Matrigel-embedded 3D culture maintains stemness and enhances hair-inductive capacity of human hair follicle epithelial stem cells

Background Human hair follicle epithelial stem cells (hHF-EpSCs) represent a promising cell source for biomedical research. However, conventional 2D culture systems show limited efficacy in maintaining their stemness and proliferative capacity. Methods In this study, we compared two 3D culture strategies: Embedding EpSCs in Matrigel (embedded culture) and seeding them on top of Matrigel (on-top culture). The formation, quantity, and size of the spheres were observed by microcscope. The cell viability and proliferation were assessed by staining Calcein AM/Propidium iodide (AM/PI) and Water-Soluble Tetrazolium 1 (Wst1) assay. The expression of EpSC specified markers such as CD200, CK19, CK15, CK10, CK5 were found via immunocytochemistry. The genes related to hair-induction, including Wnt5a, Wnt10a, Wnt10b, Lef1, Adar, and β-catenin were surveyed by reverse transcription – quantitative polymerase chain reaction (RT-qPCR) in hHF-EpSCs cultured by both methods. Results The embedded approach facilitated the formation of numerous cell aggregates with an average size below 200 μm, peaking in number on day 10 (88.33 ± 4.16 spheroids per microscopic field). In contrast, the on-top culture produced fewer aggregates (maximum 17.00 ± 1.00 per microscopic field on day 3), with larger average sizes (>200 μm). Cells cultured within Matrigel also exhibited higher viability and proliferation over 14 days. Both methods preserved the expression of key HF-EpSC markers, including CD200, CK19, CK15, CK10, and CK5. The upregulation of genes such as Wnt5a, Wnt10a, Wnt10b, Lef1, Adar, and β-catenin was observed in hHF-EpSC spheres formed by Matrigel-embedded culture after 7 days. Conclusion These findings suggest that 3D Matrigel-embedded culture is a promising approach for maintaining EpSCs, and promoting hair induction.