JP2608016B2 - Substrate material for cell culture - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a substrate material for animal cell culture containing as a main component type I collagen treated with a reducing agent. A substrate material for culturing animal cells, which is capable of enhancing the function of animal cells cultivated thereon and / or extending its life by changing the structure of the gel as a component using a reducing agent; The present invention relates to a method for culturing animal cells, characterized in that a gel is used as a substrate material, and to provide the animal cell culture substrate material inexpensively and easily.

[0002]

2. Description of the Related Art Conventionally, type I collagen has been mainly used as a substrate material for adhesion-dependent animal cell culture in the form of a coating or gel. It had not been possible to survive and was still insufficient as a substrate material.

[0003] In view of such a conventional technical background concerning a substrate for culturing animal cells, the present inventors have used, as a substrate material, for example, type I collagen from which a telopeptide has been removed, and have used a type I collagen in a medium. It has been previously reported that the addition of a specific substance makes it possible to maintain the function of cultured cells for a long period of time of 60 days or more (Japanese Unexamined Patent Publication No.
No. 4780).

However, even in this case, pepsin used for removing telopeptide from type I collagen is derived from a living body, so that it is difficult to obtain a gel having stable performance, and it is difficult to use an enzyme. Therefore, at present, there are problems such as relatively high cost.

[0005] Recently, polyhema coat dishes [Exp. Cell Res. , 189 , 87-92.
(1990)] or a proteoglycan coated dish [Exp. Cell Res. , 186 , 22
7-235 (1990)], when hepatocytes are cultured, hepatocytes gather together to form spherical aggregates (spheroids), which are suspended in the medium, and the function can be maintained for about 3 to 4 weeks. Have been reported.

However, even if these techniques are used,
Spheroids adhere to each other in the medium, hepatocytes in the central part are necrotic, and it is impossible to maintain the function for a long period of time as described above, and all the cells, especially cells in the central part of the spheroid, are fully expressed. It is difficult. In addition, there is a drawback that spheroids are sucked when the culture solution is exchanged.

[0007] As described above, although the research and development of culture techniques for enhancing the function of cultured cells and prolonging their survival time in the form of a coating or gel have been carried out, the above-mentioned problems have been solved. The situation has not yet been resolved, and there has been a strong demand for a solution in the industry.

[0008]

SUMMARY OF THE INVENTION Accordingly, the present inventors have attempted to solve the above-mentioned problems of the prior art.
Intensive studies have been conducted to provide a new type of substrate material for animal cell culture that can stably maintain the function of animal cells taken out of a living body for a long period of time at low cost, easily, and with stable quality. As a result, by using the type I collagen treated with a reducing agent in a gel form and using it as a cell adhesion substrate, it is possible to obtain a substrate material for animal cell culture that enables stable long-term maintenance of high function of animal cells. In addition, they have found that the substrate material can be provided inexpensively and easily, and have completed the present invention.

[0009] That is, an object of the present invention is to provide an inexpensive and simple substrate material for animal cell culture capable of stably maintaining the function of animal cells for a long period of time.

[0010] Another object of the present invention is to provide a substrate material for animal cell culture capable of expressing a high level of function in cultured cells, particularly primary cultured cells such as primary cultured hepatocytes, and extending the survival period thereof for a long period of time. It is to be.

It is a further object of the present invention to provide a method for culturing animal cells, particularly primary cultured cells such as primary cultured hepatocytes, which enables the cultured cells to exhibit high function and long-term survival.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, the following 1) to 5).
The present invention comprises the following. That is, the present invention is 1) a substrate material for animal cell culture, characterized by containing, as a main component, type I collagen treated with a reducing agent. Also, 2) a gel-like substrate material for animal cell culture, characterized by containing, as a main component, type I collagen treated with a reducing agent. Also,
3) The substrate material for animal cell culture according to 1) or 2) above, wherein the cultured animal cells are primary cultured hepatocytes. 4) A method for culturing animal cells, wherein a collagen gel obtained by gelling type I collagen treated with a reducing agent is used as a substrate material for culture. 5) The animal cell culture method according to 4) above, wherein the cultured animal cells are primary cultured hepatocytes.

Hereinafter, the present invention will be described in detail. The type I collagen used as a main component in the present invention includes calf dermis, pig skin, rat tail tendon, and the like, but is not particularly limited to the animal species or tissue of origin. Type I collagen can be used. As the extraction method, a method based on acid solubilization, a pepsin solubilization method, and the like are preferably used, but are not limited thereto, and a commonly used method is appropriately used.

As the reducing agent used in the present invention, any reducing agent which can be used in an aqueous solution system and which does not cause reduction of an ester or amide (peptide bond) but causes reduction of an aldehyde group can be used. As long as it has such an action, an appropriate one can be used regardless of its type.

Specific examples of the reducing agent preferably used in the present invention include sodium amalgam,
Metals such as zinc, LiAlH ₄ , NaBH ₄ , NaCN
Metal hydrogen complex compounds such as BH _{3 and the} like can be mentioned. Others
An electrolytic reduction method or a catalytic hydrogenation method using a complex catalyst can also be used.

In order to treat type I collagen with the reducing agent as described above, for example,
Examples of suitable methods include a method in which the mixture is treated with the reducing agent at a temperature in the range of 4 to 37 ° C. in the aqueous solutions of Nos. 9 to 9 and treated.
By treating type I collagen with a reducing agent in this way, aldehyde groups derived from allidine and hydroxyallidine in the collagen molecule are reduced, and at the time of gel formation, reduction with lysine and amino groups derived from hydroxylysine is achieved. Thus, the property that the formation of the crosslinks is inhibited, and as a result, a gel having a low degree of crosslinkage and low strength is formed is obtained. As described above, the pH conditions when treating with a reducing agent are pH 3-9.
The temperature condition is preferably about 4-37 ° C. As described above, the present invention has a feature that the processing operation such as the pH condition and the temperature condition at the time of treatment with the reducing agent has a wide allowable range, so that the processing operation can be performed easily and stably.

The substrate material for animal cell culture of the present invention contains such a reduced collagen as a main component, and can be either one using the reduced collagen alone or one containing an appropriate substrate material. Can be used. Further, the substrate material for animal cell culture of the present invention is characterized in that the reduced component collagen, which is a main component thereof, is gelled and used as a cell culture substrate in the form of a collagen gel.

The gel is formed by preparing a collagen solution having a desired concentration containing type I collagen which has been treated with a reducing agent as a main component, and adding the solution to a phosphate buffer, Hepes-collagen.
Neutralize with hydrochloric acid, etc., at 20 ° C or higher, preferably 3
Upon standing at 7 ° C., it gels in a few hours. The obtained gel is sufficiently washed with a phosphate buffer or a culture solution to remove the reducing agent, and then used as a substrate material for cell culture. If the gel is to be stored for a long period, the gel is washed with an acid solution having a pH of about 3 after washing. By dissolving the collagen gel and storing it at about 4 ° C., it can be stored for a long time.

Type I treated with the reducing agent of the present invention
A method of culturing animal cells using a substrate material for animal cell culture containing collagen as a main component only requires seeding the cells on a collagen gel.
In one day, the cells adhere to the top surface of the collagen gel. Animal cells can be applied to hepatocytes, mammary cells, kidney cells, and other appropriate animal cells, and the types thereof are not particularly limited.

In order to confirm the usefulness of the substrate material for animal cell culture containing the type I collagen treated with the reducing agent of the present invention as a main component, collagen extracted by acid extraction having a telopeptide and telopeptide treated with pepsin were used. Using three types of collagen, collagen from which peptide has been removed and collagen which has telopeptide-containing collagen treated with a reducing agent, collagen gels are made, respectively.
As a result of culturing animal cells such as hepatocytes, mammary gland cells, and kidney cells on the upper surface of those collagen gels, these animal cells were cultured on the upper surface of the collagen gel treated with a reducing agent, similarly to the upper surface of the collagen gel treated with pepsin. Has been shown to maintain function at a high level over a long period of time.

In addition, it has been revealed that type I collagen treated with the reducing agent is particularly effective for maintaining long-term functions of hepatocytes such as primary cultured hepatocytes, which have conventionally been difficult to culture.

[0022]

The present invention will be described more specifically below with reference to examples of the present invention. A 0.3% solution of type I collagen (pH 3) containing a telopeptide eluted from calf dermis with a hydrochloric acid solution of pH 3, a 10-fold concentration of phosphate buffered saline, and a 1% aqueous solution of sodium borohydride were added to
The mixture was mixed at a ratio of 0: 1.2: 1 and stirred at 4 ° C. for about 1 day. Thereafter, the mixture was heated to 37 ° C. and allowed to stand for about 1 day to gel the collagen. Then, only the collagen was separated by centrifugation and dissolved in 1 mM hydrochloric acid to obtain a reduced collagen solution.

A 0.3% solution of reduced collagen (pH 3) obtained as described above, a 10-fold concentration of phosphate buffered saline, and a reconstitution buffer (2.2% NaHCO 3)
₃ , 4.77% Hepes, 0.05% NaOH solution)
Was mixed at a ratio of 8: 1: 1, spread on the bottom of a plastic culture dish to a thickness of 1 to 2 mm, gelled in a 37 ° C. incubator, washed with an L-15 medium, and cultured. It was used for.

Hepatocytes were separated from rats by the collagenase perfusion method, and 5 × 10 ⁵ / c on the collagen gel.
The cells were seeded at a concentration of m ² and cultured at 37 ° C. in a 5% carbon dioxide gas incubator. The culture solution was L-15 medium, fetal calf serum 10%, insulin 10 ^-7 M, dexamethasone 1
0 ^-7 M, EGF (Epideral GrowthF
actor) 10 ng / ml, proline 30 μg / m
1, using dimethyl sulfoxide 2%,
The culture was added to a height of 1-2 mm above the gel.
The culture medium was exchanged and sampled periodically, and the amount of albumin secreted by the hepatocytes was measured based on the change in the amount of albumin in the culture medium.

As a comparative example, an untreated 0.3% type I collagen solution (pH 3), a 10-fold concentration of phosphate buffered saline, and a reconstitution buffer were mixed at a ratio of 8: 1: 1. Then, in the same manner as described above, gelation was performed on the bottom of the plastic culture dish, and hepatocytes were cultured. Hepatocytes were also cultured on plastic culture dishes coated with a 0.03% reduction type I collagen solution (pH 3).
As a result, as shown in FIG. 1, long-term function maintenance of hepatocytes was possible only on the type I collagen gel subjected to the reduction treatment. As described above, it was found that the cultured cells can maintain the same level of function as that of the living body for a long period of time only on the reduced type I collagen gel.

Further, as described above, using the untreated type I collagen and the reduced type I collagen,
Each was gelled and placed in a plastic container with a thickness of 2
A gel of about cm was produced. When an 8 mmφ metal disk was pressed against this gel and the strength at the time of breakage of the gel was measured, as shown in FIG. 2, in the case of reduced type I collagen, the gel had a very low strength and the action of the reducing agent As a result, it was found that the properties of the collagen gel were changed.

[0027]

As described in detail above, the present invention relates to a gel-like substrate material for animal cell culture characterized by containing a type I collagen treated with a reducing agent as a main component, The substrate material for culture of the present invention enables animal cells to be cultured at a high level while maintaining their functions for a long period of time. Is useful for maintaining long-term function of hepatocytes.

Further, the treatment with the reducing agent has the following advantages as compared with the conventional pepsin treatment. (1) An inexpensive reducing agent can be used as compared with the enzyme pepsin derived from a living body. (2) In the case of pepsin treatment, it is necessary to adjust the pH of the treatment solution to around 3, and inevitably the problem of inactivation due to variation in enzyme activity, autolysis, denaturation, etc. occurs. The permissible pH range is quite wide, about pH 3 to 9, so that the operation is simple and the properties are stable. (3) Since the reducing agent can be obtained in large quantities with industrially uniform quality, it is advantageous in the case of mass production.

[Brief description of the drawings]

FIG. 1 is a graph showing the amount of albumin secretion measured in Examples.

FIG. 2 is a graph showing the gel strength of a collagen gel measured in an example.

Claims (5)

(57) [Claims] 1. A substrate material for animal cell culture, comprising as its main component type I collagen treated with a reducing agent. 2. A gel-like substrate material for culturing animal cells, which comprises type I collagen treated with a reducing agent as a main component. 3. The substrate material for animal cell culture according to claim 1, wherein the animal cells to be cultured are primary cultured hepatocytes. 4. A method for culturing animal cells, wherein a collagen gel obtained by gelling type I collagen treated with a reducing agent is used as a substrate material for culture. 5. The animal cell culture method according to claim 4, wherein the animal cells to be cultured are primary cultured hepatocytes.