Cnt prime 3d barrier culture medium

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.

Punch biopsies taken from the dorsal skin of the mice were trypsinized, and the obtained epidermal sheets were incubated in 250 μl CnT-Prime 3D Barrier culture medium (CELLnTEC Advanced Cell Systems) in duplicate at 37 °C and 5% carbon dioxide. After 24 hours, the medium was collected, deproteinized using a 10 kDa MWCO spin filter, and assayed for glucose and pyruvate concentration using a colorimetric assay kit (Sigma-Aldrich). The concentration of lactate in the medium was evaluated as previously described (Limonciel et al., 2011 (link)). Fresh medium was used in all the assays as a reference. Results were normalized by the total protein content of the tissue.