Medium for long-term proliferation and development of cells

• US 5,405,772 A
• Filed: 06/18/1993
• Issued: 04/11/1995
• Est. Priority Date: 06/18/1993
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Ponting, Ian L. O.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Larson, Kristin
• Application Number:
  US08/079,719
• Time in Patent Office: 662 Days
• Field of Search: 435/240.3, 435/240.31
• US Class Current: 435/378
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides or bases
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2502/1394 Bone marrow stromal cells; whole marrow
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)

Abstract

A serum-free or serum-depleted medium for the short- and long-term proliferation and development of cells, particularly hematopoietic cells and bone marrow stromal cells, the medium comprising cell proliferation and development effective amounts of:

a standard culture medium such as Iscove'"'"'s modified Dulbecco'"'"'s medium; serum albumin; transferrin; a source of lipids and fatty acids; cholesterol; a reducing agent; pyruvate; a glucocorticoid (when the cells to be cultured are hematopoietic cells); nucleosides for synthesis of DNA and RNA; growth factors that stimulate the proliferation and development of stromal cells and cells from a variety of tissues or organs, such as epidermal growth factor, fibroblast growth factor, platelet derived growth factor, and insulin; and extracellular matrix materials, such as collagen, fibronectin, and laminin.

Claims

1. A medium for the long-term proliferation and development of cells, which comprises the following ingredients in the indicated amounts:

space="preserve" listing-type="tabular">______________________________________ (a) a standard culture medium 0.8-1.09 x, (b) serum albumin 3-50 mg/ml, (c) transferrin 25-1000 ug/ml, (d) lipids and fatty acids 5-100 ug/ml, (e) cholesterol 3-30 ug/ml, (f) reducing agent 30-300 uM, (g) pyruvate 30-500 ug/ml, (h) nucleosides 5-30 ug/ml, ______________________________________

(i) at least one growth factor selected from the group consisting of;

space="preserve" listing-type="tabular">______________________________________ (1) epidermal growth factor 5-200 ng/ml (2) fibroblast growth factor 0.5-40 ng/ml (3) platelet-derived growth factor 2-200 ng/ml; and

(4) insulin 2-100 ug/ml. ______________________________________

(j) at least one extracellular matrix material selected from the group consisting of;

space="preserve" listing-type="tabular">______________________________________ (1) collagen IV 2-100 ug/cm.sup.2 ; and

(2) fibronectin 0.5-100 ug/cm.sup.2 ;

______________________________________

wherein said medium for the long-term proliferation and development of cells is serum-free or serum-depleted and is capable of culturing cells comprising adipocytes, macrophages, endothelial cells, fibroblasts, and hematopoictic progenitor cells.

2. The medium according to claim 1, further comprising a glucocorticoid.

3. The medium according to claim 1, wherein said glucocorticoid is hydrocortisone.

4. The medium according to claim 1, further comprising stromal cells.

5. The medium according to claim 1, further comprising an inhibitor of CSF-1 activity.

6. The medium according to claim 1, wherein said cells to be cultured are incapable of producing CSF-1.

7. The medium according to claim 6, wherein said cells to be cultured are obtained from a B6C3Fe-a/a-op/op mouse strain.

8. The medium according to claim 1, wherein said standard culture medium is Iscove'"'"'s Modified Dulbecco'"'"'s Medium.

9. The medium according to claim 1, wherein said serum albumin, transferrin and growth factors are each of human, mouse or bovine origin.

10. The medium according to claim 1, wherein said nucleosides are one or more selected from the group consisting of adenosine, guanosine, cytidine, uridine, 2'"'"'-deoxyadenosine, 2'"'"'-deoxyguanosine, 2'"'"'-deoxycytidine and thymidine.

11. The medium according to claim 1, wherein all of said extracellular matrix materials are included.

12. The medium according to claim 1, wherein said cells are hematopoietic or lymphopoietic.

13. The medium according to claim 1, wherein said cells are from mammalian organs or tissues.

14. The medium according to claim 1, wherein said cells are primary cells or cell lines.

15. A method of long-term culture of cells which comprises culturing the cells for 8 to 20 weeks in the medium according to claim 1.

16. A method of short-term culture of cells which comprises culturing the cells for 2 to 7 weeks in the medium according to claim 1.