The isolation and maintenance of the human pilosebaceous unit

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Abstract
We have previously developed methods for the isolation and maintenance of human sebaceous glands and hair follicles. However, in long-term culture the maintenance of both is suboptimal. This may be due to a lack of stem cells, which are thought to be located in the bulge area of the hair follicle, and this region is not present in either model. Isolation of the entire pilosebaceous unit would retain this region, and may lead to improved maintenance of both structures. We describe a method for the isolation of viable, individual, pilosebaceous units by microdissection from human scalp face-lift skin. The viability of isolated pilosebaceous units has been determined by light microscopy, patterns of DNA synthesis by [methyl-3H] thymidine autoradiography, and lipogenesis by [U-14C] acetate uptake into lipids. When maintained for 7 days in supplemented Williams E medium, isolated pilosebaceous units showed a significant increase in length. This was due to the production of a keratinized hair fibre which grew at the in vivo rate of 0.3 mm/day. Light microscopy and [methyl-3H] thymidine autoradiography confirmed that after 7 days maintenance the hair follicle retained apparently normal morphology and patterns of DNA synthesis. However, the morphology of the sebaceous gland on maintenance was more variable, generally showing luminal keratinization. Moreover [methyl-3H] thymidine autoradiography of sebaceous glands showed a marked reduction on maintenance. The rates and patterns of lipogenesis by the whole pilosebaceous unit were, respectively, lower and different from those seen with isolated human sebaceous glands: this indicates that the bulk of pilosebaceous lipogenesis is derived from the hair follicle. Rates of recovery of [14C] from 2 mM-[U-14C] sodium acetate into thin-layer chromatography plates after 7 days maintenance decreased, although this was not statistically significant, indicating that rates of lipogenesis may fall on maintenance. Pilosebaceous units were maintained for 7 days on Gelfoam (an absorbable gelatin sponge) at the media-air interface. Initial results show a marked improvement in sebaceous gland morphology. It is possible, therefore, to obtain viable human pilosebaceous units by microdissection, and to maintain them in vitro for up to 7 days, with apparently full retention of hair follicle function, but only partial retention of sebaceous gland function.