A scalable gelatin sponge-based 3D culture platform for human umbilical cord mesenchymal stem cell-derived conditioned medium promotes hair follicle regeneration

Alopecia is common and psychosocially burdensome. Promoting the telogen-to-anagen transition is a key therapeutic strategy. Mesenchymal stem cell-derived conditioned medium (MSC-CM), a complex mixture of trophic factors, is a promising cell-free approach. A scalable 3D culture platform using clinical-grade gelatin sponge (GS) scaffolds was developed to expand human umbilical cord-derived MSCs (hUC-MSCs) and produce GS-cultured hUC-MSC-derived conditioned medium (GS-MSC-CM), which was concentrated by lyophilization. Efficacy was tested in a depilation-induced hair cycle synchronization model in C57BL/6 mice, with topical minoxidil as a positive control. Outcomes included macroscopic photography, skin grayscale analysis, histomorphometry, and immunofluorescence. In vitro, GS-MSC-CM effects on human dermal papilla cell (DPC) migration, proliferation, and gene expression were assessed by scratch assay, CCK-8, and qRT-PCR. The 3D system supported high-density hUC-MSC growth and viability for at least 15 days. GS-MSC-CM significantly accelerated hair regrowth and increased hair follicle density in vivo. Under their respective routes, its efficacy trend was similar to minoxidil, though different administration routes preclude direct comparison. Histologically, GS-MSC-CM enhanced dermal papilla alkaline phosphatase (ALP) activity and upregulated the expression of the hair follicle stem cell marker K15 and the proliferation marker PCNA. In vitro, GS-MSC-CM stimulated DPC migration, proliferation, and ALP gene expression. GS-MSC-CM promotes the telogen-to-anagen transition and hair follicle regeneration, supporting further development as a scalable, cell-free option for alopecia.