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The aim of the study was to elucidate the structure of dark-looking spheroids, which appeared in the cultures of hair follicles isolated from rat dermis. These spheroids were particularly numerous when if a keratinocyte growth medium with a reduced amount of supplements was used. Light microscopic observations disclosed that these spheroids consisted of fat droplets covered by cells reacting with the antitrichohyalin AE 15 monoclonal antibody. These fat droplets originated from subcutaneous adipocytes damaged during isolation and remained in cultures. The morphological appearance of cells covering fat droplets studied at the EM level corresponded with that of the cells in the Henle layer of the intact hair follicles in the dermis. EM observations further suggested that in cultures the Henle layer detached from the Huxley layer and that the fat droplets penetrated under the Henle layer resulting in formation of a spheroid. Since from 2 cm2 of rat skin more than 400 spheroids were regularly obtained, it seems conceivable that they could be exploited for isolation of the Henle layer cells.