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Tissue engineering of human hair follicles: long-term culture of human skin flaps is a powerful method to study follicle-formation of in-vitro generated follicle-inducing cells

By: Prof. Walter Krugluger, Karl Moser, Claudia Moser, Dr. Joerg Hugeneck, Katharina Laciak; all Moser Medical Group, Austria


Introduction: Tissue engineering of human hair follicles might be a powerful method to multiply human hair follicles in the future. Today, attempts for the in vitro expansion of follicle-inducing stem cells has mainly been done in the rodent system, where in vivo assays were established to study the behavior of in vitro generated follicle-inducing cells. In humans, no satisfactory methods exist, which enables the investigation of follicle formation by in vitro generated candidate cells. In this project, we establish an in vitro method, based on long-term culture of human skin flaps of different origin, to study the follicle-forming capacity of in vitro generated follicle-inducing cells.

Methods: Skin long-term cultures were set up from skin specimens from different regions of the body obtained during plastic surgery. The specimens were cut in pieces of approximately 0.5 cm² and subcutaneous tissue was removed from the specimens. Different semi-solid culture systems, culture media and growth factor combinations were tested for their ability to maintain skin architecture and cell viability.

To study the follicle-forming potency of in vitro expanded human hair follicle cells, cells from different parts of the follicular unit were expanded by tissue culture techniques. The resulting cell populations obtained from the dermal papilla, outer root sheath and other parts of the follicle were harvested and injected in the skin specimens for long-term culture. After culture for several weeks, specimens were subjected for historical analysis.

Results: Histological evaluation of cultured skin specimens after 6 or 8 weeks of culture demonstrated intact skin architecture. Furthermore, in situ labeling of skin cells with vital-dyes demonstrated high viability of the cells.

Skin specimens injected with potential follicle-inducing stem cells were cultured for 6 to 8 weeks in the system. Histological evaluation of the specimens revealed induction of follicle-like structures by in vitro expanded hair follicle cells.

Conclusion: The long-term culture systems for human skin flaps developed in our laboratory allows the investigation of inductive properties of follicle-inducing cells in an in vivo-like system. This system enables the investigation of cell-cell and/or cell-matrix interactions as well as the influence of locally administered growth-hormones or cytokines on follicle formation.