Selective cultivation of normal human sebocytes in vitro; a simple modified technique for a better cell yield

Selective cultivation of normal human sebocytes is essential for better understanding of drug pharmacokinetics and diseases of the pilosebaceous apparatus. In the present study, sebocytes are selectively cultivated in vitro using modified MCDB 153 medium to which cholera toxin (1 x 10(-9) M), crude bovine pituitary extract (70 micro g/ml), epidermal growth factor (10 ng/ml), basic fibroblast growth factor (2 ng/ml), hydrocortisone (1.4 x 10(-6) M), insulin (10 micro g/ml), fetal bovine serum (10%), and antibiotics were added. To maintain contact of the floating sebaceous lobules with culture plate, two methods have been adopted. (i) Using little amount of culture medium that barely covers the gland lobules with frequent dropping of the medium to replace the loss by evaporation (almost every 2 h). (ii) Placing a sterile glass slide cover over the gland lobules in the presence of enough culture medium. Both methods are performed for the first 72 h of inoculation, when cells are seen outgrowing from sebaceous lobules. Both populations show the characteristic morphology of sebocytes in culture, namely polygonal shape with abundant cytoplasm resembling basal keratinocytes. As the culture grows older, a vacuolated refractile cytoplasm becomes evident. A comparison of both methods revealed a significant percentage increase of sebocytes obtained from covered lobules being 144% (P = 0.02) on day 4, 162% (P = 0.009) on day 8, and 173% (P < 0.001) on day 12 of incubation. No further proliferation is measured thereafter. Cells obtained from both methods also showed no difference in lipogenesis (Oil-Red stain) or in the expression of the specific epidermal membrane antigen as shown by monoclonal antibody labeling (alkaline phosphatase anti-alkaline phosphatase-technique). To conclude, covering the sebaceous glands during the first 72 h of primary culture provides an excellent contact with the culture plate and hence a significant better yield of sebocytes more suitable for large experimental work.