Immortalization of epithelial cells and methods of use

• US 9,657,272 B2
• Filed: 09/26/2014
• Issued: 05/23/2017
• Est. Priority Date: 11/12/2010
• Status: Active Grant

• Current Assignee: Georgetown University
• Original Assignee: Georgetown University
• Inventors: Schlegel, Richard, Liu, Xuefeng
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US14/498,089
• Publication Number:
  US 20150099267A1
• Time in Patent Office: 970 Days
• Field of Search: 435375, 435377, 435 4051, 435325
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides or bases
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0625 Epidermal cells, skin cells; Cells of the oral mucosa
• CPC Class Codes: C12N 5/0631 Mammary cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0683 Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells spermatogonia C12N5/061
• CPC Class Codes: C12N 5/0688 Cells from the lungs or the respiratory tract
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 : Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism

Abstract

The present invention is directed towards methods of culturing non-keratinocyte epithelial cells, with the methods comprising culturing non-keratinocyte epithelial cells in the presence of feeder cells and a calcium-containing medium while inhibiting the activity of Rho kinase (ROCK) in the feeder cell, the non-keratinocyte epithelial cells or both during culturing.

Claims

1. A cell culture composition comprising

a) a population of conditionally immortalized, non-keratinocyte epithelial (NKE) cells in a calcium-containing medium, wherein the NKE cells are not embryonic stem cells,

b) feeder cells in the calcium-containing medium, and

c) at least one inhibitor of Rho kinase (ROCK inhibitor) in the calcium-containing medium.

2. The cell culture composition of claim 1, wherein the conditionally immortalized NKE cells are derived from a population of non-immortalized NKE primary cells obtained from epithelial tissue.

3. The cell culture composition of claim 1, wherein the conditionally immortalized NKE cells are derived from cells selected from the group consisting of primary squamous cells, primary columnar cells, primary adenomatous cells and primary transitional epithelial cells.

4. The cell culture composition of claim 2, wherein the conditionally immortalized NKE cells are capable of maturing only into the type of tissue from which the non-immortalized NKE cells were obtained, wherein the non-immortalized NKE cells are selected from the group consisting of prostate epithelial cells, mammary epithelial cells, hepatocytes, pancreatic islet cells, airway epithelial cells, kidney epithelial cells, bladder epithelial cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, retinal epithelial cells, neuroendocrine epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid epithelial cells, parathyroid epithelial cells, adrenal epithelial cells, thymus epithelial cells, gall bladder epithelial cells and pituitary epithelial cells, after the conditionally immortalized NKE cells are placed in a subsequent environment that promotes differentiation thereto.

5. The cell culture composition of claim 1, wherein the calcium-containing medium comprises serum.

6. The cell culture composition of claim 1, wherein the at least one ROCK inhibitor is a small molecule inhibitor of Rho kinase 1 (ROCK 1), a small molecule inhibitor Rho kinase 2 (ROCK 2), a RNAi molecule directed to ROCK 1, a RNAi molecule directed to ROCK 2 or a combination thereof.

7. The cell culture composition of claim 6, wherein the small molecule inhibitor is chosen from Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

15. The cell culture composition of claim 1, wherein the population of conditionally immortalized NKE cells has an average telomere length of about 4 kb or less and undergoes cell division.

17. The cell culture composition of claim 15, wherein the population of conditionally immortalized NKE cells has an average telomere length of about 2 kb or less.

18. The composition of claim 1, wherein the non-immortalized NKE cells are derived from a population of tumor primary NKE cells.

19. The cell culture composition of claim 2, wherein the non-immortalized NKE cells are obtained from differentiated epithelial tissue.

8. A cell culture composition comprising

a) a population of conditionally immortalized, non-keratinocyte epithelial (NKE) cells in a calcium-containing medium, wherein the conditionally immortalized NKE cells are not embryonic stem cells, and wherein the calcium-containing medium is feeder-cell conditioned medium, and

b) at least one inhibitor of Rho kinase (ROCK inhibitor) in the calcium-containing medium.

9. The cell culture composition of claim 8, wherein the conditionally immortalized NKE cells are derived from a population of non-immortalized NKE primary cells obtained from epithelial tissue.

10. The cell culture composition of claim 8, wherein the conditionally immortalized NKE cells are derived from cells selected from the group consisting of primary squamous cells, primary columnar cells, primary adenomatous cells and primary transitional epithelial cells.

11. The cell culture composition of claim 9, wherein the conditionally immortalized NKE cells are capable of maturing only into the type of tissue from which the non-immortalized NKE cells were obtained, wherein the non-immortalized NKE cells are selected from the group consisting of prostate epithelial cells, mammary epithelial cells, hepatocytes, pancreatic islet cells, airway epithelial cells, kidney epithelial cells, bladder epithelial cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, retinal epithelial cells, neuroendocrine epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid epithelial cells, parathyroid epithelial cells, adrenal epithelial cells, thymus epithelial cells, gall bladder epithelial cells and pituitary epithelial cells, after the conditionally immortalized NKE cells are placed in a subsequent environment that promotes differentiation thereto.

12. The cell culture composition of claim 8, wherein the calcium-containing medium comprises serum.

13. The cell culture composition of claim 8, wherein the at least one ROCK inhibitor is a small molecule inhibitor of Rho kinase 1 (ROCK 1), a small molecule inhibitor Rho kinase 2 (ROCK 2), a RNAi molecule directed to ROCK 1, a RNAi molecule directed to ROCK 2 or a combination thereof.

14. The cell culture composition of claim 8, wherein the small molecule inhibitor is chosen from Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

16. The cell culture composition of claim 8, wherein the population of conditionally immortalized NKE cells has an average telomere length of about 4 kb or less and undergoes cell division.

20. The cell culture composition of claim 8, wherein the non-immortalized NKE cells are derived from a population of tumor primary NKE cells.

21. The cell culture composition of claim 9, wherein the non-immortalized NKE cells are obtained from differentiated epithelial tissue.