Regenerative therapy · 2026
Matrigel-embedded 3D culture maintains stemness and enhances hair-inductive capacity of human hair follicle epithelial stem cells.
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Dao Thuy Thi-Thanh, Trinh Nhu Thi-Linh, Tran Thi Xuan-Khanh, Dao Kim Hoang-Thien, Dang Loan Thi-Tung
Аннотация
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.
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.