Immortalization of Epithelial Cells and Methods of Use

• US 20130309681A1
• Filed: 11/11/2011
• Published: 11/21/2013
• Est. Priority Date: 11/12/2010
• Status: Active Grant

• Current Assignee: Georgetown University
• Original Assignee: Georgetown University
• Inventors: Schlegel, Richard, Liu, Xuefeng
• Granted Patent: US 9,279,106 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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

1-58. -58. (canceled)

59. 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.

60. The method of claim 59, wherein the non-keratinocyte epithelial cells are selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

61. The method of claim 60, 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.

62. The method of claim 59, wherein the calcium-containing medium comprises serum or a serum replacement.

63. The method of claim 59, wherein the feeder cells are proliferating or non-proliferating fibroblasts.

64. The method of claim 59, 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.

65. The method of claim 64, wherein the small molecule ROCK 1 or ROCK 2 inhibitor is selected from the group consisting of Y-27632, HA1100 hydrochloride, HA1077 and GSK429286.

66. The method of claim 59, 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.

67. The method of claim 59, further comprising

a) passaging the non-keratinocyte epithelial cells after inhibiting ROCK, and

b) placing the passaged cells in cell culture environment in which ROCK is not being inhibited.

68. The method of claim 67, wherein the environment in which ROCK is not being inhibited is a three-dimensional cell culture environment.

69. A population of conditionally immortalized non-keratinocyte epithelial cells.

70. The cell population of claim 69, wherein the conditionally immortalized non-keratinocyte epithelial cells are derived from normal cells or tumor cells.

71. The cell population of claim 70, wherein the conditionally immortalized non-keratinocyte epithelial cells are derived from cells selected from the group consisting of squamous cells, columnar cells, adenomatous cells and transitional epithelial cells.

72. The cell population of claim 71, wherein the conditionally immortalized non-keratinocyte epithelial cells are capable of differentiating into 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, after the conditionally immortalized non-keratinocyte epithelial cells are placed in a subsequent environment that promotes differentiation thereto.

73. A method of identifying a candidate treatment for a subject in need of treatment of a condition that is marked by the presence of abnormal non-keratinocyte epithelial (NKE) cells, the method comprising

a) obtaining a sample of the abnormal NKE cells from the subject,

b) culturing the abnormal NKE cells in the presence of feeder cells, a calcium-containing medium and at least one Rho kinase (ROCK) inhibitor, to produce a population of abnormal NKE cells in vitro,

c) determining a response profile of at least a portion of the abnormal NKE cells in vitro, and

d) identifying a candidate treatment for the subject based on the determined response profile.

74. The method of claim 73, wherein the response profile is at least partially determined by a procedure selected from the group consisting of identifying the sequence of at least one portion of DNA extracted from the abnormal NKE cells in vitro, identifying at least one mRNA that is produced in the abnormal NKE cells in vitro, identifying at least one mRNA that is not produced in the abnormal NKE cells in vitro, identifying one or more proteins that the abnormal NKE cells in vitro express, and identifying one or more proteins that the abnormal NKE cells in vitro do not express.

75. The method of claim 73, wherein the response profile is at least partially determined by subjecting the abnormal NKE cells in vitro to a chemotherapeutic agent and determining the therapeutic index of the chemotherapeutic agent on the abnormal NKE cells in vitro.

76. The method of claim 73, wherein the abnormal NKE cells from the subject are obtained from the circulation of the subject.

77. A method of identifying an abnormal non-keratinocyte epithelial (NKE) cell in a subject, the method comprising

a) culturing at least one candidate abnormal NKE cell isolated from the subject in the presence of feeder cells, a calcium-containing medium and at least one Rho kinase (ROCK) inhibitor, to produce a population of candidate abnormal NKE cells in vitro,

b) determining a tissue origin profile of at least a portion of the population of candidate abnormal NKE cells in vitro, and

c) comparing at least one feature of the candidate abnormal NKE cells to the same feature of normal NKE cells that are obtained from the same tissue as that of the determined tissue origin profile of the candidate abnormal NKE cells to determine if there is a difference between the candidate abnormal NKE cells and the normal NKE cells,

d) wherein a difference indicates that the candidate abnormal NKE cells are abnormal compared to normal NKE cells.

78. The method of claim 77, wherein the candidate abnormal NKE cells are obtained from the circulation of the subject.

79. The method of claim 77, wherein the tissue origin profile is at least partially determined by a procedure selected from the group consisting of identifying at least one mRNA that is produced in the candidate abnormal NKE cells in vitro, identifying at least one mRNA that is not produced in the candidate abnormal NKE cells in vitro, identifying one or more proteins that the candidate abnormal NKE cells in vitro express and identifying one or more proteins that the candidate abnormal NKE cells in vitro do not express.