Immortalization of epithelial cells and methods of use

• US 9,279,106 B2
• Filed: 11/11/2011
• Issued: 03/08/2016
• 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:
  US13/885,078
• Publication Number:
  US 20130309681A1
• Time in Patent Office: 1,579 Days
• Field of Search: 435/375, 435/377
• US Class Current: 1/1
• 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, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• 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 together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• 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 of continuously culturing non-keratinocyte epithelial cells, the method comprising

a) culturing the non-keratinocyte epithelial cells in the presence of feeder cells and a calcium-containing medium, and

b) inhibiting the activity of Rho kinase (ROCK) in the feeder cell, the non-keratinocyte epithelial cells or both during culturing,

wherein the continuously cultured non-keratinocyte epithelial cells are not embryonic or adult stem cells and the continuously cultured non-keratinocyte epithelial cells become conditionally immortalized.

2. The method of claim 1, wherein the non-keratinocyte epithelial cells are selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

3. The method of claim 2, wherein the non-keratinocyte epithelial cells are 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.

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

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

6. The method of claim 1, wherein inhibiting the activity of ROCK comprises culturing the non-keratinocyte epithelial cells in the presence of a small molecule inhibitor of Rho kinase 1 (ROCK 1), Rho kinase 2 (ROCK 2) or both.

7. The method of claim 6, wherein the small molecule ROCK 1 or ROCK 2 inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

8. The method of claim 1, wherein inhibiting the activity of ROCK comprises genetic manipulation of the expression of Rho kinase 1 (ROCK 1), Rho kinase 2 (ROCK 2) or both.

9. The method of claim 1, further comprising passaging the non-keratinocyte epithelial cells after inhibiting ROCK, and placing the passaged cells in cell culture environment in which ROCK is not being inhibited, wherein the cells are no longer conditionally immortalized after the cells are placed in the environment in which ROCK is not being inhibited.

10. The method of claim 9, wherein the environment in which ROCK is not being inhibited is a three-dimensional cell culture environment.