103 In Vitro Development of Hair Follicles Reorganized from Single-Cell Suspension of Embryonic Mouse Upper Lips

T. Matsuzaki, M. Imai, A. Wada, M. Iida, and S. Ihara. Department of Biological Science, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan

It is known that hair follicles are able to be reconstructed in vitro from dissociated vibrissal hair germs of embryonic rats by rotation culture followed by flotation culture (two-step culture method). We applied this method to mice in order to take advantages of using a lot of genetically modified mice. Upper lips were collected from E13 or 14 of C57BL/6 mice, and treated with trypsin for 12 min. In some experiments, collagenase digestion preceded the trypsin treatment. Dissociated cells were cultured in rotating dishes and then resultant cell-aggregates were cultured by floating on the surface of a medium. Gene-transfer efficiency was examined by adding retrovirus vectors carrying a lac-Z gene into rotation culture. In other experiments, we mixed upper lip cells of GFP-transgenic mice into rotation culture to chase cell-lineage during follicle development. Lac-Z gene transcripts were detected by X-gal staining, and GFP-positive cells were detected under fluorescent microscope after proper fixation. Hair follicles were formed in the aggregates and differentiated normally in 7 days of cultivation. Incidence of hair formation was related to duration of tryptic digestion, i.e. longer treatment resulted in poor aggregation of cells and in failure of hair formation. Lac-Z retrovirus vectors were integrated into dissociated cells very efficiently. Addition of Lac-Z retrovirus vectors or GFP-transgenic cells did not cause any abnormality or retardation of development. Thus both techniques seem to be applied to the two-step culture. However, cell dissociation procedure was thought not enough, because X-gal-positive cells were frequently found in dermal tissues but less in epithelial ones, and GFP-positive keratinocytes did not mix well with GFP-negative ones. Collagenase treatment was very effective to increase the number of single cells without prolonged tryptic digestion. The cells isolated by collagenase/trypsin digestion formed hair follicles in which GFP-positive keratinocytes were randomly distributed in epithelial tissues reorganized through the two-step culture.