Generation and integration of hair follicle-primed spheroids in bioengineered skin constructs

Skin is a complex organ made up of different cell layers, appendages, connective tissues, and immune repertoires. These different components interact extensively to maintain the overall functions of the integumentary system. In particular, appendages such as hair follicles critically contribute to the skin's function in thermoregulation, sensory perception, and homeostatic regeneration. Despite a strong need for better skin regenerative therapeutics, efforts to bio-engineer highly functional appendage-containing human reconstituted skinin vitrohave not yielded much success. Here, we report methods in generating and incorporating hair follicle-primed heterotypic spheroids into epidermal-dermal skin constructs that induced invaginating outgrowths with follicle-like organization and lineage gene expression. By co-culturing epithelial keratinocytes (KCs) with dermal papilla (DP) cells in low attachment plates, we established the media and culture conditions that best supported the viability, signalling and remodelling of the cell aggregates to form 3D KC-DP spheroids with the expression of both DP inductiveness and hair follicle lineage genes. We show that long-term growth and maturation of KC cells in these spheroids was supported by incorporation into epidermal-dermal constructs but not in scaffold-less media. When cultured, the bio-fabricated constructs developed invaginations from the integrated spheroids with follicle-forming potential. The generation of these constructs is a step towards the development of functional hair-bearing skin mimetics.