Effect of Oxygen on the Growth of Human Epidermal Keratinocytes

We studied the growth of secondary cultures of neonatal human keratinocytes at oxygen concentrations between 1 and 89%. Keratinocytes were grown in MCDB medium with 5% fetal bovine serum, 10 ng/mI epidermal growth factor, 5 μg/ml insulin, 0.5 μg/mI hydrocortisone, and 0.1mM ethanolamine and phosphorylethanolamine. Medium in the flasks was equilibrated with gas mixtures containing 5% CO2, various percentages of oxygen from 0–95% and nitrogen to balance. Cells were seeded at 104 cells/cm2 in sealed flasks (25cm2). These were incubated at 37°C in incubators maintained at the experimental oxygen tensions. Cells grew best at PO2 (partial pressure of oxygen) 133mm Hg (18% O2), with a mean population doubling time of 2.8 days. Growth was retarded by 60% at PO2 38mm Hg (5% O2) and by 98% at PO2 7mm Hg (1 % O2) However, the oxygen tension that resulted in the best plating efficiency was at PO2 12mm Hg (2% O2). When oxygen tensions were shifted to 78–133mm Hg, cells seeded under low oxygen tensions began to proliferate. These data suggest that a better harvest of keratinocytes is obtained when cells are seeded under low oxygen tension and then shifted to ambient oxygen tensions. At high oxygen tensions, above 20%, growth was inhibited by 75% at PO2 241mm Hg (34% O2) and 98% at PO2 374mm Hg (52% O2). At PO2 637mm Hg (89% O2) no cell growth occurred. These findings showed that high oxygen tensions, above 20%, have no beneficial effect on the growth of keratinocytes.