In this study, the immortalized human keratinocyte line HaCaT (Cytion, 300493; passage 3) and the human dermal fibroblast line HDF (ATCC, PCS-201-010; passage 5) were employed. HDF cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA), whereas HaCaT cells were cultured in DMEM/F-12 (Gibco, USA). All media were supplemented with 10 % fetal bovine serum (Biological Industries, Cat. No. 04-001-1A), 2 mmol L⁻¹ L-glutamine (Invitrogen, USA), and 100 U mL⁻¹ penicillin plus 100 µg mL⁻¹ streptomycin (Invitrogen, USA). Cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO₂. Both lines were confirmed to be mycoplasma-free using the Microsart® AMP Mycoplasma Kit (Sartorius, SMB95-1001); representative agarose-gel electrophoresis results are shown in Supplementary Figure 1. HaCaT and HDF cells were seeded into 24-well plates at 5 × 10⁴ or 1 × 10⁵ cells cm⁻². Twenty-four hours after seeding, L-ascorbic acid (L-AA; Sigma-Aldrich, USA) was added as described below.

L-ascorbic acid (Sigma-Aldrich, USA) stock solutions were prepared at 10 mg mL⁻¹ in sterile PBS (pH 7.0), filtered through a 0.22-µm syringe filter, aliquoted under minimal light, and stored at −20 °C. Working dilutions (25, 50, and 100 µg mL⁻¹) were prepared immediately before use and added to pre-warmed medium. To minimize oxidation, all L-AA handling was performed under light protection, and aliquots were thawed only once. Culture medium was replaced every 48 h with freshly prepared L-AA-supplemented medium. Control groups without L-AA were also included. In total, 16 experimental groups were established, as summarised in Table 1.

Cell morphology was monitored daily under an inverted phase-contrast microscope for 7 days. Half of the medium was replaced every 48 h, and at the end of the culture period sheet formation was evaluated microscopically. Formed sheets were gently washed with PBS, mechanically detached with a sterile pipette, and transferred to clean 6-well plates. To promote adhesion to a new surface, fresh medium was applied dropwise every 30 min for 3 h, after which the sheets were monitored for an additional 3 days. Cell sheet formation was defined according to both morphological and mechanical criteria: sheets were considered positive when they could be detached as an intact, continuous layer with a minimum diameter of ≥ 5 mm without enzymatic digestion, and when structural integrity was preserved under phase-contrast microscopy. Cultures that remained as fragile monolayers or detached in fragments were not classified as cell sheets. All groups (0, 25, 50, 100 µg mL⁻¹) were evaluated by MTT assay and phase-contrast microscopy. Groups that did not form cohesive sheets (25 µg mL⁻¹ and 100 µg mL⁻¹) were not processed for crystal violet staining or qRT-PCR.

Cell viability and metabolic activity were evaluated using the MTT assay on days 1, 3, 5, and 7 of the culture period. A 5 mg mL⁻¹ MTT solution was added to each well, and plates were incubated at 37 °C for 3 h in a 5 % CO₂ incubator. Following incubation, isopropanol containing 0.04 % (v/v) hydrochloric acid (Merck, Germany) was added to solubilize the formazan crystals 18. Absorbance was measured on a microplate spectrophotometer (Thermo Scientific Multiskan GO, USA) at 570 nm with a reference wavelength of 690 nm.

To evaluate cell morphology and proliferation, crystal violet staining was performed on days 3 and 7. Before staining, the culture medium was removed and the cells were fixed with a 1:1 acetone:methanol solution (Merck, Germany) for 10 min at room temperature. Fixed cells were stained with 0.1 % crystal violet (Sigma-Aldrich, USA) for 30 min at room temperature. After staining, excess dye was removed by washing with distilled water 19. Plates were air-dried and examined with an inverted phase-contrast microscope (Zeiss, Germany) to assess morphology, proliferation, and adhesion.

On day 7, gene expression analysis was performed to evaluate epidermal and dermal differentiation as well as cell proliferation. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany), and 1 µg RNA per sample was reverse-transcribed with iScript™ Reverse Transcription Supermix (Bio-Rad, USA). For HDF cells, RNA was isolated from mechanically detached cohesive sheets (50 µg mL⁻¹, 50 k seeding). For HaCaT cells, because no sheets were obtained, RNA was extracted from confluent adherent monolayers at the indicated seeding densities and L-AA concentrations. To assess epidermal differentiation, KRT14 and IVL transcripts were quantified in HaCaT cells; Ki-67 expression was determined to assess proliferation. In HDF cells, the extracellular matrix genes COL1A1, COL3A1, and FN1 were analysed. Relative quantification was performed using the 2^−ΔΔCt method. GAPDH and ACTB served as reference genes and displayed stable expression across all groups. All qRT-PCR reactions were conducted with iTaq™ Universal SYBR® Green Supermix (Bio-Rad, USA) according to the manufacturer’s instructions. Threshold cycle (Ct) values were recorded and relative expression levels calculated. All primers were newly designed to span exon–exon junctions and therefore avoid genomic DNA amplification; sequences were generated with NCBI Primer-BLAST based on RefSeq mRNA entries. Primer sequences and target gene information are summarised in Table 2.

All microscopic images were obtained with an inverted phase-contrast microscope (Zeiss, Germany) at 10× and 20× magnification. Scale bars were included in every panel and adjusted for clarity. Images were exported in TIFF format at 300 dpi. All illustrations are original and were generated during the course of this study. Only uniform adjustments of brightness and contrast were applied to entire images; no selective manipulations were performed. Macroscopic images of detached cell sheets were captured with a digital camera to illustrate gross morphology; consequently, no scale bar was applied.

Data are presented as mean ± standard deviation (SD) from three independent experiments per group. HaCaT and HDF cell lines were evaluated separately. The effects of different L-AA concentrations and cell densities on cell viability and gene expression were analysed for each line. Individual and interactive effects of ascorbic acid concentration and cell density were examined by two-way analysis of variance (two-way ANOVA). When significance was detected, Tukey’s multiple-comparison test was applied. Gene expression levels were calculated from Ct values using the ΔΔCt method. Differences with p < 0.05 were considered statistically significant. Full numerical MTT data (raw absorbance values, mean ± SD, and post-hoc p-values) are provided in Supplementary Tables S1–S4.

Cell-sheet formation was assessed in selected groups by culturing HaCaT and HDF cells at various seeding densities in the presence of escalating concentrations of L-ascorbic acid (L-AA). Cell morphology was inspected daily under an inverted microscope for 7 days, and sheet formation was scored according to the degree of confluence (Figures 1 and 2). In HDF cultures seeded at 50 000 cells cm⁻² and supplemented with 50 µg mL⁻¹ L-AA, a compact, well-defined sheet became apparent on day 7 (Figure 3). These sheets satisfied the predefined criteria of intact detachment (≥ 5 mm diameter) and microscopic structural integrity. Following gentle mechanical detachment, the sheets were successfully transferred onto fresh culture substrates, to which they adhered and remained stable during subsequent incubation. High-resolution microscopy revealed a dense, homogeneous architecture throughout both central and peripheral regions, confirming structural integrity (Figure 3A,B). Representative images acquired on day 3 are shown in Figure 3C to illustrate early morphological changes and proliferation. A macroscopic photograph obtained after successful transfer of an intact sheet further demonstrates its cohesiveness (Figure 3D). Annotations highlight the uniform cell distribution and extracellular-matrix-rich appearance of the construct.

Unlike HDFs, HaCaT cells failed to generate discernible sheets under any condition during the 7-day observation period. The highest proliferation rate was recorded in the group seeded at 100 000 cells cm⁻² and treated with 50 µg mL⁻¹ L-AA. All cultures were maintained by exchanging half of the medium every 48 h to satisfy metabolic demands (Figure 2). Enhanced proliferation in the 100 000-cell wells receiving 50 µg mL⁻¹ L-AA implies a pro-growth, non-differentiating effect of this dose. Conversely, cytotoxicity—manifested by decreased cell density and morphological alterations—was evident at 100 µg mL⁻¹. At 25 µg mL⁻¹, both cell types resembled untreated controls and failed to form cohesive sheets. Because of the pronounced toxicity, the 100 µg mL⁻¹ groups were omitted from crystal-violet and qRT-PCR assays, although viability and morphology data are presented in the main figures.

An MTT assay performed on days 1, 3, 5, and 7 demonstrated that the metabolic activity of both cell types was dependent on L-AA concentration and seeding density. In HDFs, metabolic activity peaked on day 5 in the 50 µg mL⁻¹ groups, with viability significantly exceeding that of the 25 and 100 µg mL⁻¹ groups (p < 0.05). In contrast, viability declined markedly in cultures exposed to 100 µg mL⁻¹ L-AA, indicating dose-dependent toxicity (Figure 4).

In HaCaT cells, proliferation increased from day 3 onward in high-density cultures supplemented with 50 µg mL⁻¹ L-AA, whereas the 100 µg mL⁻¹ groups showed no such rise (Figure 5). Overall, low- to moderate L-AA concentrations promoted time-dependent increases in viability, but this effect was abrogated at 100 µg mL⁻¹. The magnitude of the metabolic response depended on both L-AA dose and initial cell number.

Crystal-violet staining was used to visualise sheet morphology and cell density. After 7 days, only the HDF group seeded at 50 000 cells cm⁻² and treated with 50 µg mL⁻¹ L-AA produced a uniform, compact sheet that retained its morphology after transfer (Figure 6). Preservation of central-zone integrity confirmed successful sheet formation. Stepwise supplementation of culture medium at 30-min intervals facilitated re-spreading of the temporarily contracted sheet on the new substrate.

Because HaCaT cells did not form sheets, RNA was extracted from confluent monolayers, whereas HDF RNA was isolated from detached sheets. Expression data were normalised to the geometric mean of GAPDH and ACTB, which remained stable across all conditions. Relative expression was calculated by the 2^−ΔΔCt method. In HDF sheets (50 000 cells cm⁻², 50 µg mL⁻¹ L-AA, day 7) COL1A1, COL3A1, and FN1 were significantly up-regulated versus untreated controls (p < 0.05), with COL1A1 displaying the greatest fold increase (Figure 7A), indicating enhanced extracellular-matrix synthesis under optimal L-AA supplementation.

In confluent HaCaT monolayers (100 000 cells cm⁻², 50 µg mL⁻¹ L-AA), KRT14, IVL, and Ki-67 transcripts were elevated on day 7, with IVL showing the most pronounced induction (Figure 7B). These findings suggest that, in keratinocytes, 50 µg mL⁻¹ L-AA stimulates proliferation and early differentiation without promoting sheet cohesion. Collectively, L-AA modulates gene expression and functional outcomes in a dose- and cell-type-specific manner: fibroblasts respond by producing cohesive, ECM-rich sheets, whereas keratinocytes exhibit increased proliferation and differentiation marker expression in the absence of sheet formation.

Hatice Demir conceptualized the study, designed the methodology, conducted the experiments, curated and analyzed the data, created the visualizations, and wrote the original draft of the manuscript. Arzu Çöleri Cihan contributed to the conceptualization and methodology, supervised the research, reviewed and edited the manuscript, and was responsible for project administration and funding acquisition. All authors read and approved the final manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.  

The authors declare that they have not used generative AI (a type of artificial intelligence technology that can produce various types of content including text, imagery, audio and synthetic data. Examples include ChatGPT, NovelAI, Jasper AI, Rytr AI, DALL-E, etc) and AI-assisted technologies in the writing process before submission.

The authors declare that they have no competing interests.