Comparative Analysis of Ascorbic Acid-Induced Cell Sheet Formation in HaCaT Keratinocytes and Human Dermal Fibroblasts

Introduction: This study examines the effects of varying L-ascorbic acid (L-AA) concentrations and initial seeding densities on cell-sheet formation by human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). Both cell types were seeded at either 50,000 or 100,000 cells cm⁻² and exposed to 0, 25, 50, or 100 µg mL⁻¹ L-AA. Cell-sheet formation was assessed morphologically; cell viability was quantified by an MTT assay; and gene expression was measured by qRT-PCR using newly designed primers. Methods: In HDF cultures, the 50 µg mL⁻¹ group at a seeding density of 50,000 cells cm⁻² produced the most cohesive and viable sheets, accompanied by a marked up-regulation of COL1A1, reflecting enhanced extracellular-matrix synthesis. At 25 µg mL⁻¹, HDF morphology and viability were similar to those of the untreated control, with no cohesive sheet formation, whereas 100 µg mL⁻¹ induced pronounced cytotoxicity. Results: In HaCaT cultures, no cohesive sheets were observed at any concentration. Nonetheless, at 50 µg mL⁻¹ and 100,000 cells cm⁻², confluent HaCaT monolayers displayed increased expression of keratin 14 (KRT14), involucrin (IVL), and Ki-67, indicating enhanced proliferation and early differentiation despite absent sheet integrity. Conclusion: Collectively, these findings demonstrate that L-AA promotes sheet formation in fibroblasts but not in keratinocytes under two-dimensional monolayer conditions. The limited HaCaT response suggests that co-culture or three-dimensional systems may be required for epithelial stratification. This study underscores the importance of tailoring L-AA concentrations and cell densities to each cell type in skin tissue engineering.