Cnt 09

Keratinocytes were harvested by agitating the epidermis, then cultured using CellnTec (CnT-09) media. Cells were cultured on T25 flasks until approximately 60% confluent. These cells were trypsinzied using TrypLE and re-seeded on a new flask and let grow for overnight. The media was changed and all cells which were not attached were thrown away with the media. The cells were grown until approximately 60% confluent. This process was repeated. This process only allows for basal keratinocytes to proliferate in culture, as other cell types have a much slower growth process and will be rapidly overgrown by keratinocytes [19 (link)]. The exception to this is fibroblasts. If there was any fibroblast contamination in the keratinocytes, differential trypsinization was performed to detach the fibroblasts and not keratinocytes. After proliferation these were frozen in cell culture freezing media (Gibco 12638-010) overnight at −80 °C then kept in liquid nitrogen for storage. Cells were seeded onto Lab-TeK II Chamber Slides (Thermo Fisher 154526, Waltham, MA, USA) at 1 × 10⁵ cells per well. Once cells reached confluency, this time point was labeled as Day 0 (D0). CPEK cells were purchased from CellnTec (CPEK). These cells were grown with CellnTec (CnT-09) media. They were seeded at the same concentrations as the primary keratinocytes.

The skin samples were washed in buffer, and epidermis was separated from the dermis mechanically after incubation in dispase II solution (2.4 U/mL, CnT‐DNP‐10, CellnTec, Bern, Switzerland) at 37°C. Subsequently, the epidermis was cut into small pieces and incubated in 0.25% trypsin with 0.05% ethyl‐enediaminetetraacetic acid (EDTA; Trypsin‐EDTA Solution, Sigma Aldrich, Saint Louis, Missouri, USA). Enzymatic activity was stopped using medium containing 10% fetal bovine serum (FBS, Gibco‐Thermo Fisher Scientific, Waltham, Massachusetts, USA). After centrifugation (200g, 7 minutes, 4°C), the cell pellet was resuspended in an appropriate culture medium in a 6‐well culture plate with addition 1% of streptomycin/penicillin solution (Sigma Aldrich, Saint Louis, Missouri, USA). The keratinocytes were cultivated in Epidermal Keratinocyte Medium (CnT‐09, CellnTec, Bern, Switzerland) supplemented with CnT‐IsoBoost Supplement (CnT‐ISO‐50, CellnTec, Bern, Switzerland), whereas fibroblasts were cultivated in DMEM medium (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland) supplemented with 10% FBS (Gibco‐Thermo Fisher Scientific, Waltham, Massachusetts) and 2 mM of L‐glutamine (Sigma Aldrich, Saint Louis, Missouri, USA) at 37°C and under 5% CO₂.