Epithelial cell cultures for in vitro testing

• US 6,383,805 B1
• Filed: 04/11/2000
• Issued: 05/07/2002
• Est. Priority Date: 08/29/1997
• Status: Expired due to Fees

• Current Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Original Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Inventors: Latimer, Jean J.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US09/547,906
• Time in Patent Office: 756 Days
• Field of Search: 435/325, 435/371, 435/378, 435/379, 435/380, 435/383, 435/392, 435/404, 435/408
• US Class Current: 435/325
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0631 Mammary cells
• CPC Class Codes: C12N 5/0693 Tumour cells; Cancer cells

Abstract

A method and a medium for culturing epithelial cells of both normal and malignant origin is provided. The method entails physically disaggregating tissue samples, placing the resulting fragments onto a surface comprised of basement membrane matrix components, and culturing the tissue in a medium containing preselected fetal and newborn calf sera and rat sera. Both primary explant cell cultures and cell lines, which are long-lived and particularly suitable for further study, are produced. The cultured primary explant cells undergo differentiation to form complex structures resembling those seen in vivo.

Claims

1. Cultured primary explant cells that are derived from an epithelial tissue source selected from the group consisting of breast, endometrium and ovary, wherein said cells are produced by a method comprising:

(a) providing physically disaggregated epithelial tissue from said source,

(b) placing the disaggregated tissue on a solid surface of basement membrane matrix components, wherein the final concentration of protein in said basement membrane matrix is about 5-50 mg/ml, and

(c) culturing the tissue in cell culture medium comprising about 2.5% to about 40% by concentration fetal bovine serum, about 2.5% to about 40% by concentration newborn calf serum, and about 1% to about 10% by concentration rat serum,

wherein

(i) said cultured primary explant cells are obtained from breast epithelial tissue and form epithelial-cell domes, which cultures and domes persist for at least six weeks as primary, untrypsinized cultures,

(ii) said cultured primary explant cells are obtained from endometrium epithelial tissue and either form epithelial-cell domes, which cultures and domes persist for at least six weeks as primary, untrypsinized cultures, or grow as single cells with a rounded cobblestone conformation, or

(iii) said cultured primary explant cells are obtained from ovary epithelial tissue and grow as single cells with a rounded cobblestone conformation.

2. The cells of claim 1, wherein the final concentration of protein in said basement membrane matrix is about 10-20 mg/ml.

3. Cultured primary explant cells according to claim 1 that (i) are derived from breast epithelial tissue.

4. The cells of claim 3, wherein said breast epithelial tissue is from a non-tumor source.

5. The cells of claim 4, wherein the cultured primary explant cells have formed branching duct-like structures.

6. Cultured primary explant cells according to claim 1 that are derived from endometrium epithelial tissue.

7. Cultured primary explant cells according to claim 1 that (i) are derived from ovarian epithelial tissue.

17. A method of producing a cell line derived from an epithelial tissue source selected from the group consisting of breast, endometrium, and ovary, wherein the method comprises:

(a) providing cultured primary explant cells of claim 1,

(b) treating the primary explant culture, or a secondary explant culture obtained from the primary explant culture, with trypsin such that free cells arise,

(c) placing said free cells into a fresh culture chamber,

(d) passaging the cultured cells for at least 14 passages in order to establish a cell line.

18. The method of claim 17, wherein the total concentration of sera is no greater than about 45%, and wherein said fetal bovine serum and said newborn calf serum are selected by pretesting the effects of the sera on the growth and differentiation of cultured embryonic stem cells in the presence of LIF, such that sera that cause spontaneous differentiation of said stem cells are rejected.

24. The method of claim 17, wherein the epithelial tissue is obtained from non-tumor tissue.

27. The method of claim 17, wherein the epithelial tissue source is breast epithelial tissue from a non-tumor source.

28. The cells of claim 3, wherein the epithelial source is normal breast tissue.

8. A method of culturing excised epithelial tissue, comprising:

(a) providing physically disaggregated epithelial tissue,

(b) placing the disaggregated tissue on a solid surface of basement membrane matrix components, wherein the final concentration of protein in said basement membrane matrix is about 5-50 mg/ml, and

(c) culturing the disaggregated tissue in culture medium comprising about 2.5% to about 40% by concentration fetal bovine serum, about 2.5% to about 40% by concentration newborn calf serum, and about 1% to about 10% by concentration rat serum.

9. The method of claim 8, wherein said basement membrane matrix components are at a final concentration of about 10-20 mg/ml.

10. The method of claim 8, wherein the epithelial tissue is obtained from a tissue source selected from the group consisting of breast, endometrium, and ovary.

11. The method of claim 8, wherein the epithelial tissue is obtained from non-tumor tissue.

12. The method of claim 8, wherein the matrix proteins are extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

19. The method of claim 8, wherein the total concentration of sera is no greater than about 45%, and wherein said fetal bovine serum and said newborn calf serum are selected by pretesting the effects of the sera on the growth and differentiation of cultured embryonic stem cells in the presence of LIF, such that sera that cause spontaneous differentiation of said stem cells are rejected.

13. A method of producing a culture of primary explant cells derived from an epithelial tissue source selected from the group consisting of breast, endometrium, and ovary, wherein the method comprises:

(a) obtaining physically disaggregated epithelial tissue from a subject,

(b) placing the disaggregated tissue on a solid surface of basement membrane matrix components, wherein the final concentration of protein in said basement membrane matrix is about 5-50 mg/ml, and

(c) culturing the disaggregated tissue in an initial culture medium comprising about 2.5% to about 40% by concentration fetal bovine serum, about 2.5% to about 40% by concentration newborn calf serum, and about 1% to about 10% by concentration rat serum to produce a primary explant culture, and

(d) removing the initial culture medium and replacing it with fresh medium having the same composition as the initial culture medium to maintain said primary explant culture.

14. The method of claim 13, wherein said basement membrane matrix components are at a final concentration of about 10-20 mg/ml.

20. The method of claim 13, wherein the total concentration of sera is no greater than about 45%, and wherein said fetal bovine serum and said newborn calf serum are selected by pretesting the effects of the sera on the growth and differentiation of cultured embryonic stem cells in the presence of LIF, such that sera that cause spontaneous differentiation of said stem cells are rejected.

22. The method of claim 13, wherein the epithelial tissue is obtained from non-tumor tissue.

25. The method of claim 13, wherein the epithelial tissue source is breast epithelial tissue from a non-tumor source.

15. A method of producing a secondary explant culture derived from an epithelial tissue source selected from the group consisting of breast, endometrium, and ovary, wherein the method comprises:

(a) obtaining physically disaggregated epithelial tissue from a subject,

(b) placing the disaggregated tissue in a culture chamber on a solid surface of basement membrane matrix components, wherein the final concentration of protein in said basement membrane matrix is about 5-50 mg/ml,

(c) culturing the disaggregated tissue in an initial culture medium comprising about 2.5% to about 40% by concentration fetal bovine serum, about 2.5% to about 40% by concentration newborn calf serum, and about 1% to about 10% by concentration rat serum to produce a primary explant culture, and

(d) removing the initial culture medium and placing it into a fresh culture chamber, such that detached cells from the primary explant culture present in the culture medium establish a secondary explant culture in said fresh culture chamber.

16. The method of claim 15, wherein the final concentration of protein in said basement membrane matrix is about 10-20 mg/ml.

21. The method of claim 15, wherein the total concentration of sera is no greater than about 45%, and wherein said fetal bovine serum and said newborn calf serum are selected by pretesting the effects of the sera on the growth and differentiation of cultured embryonic stem cells in the presence of LIF, such that sera that cause spontaneous differentiation of said stem cells are rejected.

23. The method of claim 15, wherein the epithelial tissue is obtained from non-tumor tissue.

26. The method of claim 15, wherein the epithelial tissue source is breast epithelial tissue from a non-tumor source.