JPH04320681A - Culture of animal cell - Google Patents

Abstract

PURPOSE:To promote multiplication, to improve number of cells reached and to improve production of useful substance in culture of cells of adhesion dependent animal. CONSTITUTION:In culturing cells of adhesion dependent animal by using micro carrier, hollow fiber or microcapsules, ascorbic acid or a derivative thereof is added to the cells and the cells are cultured.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for culturing adhesion-dependent animal cells.

0002

BACKGROUND OF THE INVENTION Useful substances are produced using diploid cells, tumor cells and genetically modified animal cells. As a culture method, a method using a roux bottle or a roller bottle is known, but it is considered extremely difficult to perform large-scale culture using this method. That is, in this method, the cells only grow in a single layer on the bottom of the roux bottle or the inner surface of the roller bottle, and therefore a large number of bottles are required for mass culture, making it unsuitable. Furthermore, it is almost impossible to control the pH, dissolved oxygen concentration, etc. in the culture medium to a constant level. Therefore, methods have been developed that allow for large quantities and constant control of culture conditions. Methods include culturing adhesion-dependent animal cells on microcarriers or hollow fibers, or immobilizing and culturing them in microcapsules. These methods are
When scaling up, the number of cells reached in the main culture often decreases because the proliferation rate decreases and the number of seed cells becomes insufficient.

0003

[Problems to be Solved by the Invention] The purpose of the present invention is to provide a culture system that stabilizes the culture system and improves the production of useful substances by promoting proliferation and increasing the number of cells reached when culturing animal cells in large quantities. It is about providing law.

0004

[Means for Solving the Problems] The above object is achieved by the following present invention. That is, the present invention provides a method for culturing adhesion-dependent animal cells on microcarriers or hollow fibers, or immobilizing them on microcapsules.
A method for culturing animal cells characterized by adding ascorbic acid or a derivative thereof. Adhesion-dependent cells refer to human or animal-derived fibroblasts, vascular endothelial cells, epithelial cells, and various cancer cells. It also includes genetically modified adhesion-dependent cells. Among these, fibroblasts are particularly preferred.

[0005] The microcarrier used in the present invention is
The matrix material is made of a synthetic resin such as collagen, gelatin, cellulose, crosslinked dextran, or polystyrene, and preferably has a negatively charged charged group such as dimethylaminopropyl, dimethylaminoethyl, trimethylhydroxyaminopropyl. Matrix materials coated with collagen or gelatin are also used. As a commercial product, “Cytodex-1” is a product with dimethylaminoethyl added to cross-linked dextran.
(Pharmacia) and "Cytodex-3" (Pharmacia), which is a cross-linked dextran coated with denatured collagen. As the hollow fiber, there is one using modified cellulose. A commercially available product is "Vitafiber" (Amicon). Microcapsules are created by embedding cells inside using collagen or sodium alginate, which form a water-permeable gel (A. Klausne
r, Bio/technol. , 1,736 (1983
).

To culture adhesion-dependent animal cells, seed cells are inoculated onto microcarrier beads or hollow fibers, or microcapsule-immobilized cells are prepared and cultured. The medium composition, serum concentration, etc. are appropriately selected depending on the type of cells to be cultured, cell concentration, etc. Ascorbic acid or an ascorbic acid derivative is added to the prepared medium. A conventional medium can be used in the present invention. It is preferable to appropriately select a medium suitable for the cells. During cultivation,
The culture medium is appropriately replaced with a medium containing ascorbic acid or an ascorbic acid derivative, and cultured for several to 20 days. Continue culturing until cells are confluent. Ascorbic acid is rapidly oxidized and decomposed in a neutral solution at 37° C. under oxygen conditions. Therefore, when ascorbic acid is used for culture, it is preferable to add it frequently, such as daily. The ascorbic acid used for culture is preferably a derivative that is stable under culture conditions and has ascorbic acid action. For example, L-ascorbic acid phosphate ester (Hata et al., 1988, Abstracts of the 35th Collagen Study Group, p. 8)
5-89) and L-ascorbic acid glucoside. Ascorbic acid or a derivative thereof is added at a concentration of 0.05 to 10 mM. Preferably, it is used at 0.5-3mM. For each derivative, it is preferable to appropriately select a concentration suitable for culture.

[0007]

[Examples] The present invention will be specifically explained below with reference to Examples, but the present invention is not limited by these. Example 1 Fetal bovine serum 5%, cross-linked dextran microcarriers with diethylaminoethyl groups (“Cytodex-1
Eagle MEM containing 5g/L (Pharmacia)
System medium: 0.5 ascorbic acid phosphate per 2L
With or without addition of mM, human fibroblast cells were
The cells were cultured for 6 days at 37° C., pH 7.2, and 20% saturated oxygen concentration with gentle stirring in a spinner flask inoculated at a rate of 10 5 cells/ml. On the way, 1st day, 3rd day,
The medium was replaced on the 5th day. The number of cells reached is 3.2 x 106 cells/ml without addition, and 3 with 0.5mM addition.
．． It was 9 x 106 cells/ml.

[0008]

[Effects of the Invention] According to the present invention, microcarriers,
In adhesion-dependent animal cell culture using hollow fibers or microcapsules, cell proliferation can be promoted and the number of cells reached can be increased. Therefore, it is possible to stabilize the culture system when scaling up to an industrial production scale, and as a result, the production of useful substances can be improved.

Claims (1)

[Claims] [Claim 1] A method for producing animal cells characterized in that adhesion-dependent animal cells are cultured in a microcarrier or hollow fiber, or immobilized in a microcapsule, with the addition of ascorbic acid or a derivative thereof. Culture method.