Substratum for cell culture and a method for culturing and isolating cells using same

• US 4,559,304 A
• Filed: 08/08/1983
• Issued: 12/17/1985
• Est. Priority Date: 08/09/1982
• Status: Expired due to Fees

• Current Assignee: Koken Limited
• Original Assignee: Koken Limited
• Inventors: Miyata, Teruo, Akaike, Toshihiro, Kasai, Shunji
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Heck, Jean A.
• Application Number:
  US06/521,083
• Time in Patent Office: 862 Days
• Field of Search: 435/240, 435/948, 260/123.7, 260/112 B
• US Class Current: 435/392
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: Y10S 435/948 using viruses or cell lines

Abstract

A substratum for cell culture which comprises a chemically modified collagen rich in either positive or negative charges when under culture conditions. The substratum is prepared by modifying the amino groups or carboxyl groups of collagen. The chemically modified collagen enhances the adherence and proliferation of animal cells much more actively than unmodified collagen in the presence or absence of bovine fetus serum. The cultured animal cells can be detached efficiently from the chemically modified collagen. This allows for highly selective isolation and recovery of the cultured animal cells which can be accomplished without incurring any injury from the chemically modified collagen.

Claims

1. In a mammalian cell culture containing a substratum and nutrient medium, the improvement comprising a substratum composed of collagen which has been modified by chemical means to increase the charge of the collagen to a degree sufficient to enhance mammalian cell attachment.

2. The cell culture according to claim 1, wherein the collagen is modified by chemical means to increase the negative charge of the collagen to a degree sufficient to enhance mammalian cell attachment.

3. The cell culture according to claim 1, wherein the collagen is modified by chemical means to increase the positive charge of the collagen to a degree sufficient to enhance mammalian cell attachment.

4. The cell culture according to claim 2, wherein the chemically modified collagen is succinylated collagen.

5. The cell culture according to claim 3, wherein the chemically modified collagen is esterified collagen.

6. The cell culture according to claim 4, wherein the succinylated collagen is succinylated more than 20 mol% of total amino groups of collagen.

7. The cell culture according to claim 3, wherein the esterified collagen is esterified more than 20 mol% of total carboxyl groups of collagen.

8. An improved method for culturing and isolating mammalian cells wherein the cells are cultured under conventional culture conditions, according to the type of cell being cultured, and collagen is used as a substratum for attaching the cells during culturing, the improvement comprising:

adhering the mammalian cells to collagen which has been modified by chemical means to increase the charge of the collagen, and

incubating the mammalian cells and treating the animal cells with bivalent cation chelating agent to isolate and recover the cells.

9. A method according to claim 8, wherein macrophages are incubated in the mixed system with lymphocytes and only macrophages are selectively isolated and recovered.

10. A method according to claim 8, wherein the bi-valent catin chelating agent is EDTA or EGTA.

11. A method according to claim 8, wherein the adhesion and incubation of mammalian cells are carried out in the presence of bovine fetus serum or fibronectin.