation, flat bottom,
After adding 0.2 ml each to the wells of a tissue culture laboratory and homogenizing the mixture, the mixture was immediately transferred to a 37° C. incubator, incubated for two weeks, and solidified into agar.

Fresh platelet-containing plasma (5x
105/mm3) was added for 0.2 d and shaken lightly.

At this later elapsed time, above the well? The number of platelets contained in uH was calculated using an automatic white cell counter (Ortho Inc. %, 14
) to determine the relationship between elapsed time and platelet adsorption rate, and at the same time, the collagen gel was scanned and observed using a deuteron microscope to examine platelet adhesion and aggregation status. The results are shown in Table 1 and Figure 1.

The results revealed that after 60 minutes, 80% of the platelets had adhered and aggregated due to reaction with collagen.

The adsorption of platelet-derived growth factor, which is thought to be released by rough aggregation, onto collagen gel was investigated using platelet factor 4 (PF~4) and β-thromboglobulin (β-thromboglobulin) in the supernatant.
-TG) as a marker, it was found that most of the collagen was adsorbed. The results are shown in Table 2.

Comparative Examples 1 to 2 For comparison, wells that were not incubated at 37°C after adding collagen (Comparative Example 1) and wells that were not added with collagen (Comparative Example 2) were treated in the same manner as in Example 1. Platelet-containing plasma was added to the gel, and the platelet adsorption rate and the degree of adsorption of platelet-derived growth factor to the gel using P[-4 and β-TG as markers were measured. The results are shown in Tables 1 and 2 and FIG.

(Leaving space below) Table 1 Platelets @ Adhesion rate (%) 5 minutes 20 minutes 60 minutes Example 1 29 51 80 Comparative example 1
20 22 24 Comparative example 2 15
18 20 Table 2 Concentration in reaction supernatant (no/ml) Marker O minute 60 minutes Example 1 PF-42000140 Comparative example 1 PF-420001610 Comparative example 2 PF-
42000 1760 Example 1 β-TG 7400 510 Comparative Example 1
β-TG 7400 5830 Comparative Example 2 β-
TG 7400 6840 Example 2 Collagen solution (Kokenso, 0.3%, for tissue culture)
．． After dialysis with 02M phosphate buffer, pH 7.4, at room temperature overnight, the mixture was transferred to a stainless steel vat and freeze-dried for 24 hours. Further add 24% to 1% glutaraldehyde solution.
After soaking for a period of time, the target collagen matrix was obtained by washing thoroughly with water.

Next, a mixed solution of 20% polyether-type urethane (Leathermin 2045R, manufactured by Dainichiseika Kaisha, Ltd.) in tetrahydrofuran/dimethylformamide was applied onto the silicone release paper using a precision coating tool (for example, an applicator) to form a film. Immediately after coating, the above-mentioned Lagen matrix was placed on the wet layer, and after being left at room temperature for 10 minutes,
Cured in oven at 60'C (at least 1 hour).

Fresh rat platelet-containing plasma prepared in advance (50%
x 105/mm3) and adjust YJ in the same manner as in Example 1.
Collagen mixed solution (0.3% collagen solution, 9
Double-concentrated Hank's solution and Kyoto water were mixed at a ratio of 3:1:5 at 4°C, and the mixture was soaked at 37°C for 2 weeks. After 60 minutes, the collagen matrix was thoroughly washed with phosphate buffer solution, I) H7,4.

Example 3 1 cm of the full thickness of the dorsal skin of a 5-week-old rat was removed and DI
A wound was formed. The cell culture substrate obtained in Example 2 (1 c scrap area) was attached to this wound site, gauze and Elasdik band impregnated with physiological saline were applied, and the edges contained antibiotics. Tissue reactions were examined one week after applying Vaseline.

The results are shown in Table 3.

Comparative Examples 3 to 6 For comparison, various materials shown below were applied to the ΩHa site of a rat in the same manner as in Example 3, and the tissue reaction after one week was examined. The results are shown in Table 3. The coating material used was freeze-dried pork skin in Comparative Example 3, and enzyme-based collagen derived from cowhide in Comparative Example 4! 2! ! Comparative Example 5 is a collage nonwoven fabric prepared from atelocollagen that has been subjected to 11I treatment. Detach with a formalized polyvinyl alcohol sponge.

Table 3 Tissue response 0 Example 3 ++ + + ++
Comparative example 3 + 10+++++ u4 +++ +++ +rt 5
++ Shido Jujujun 5 +++
+10+6 responses were evaluated in 3 levels: +...weak, 10+...moderate, and 10+10 strong.

As shown in FIG. 3, in Comparative Example 3, the exudate reaction was weak and the infiltrating cell reaction was moderate, but there was a very strong foreign body cell reaction and little granulation formation. In Comparative Example 4, the exudate reaction was weak and the infiltrating cell reaction was moderate, but the foreign body cell reaction was somewhat strong and granulation formation was small. Therefore, these biomaterials are considered to have a fairly strong reaction with foreign proteins from tissues. on the other hand,
In the case of Comparative Example 5, the exudate reaction and the infiltrated cell reaction were moderate, but the foreign body cell reaction was caused around the fibers, and the granulation reaction was strong and surrounded the tissue.

In the case of Comparative Example 6, the exudate reaction was strong, the infiltrating cell reaction and foreign body cell reaction were weak, and granulation formation was small.

On the other hand, in the case of Example 6 of the present invention, cell proliferation was promoted and the formation of granulations and figures with capillaries was observed.

IV. Effects of the Invention As described above, the present invention is a cell culture substrate characterized by having a collagen-containing substrate supporting platelet-derived cell growth factors. It strongly supports cell proliferation when applied to intraluminal cell culture, and is highly biocompatible and non-immunogenic, so it can be used, for example, as a wound dressing, to promote early granulation. It brings about layer formation or epidermis formation and promotes healing. In addition, if the collagen in the cell culture substrate of the present invention is made into oriented fibers or cross-linked, it will better support cell growth factors, and it will also be able to support collagenase, etc. It becomes possible to suppress decomposition by enzymes. Furthermore, the cell culture substrate of the present invention includes a support layer having air permeability, such as polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer, polyvinyl chloride, polyester, or fluorine-based It can also be lined with a support layer made of resin, which makes it possible to minimize the occurrence of body fluid leakage, bacterial infection, etc. when applied, for example, as a wound dressing.

Generally speaking, in the cell culture substrate of the present invention, if the collagen-containing substrate has pores with a pore diameter of 1 to 100 μm, cell proliferation will be better.

The present invention also includes adding a balanced salt solution to the collagen solution, then incubating it at about 37°C to gel it, and then adding a solution containing fresh platelet components to this gel and incubating it. Since the method for manufacturing a cell culture substrate is characterized by the following, it is possible to easily manufacture a cell culture substrate having the above-mentioned excellent performance by utilizing good platelet aggregation. In this production method, the balanced salt solution is Hank's solution, the platelet component-containing solution is platelet-containing plasma, and the homogenization process is performed after adding the balanced salt solution to the collagen solution. It is possible to provide a submerged substrate for cell culture.

The present invention further provides a cell culture method in which the collagen solution is freeze-dried, the resulting collagen layer is cross-linked, and the cross-linked collagen layer is then impregnated with a fresh platelet component-containing solution and incubated. Since the present invention is a method for producing a substrate for cell culture, it is possible to easily produce a substrate for cell culture having the above-mentioned excellent performance. Furthermore, this'! In method A, the crosslinking treatment uses a treatment agent selected from the group consisting of glutaraldehyde, formaldehyde, and glyoxal, and the fresh platelet component-containing solution impregnated into the collagen layer is a collagen solution. If it is preferably applied as a mixture with a collagen solution to which a balanced salt solution has been added and incubated at 37°C, it will be easier to provide an excellent cell culture substrate. .

In this manufacturing method, it is possible to easily form a collagen layer on a support layer, preferably a breathable support layer, and for example, it is possible to form a collagen layer on a support layer, preferably a breathable support layer.
It can be lined with a support made of propylene-diene terpolymer, polyvinyl chloride, polyester or fluororesin.

[Brief explanation of the drawing]

FIG. 1 is a graph showing temporal changes in the rate of platelet adsorption to the collagen layer according to an example of the method for producing a substratum for follicle culture of the present invention and a comparative example.

Claims (22)

[Claims] (1) A cell culture substrate characterized in that a substrate containing collagen supports cell growth factors derived from platelets. (2) The cell culture substrate according to claim 1, wherein the collagen is an oriented fiber. (3) The cell culture substrate according to claim 1 or 2, wherein the collagen is crosslinked. (4) The cell culture substrate according to any one of claims 1 to 3, wherein the collagen-containing substrate is formed by coating a breathable support with collagen. (5) The cell culture substrate according to any one of claims 1 to 3, wherein the collagen-containing substrate consists essentially of collagen only. (6) The collagen-containing base material is lined with an air-permeable support layer.
The cell culture substrate according to any one of Item 5. (7) The base material containing collagen is gel-like, film-like,
The cell culture substrate according to any one of claims 1 to 6, which has a woven fabric, nonwoven fabric, porous, or sponge-like form. (8) The support layer is made of polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer, polyvinyl chloride, polyester, or fluororesin. 7. The cell culture substrate according to item 7. (9) The cell culture substrate according to any one of claims 1 to 8, wherein the collagen-containing substrate has pores with a pore diameter of 1 to 100 μm. (10) The membrane pores of the cell culture substrate are 10 μm to 10 mm.
11. The cell culture substrate according to any one of claims 1 to 10, which is of a certain degree. (11) The cell culture substrate according to any one of claims 1 to 10, which is applied as a wound dressing. (12) A balanced salt solution is added to the collagen solution, and then incubated at about 37°C, and further gelled, and then a fresh platelet component-containing solution is added to this gel and incubated. A method for producing a substrate for cell culture. (13) The method for producing a cell culture substrate according to claim 12, wherein the balanced salt solution is Hank's solution. (14) The method for producing a cell culture substrate according to claim 12 or 13, wherein the platelet component-containing solution is platelet-containing plasma. (15) The cell culture substrate according to any one of claims 12 to 14, wherein the collagen solution is homogenized after adding a balanced salt solution, and then incubated at about 37°C. Method of manufacturing wood. (16) A cell culture substrate characterized by freeze-drying a collagen solution, crosslinking the resulting collagen layer, and then impregnating the crosslinked collagen layer with a fresh platelet component-containing solution and incubating it. manufacturing method. (17) The method for producing a cell culture substrate according to claim 16, wherein the crosslinking treatment uses a treatment agent selected from the group consisting of glutaraldehyde, formaldehyde, and glyogysar. (18) The method for producing a cell culture substrate according to claim 16 or 17, wherein the collagen layer is formed on a support layer. (19) The method for producing a cell culture substrate according to claim 18, wherein the support layer has air permeability. (20) The support layer is made of polyurethane, silicone, styrene-butadiene block copolymer, ethylene-propylene-diene terpolymer, polyvinyl chloride, polyester, or fluororesin.Claim 18 20. The method for producing a cell culture substrate according to item 10 or 19. (21) The cell culture substrate according to any one of claims 16 to 20, wherein the fresh platelet component-containing solution impregnated into the collagen layer is applied as a mixture with the collagen solution. manufacturing method. (22) Manufacturing the cell culture substrate according to claim 21, wherein the collagen solution to be mixed with the fresh platelet component-containing solution is one that has been incubated at 37°C with a balanced salt solution added in advance. Method.