Cnt prime

The hASCs were isolated as previously reported^{2 (link),17 (link),30 (link)}, and maintained in alpha modified Eagle’s medium (α-MEM) comprising 10% fetal bovine serum, and 10 ng/mL epidermal growth factor (PeproTech, Rocky Hill, NJ, USA). The cells were plated on fibronectin-coated dishes at a seeding density of 4 × 10³ cells/cm². The medium was replaced every 2 days. To obtain a hypoxic culture system, the cells were cultured in a gas mixture comprising 90% N₂, 5% CO₂, and 5% O₂. A ProOx C21 carbon dioxide and oxygen controller and a C-Chamber (Biospherix, Redfield, NY, USA) were used to maintain the hypoxic conditions using the gas mixture. ASCs at passages 2–4 were used for the experiment. HNDF were purchased from Lonza (Basal, Switzerland) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. HNDF at passages 3–5 were used for the experiment. The cells were plated at a seeding density of 4 × 10³ cells/cm². The medium was replaced every 2 days. HPEK were purchased from CELLnTEC (Bern, Switzerland) and maintained in CnT-Prime (CELLnTEC) culture medium according to the manufacturer’s protocol. The human skin equivalents were generated using CnT-Prime-3D Barrier culture medium (CELLnTEC) according to the manufacturer’s protocol.

Human primary keratinocytes were isolated from surplus human skin obtained through plastic surgery according to the principles and guidelines of the principles of Helsinki. From the skin, biopsies were taken and keratinocytes were isolated as described previously (Tjabringa et al. 2008 (link)). N/TERT–2G keratinocytes were a kind gift of James Rheinwald, Brigham’s Woman hospital (Dickson et al. 2000 (link)) and were cultured as monolayers in CnT–prime (CELLnTEC, Bern, Switzerland, CnT–PR) until confluent before use in HEE cultures (Smits et al. 2017 (link)). Knockout N/TERT–2G cell lines were generated through CRISPR/Cas9 and validated previously (FLG (Smits et al. 2023a ), CLDN1 (Arnold et al. 2023, accepted with minor revisions), TFAP2A (Smits et al. 2023b , accepted), AHR (Smits et al. 2023b , accepted)).

MEFs were derived from the limbs of E13.5 embryos and dermal fibroblasts (MDFs) were derived from P0-P1 mice using standard protocols. Male and female littermates were pooled to generate fibroblast cultures. We sorted these MEF and MDF cultures against CD45/Ter119/CD31/Mac-1(CD11b)/EpCAM to exclude potential contamination with hematopoietic, endothelial, macrophages and epithelial cells.
MEFs and MDFs were cultured in DMEM supplemented with 10% FBS, 1 × antibiotic/antimycotic (Invitrogen) on 0.1% gelatin-coated tissue culture plates (Millipore). iEpt cells were generated in defined basal epithelial media Cnt-Prime (CellnTec) supplemented with 100 nM dexamethasone (Sigma). iPSCs were maintained in DMEM/F12 medium supplemented with 10% knock-out serum replacement, 1x nonessential amino acids, 1 × GlutaMax, 1 × antibiotic/antimycotic, 55 μM β-mercaptoethanol (all from Invitrogen), and 1000 U ml⁻¹ mouse Leukemia Inhibitory Factor (LIF; Millipore). iPSC colonies were passaged on mitomycin-treated passage 1 wild-type MEFs and maintained in the same media with 10% FBS. Mitomycin was used at a concentration of 10 μg ml⁻¹ for 3 h.

Proprietary cell culture medium CnT-Prime was purchased from Cellntec Advanced Cell Systems AG (Bern). Trypsin-ethylenediaminetetraacetic acid (EDTA), soybean trypsin inhibitor, sodium bicarbonate, goat serum, sodium azide, Tween-20, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Triton X-100, gentamicin, bovine serum albumin (BSA), fetal bovine serum (FBS), penicillin-streptomycin, human recombinant insulin, hydrocortisone, adenine, 3,3,5-triiodo-L-thyronine (T3), dispase II, and trypan blue were purchased from Sigma Aldrich (St Louis, MO). Cholera toxin was purchased from Enzo Life Sciences (Farmingdale, USA). Epidermal growth factor, Gibco low-glucose Dulbecco’s modified Eagle’s medium (DMEM), Gibco 1:1 DMEM/F12 nutrient mix, minimal essential medium (MEM), phosphate buffered saline (PBS), Hank’s balanced salt solution, routine plastics, pipettes and Nunclon Δ surface multi-dishes were purchased from Thermo Fisher Scientific (Life Technologies) (Waltham, MA).

HGECs obtained from CELLnTEC (CELLnTEC, Berne, Switzerland) were used in this study as our established protocol [19 (link)]. They were cultured in epithelial culture medium (CnT-prime, CELLnTEC, Berne, Switzerland, changed every two days) in a humidified incubator (37 °C with 5% CO₂). The 3rd to 5th passages of HGECs were used in all experiments.