Immortalization of epithelial cells and methods of use

• US 9,951,315 B2
• Filed: 02/10/2016
• Issued: 04/24/2018
• 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:
  US15/040,783
• Publication Number:
  US 20160289644A1
• Time in Patent Office: 804 Days
• Field of Search: 435375, 435373, 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 method for continuously culturing non-keratinocyte epithelial cells, comprising:

(a) culturing non-keratinocyte epithelial (NKE) cells isolated from tissue of a mammal, or cells derived therefrom, in the presence of feeder cells or a medium conditioned with feeder cells; and

a calcium-containing medium; and

(b) inhibiting the activity of Rho kinase (ROCK) in the feeder cells, the NKE cells or both during the culturing, thereby generating a cell culture comprising a population of NKE cells, wherein;

the cell culture does not include embryonic stem cells,

the population of the NKE cells in the cell culture is capable of 60 or more population doublings, and

the NKE cells isolated from tissue of a mammal, or cells derived therefrom, not exposed to (a) and (b) are not capable of 60 population doublings.

2. The method of claim 1, wherein the NKE cells are derived from primary cells.

3. The method of claim 2, wherein the primary cells are from epithelial tissue of a human patient.

4. The method of claim 1, wherein the NKE cells are not derived from primary cells.

5. The method of claim 1, wherein the NKE cells are derived from a population of tumor NKE cells.

6. The method of claim 1, wherein the NKE cells are derived from non-immortalized NKE cells obtained from epithelial tissue.

7. The method of claim 1, wherein the NKE cells are derived from differentiated NKE cells obtained from epithelial tissue.

8. The method of claim 1, wherein the NKE cells are derived from organ-specific NKE cells obtained from epithelial tissue.

9. The method of claim 1, wherein the NKE cells are derived from cells selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

10. The method of claim 9, wherein the NKE cells are derived from cells selected from the group consisting of prostate cells, mammary cells, hepatocytes, pancreatic islet cells, pulmonary epithelial cells, kidney cells, bladder cells, stomach epithelial cells, large intestinal epithelial cells, small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymus cells, gall bladder cells and pituitary cells.

11. The method of claim 1, wherein the calcium-containing medium comprises serum or a serum replacement.

12. The method of claim 1, wherein the feeder cells are proliferating or non-proliferating fibroblasts.

13. The method of claim 1, wherein inhibiting the activity of ROCK comprises culturing the NKE cells in the presence of one or more of a small molecule inhibitor of Rho kinase 1 (ROCK 1), a small molecule inhibitor of Rho kinase 2 (ROCK 2), a RNAi molecule directed to ROCK 1, and a RNAi molecule directed to ROCK 2.

14. The method of claim 13, wherein the small molecule ROCK inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

15. The method of claim 1, wherein the population of the NKE cells in the cell culture express basal epithelial cell marker P63.

16. The method of claim 15, wherein the basal epithelial cell marker P63 is expressed in the population of NKE cells after about 30 passages.

17. The method of claim 15, wherein the basal epithelial cell marker P63 is expressed in the population of NKE cells after about 80 passages.

18. The method of claim 1, wherein the population of the NKE cells in the cell culture is capable of 80 or more population doublings.

19. The method of claim 1, wherein the population of the NKE cells in the cell culture is capable of 100 or more population doublings.

20. The method of claim 1, wherein the population of the NKE cells in the cell culture is capable of 120 or more population doublings.