Biomedical and Biotechnology Research Journal · Published 2026-04-01 · DOI 10.4103/bbrj.bbrj_23_26
Background: Human hair follicle dermal papilla cells (hHF-DPCs) play a critical role in hair follicle regeneration. Under conventional two-dimensional (2D) culture conditions, DPCs progressively lose the expression of key biological markers, suggesting that three-dimensional (3D) culture may better preserve their characteristic properties. Methods: In this study, hHF-DPCs were seeded into 0.2 mL polypropylene microtubes at three cell densities – 5 × 104, 105, and 2 × 105 cells/mL – to evaluate forced aggregation as a 3D culture approach. Aggregate formation and diameter were monitored over time. Viability was assessed by acridine orange/propidium iodide (AO/PI) staining and Trypan blue exclusion, while gene expression of alkaline phosphatase (ALP), versican, alpha-smooth muscle actin (α-SMA), and CD133 was quantified by reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Results: Results demonstrated that hHF-DPC aggregates formed within 24 h across all densities. At 5 × 104 cells/mL, spheroids formed but disaggregated by day 3, whereas higher densities (105 and 2 × 105 cells/mL) produced compact, architecturally stable spheroids maintained for 7–10 days. Necrotic cores were detected from day 3 at the two higher densities, accompanied by a reduction in viable cell number. Compared with 2D culture, 3D spheroids exhibited significant upregulation of ALP, versican, and CD133, while α-SMA expression was downregulated at both high densities (P < 0.05). Conclusions: Microtube-based forced aggregation successfully generated hHF-DPC spheroids at densities of 105 and 2 × 105 cells/mL within 24 h, with stable morphology maintained for up to 10 days and enhanced expression of DPC-specific markers compared to conventional 2D culture.
Abstract from DOAJ. Public domain (CC0 1.0).
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