Increasing cell culture population doublings for long-term growth of finite life span human cell cultures
View Patent ↗Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.
1. A method of increasing population doublings in a cell culture of human epithelial cells with finite life span, comprising the steps of:
providing a cell culture of pre-stasis human epithelial cells, and
providing a cell culture medium comprising a mixture of
(a) 30% to 60% of MM medium with up to 3% serum and (b) 40% to 70% of a different, serum-free medium comprising defined ingredients for growth of human mammary epithelial cells, further comprising (c) an effective amount of an anti-stress associated compound, whereby population doublings and finite life span of said cell culture are increased as compared to a control containing only either the MM medium with serum or a serum free medium.
2. The method of claim 1 , wherein the medium with serum further contains conditioned media.
3. The method of claim 1 , wherein the medium with serum does not contain conditioned media.
4. The method of claim 1 , wherein the serum-free medium is MCDB170.
5. The method of claim 1 , wherein the cell culture medium further comprises at least one of EGF, hydrocortisone, bovine pituitary extract, estradiol, triiodothyronine, insulin or insulin-like growth factor, or lipid rich bovine serum albumin (BSA).
6. The method of claim 1 , wherein the human epithelial cells are human mammary epithelial cells.
7. The method of claim 1 wherein the cell culture comprises 50% medium with serum and 50% serum-free medium.
8. The method of claim 1 wherein the cell culture comprises 30% medium with serum and 70% serum-free medium.
9. The method of claim 1 wherein the cell culture comprises 60% medium with serum and 40% serum-free medium.
10. The method of claim 1 , wherein the anti-stress associated compound is selected from the group consisting of oxytocin, lipid rich bovine serum albumin (BSA), angiotensin II, serotonin (5-HT), melanin concentrating hormone, histamine, bombesin and gastrin-releasing peptide (GRP), glucagons-like peptide-1 (GLP-1), cholecystokinin (CCK), dopamine, and corticotrophin releasing factor.
11. The method of claim 10 , wherein the anti-stress associated compound is oxytocin.
12. The method of claim 11 , wherein the effective amount of oxytocin is a concentration between 0.05 nM and 5.0 nM.
13. The method of claim 11 , wherein the effective amount of oxytocin is a concentration of 0.1 nM.
14. The method of claim 11 further comprising the step of providing a second anti-stress associated compound selected from the group consisting of lipid rich bovine serum albumin (BSA), angiotensin II, serotonin (5-HT), melanin concentrating hormone, histamine, bombesin and gastrin-releasing peptide (GRP), glucagons-like peptide-1 (GLP-1), cholecystokinin (CCK), dopamine, and corticotrophin releasing factor.
15. The method of claim 14 , wherein the second anti-stress associated compound is lipid rich bovine serum albumin (BSA).
16. The method of claim 10 , wherein the anti-stress associated compound is added to the primary passage of the cell culture.
17. The method of claim 10 , wherein the anti-stress associated compound is added at or after the second passage of the cell culture.
18. The method of claim 1 further comprising a step of growing the cell culture on an extracellular matrix material.
19. The method of claim 18 wherein the extracellular matrix is laminin.
20. The method of claim 1 further wherein said cell culture is cultured for at least 30 population doublings.