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The question of epidermal oxygen concentration is of controversial nature. With increasing distance to dermal microvasculature, hypoxic conditions are predominating in the epidermis and are discussed to range from 0,5% and 10% oxygen. Therefore, in vitro cultivation of keratinocytes should be performed under slight hypoxic conditions, which may reduce oxidative damage and improve keratinocytes’ growth. However, epidermal cells are often exposed to normoxic culture conditions with atmospheric oxygen concentrations of 21%. This may cause oxidative stress and DNA damage in keratinocytes. Until now, there is a lack of knowledge of specific oxygen concentrations in the epidermis. Therefore, it is difficult to provide recommendation for optimal oxygen concentration for in vitro cultured keratinocytes. Therefore, the objective of the present study was the analysis of cell viability of keratinocytes after exposure to different hypoxic concentrations compared to atmospheric culture conditions.
MATERIALS AND METHODS
Keratinocytes were isolated from bovine claws and exposed to atmospheric as well as hypoxic conditions with 1% and 5% oxygen. Subsequently, keratinocytes’ viability was measured by using LDH (lactate dehydrogenase) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assays. In a second step, differentiation patterns of the keratinocytes after exposure to hypoxia with 1% oxygen were detected by western blot analysis using the differentiation markers involucrin and loricrin.
The results show no significant differences of cell viability between exposure to 5% oxygen and atmospheric culture conditions. A decrease of cell viability with high cytotoxicity was only measured after exposure to 1% oxygen for two weeks. This effect was accompanied by slight changes in keratinocytes’ morphology. Furthermore, keratinocytes showed an increased expression pattern of involucrin and loricrin after exposure to hypoxia with 1% oxygen.
As the results show, the viability of keratinocytes was only slightly affected by the different oxygen concentrations. This might be based on keratinocytes’ ability to adapt their glucose metabolism and cell function to different environmental conditions alike the hypoxic milieu in the epidermis. One example is the upregulation of LDH to force the anaerobic glycolysis in keratinocytes, which could also be observed within this study. However, results also show that only hypoxic conditions lead to an appropriate differentiation of keratinocytes.