Proox c21

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.

BV-2 microglial cells (CHI Scientific, Wuxi, China; cat. no. 7-1502) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco/Thermo Fisher, Grand Island, NY, USA; cat. no. 8117046) supplemented with 10% fetal bovine serum (FBS) (Capricorn Scientific GmbH, Ebsdorfergrund, Germany; cat. no. FBS-52A) at 37 °C in a humidified incubator with 5% CO₂/95% air. The cells were divided into five groups: control group, exposed to 20% O₂ + 5% CO₂; high concentration of carbon dioxide group (HC group), exposed to 20% O₂ + 15% CO₂; hypoxia group, exposed to 0.2% O₂ + 5% CO₂; hypoxia + HC group, exposed to 0.2% O₂ + 15% CO₂; hypoxia + HC + Z-YVAD-FMK group, treated with Z-YVAD-FMK (10 μM) [24 (link)] (ApexBio, Boston, MA, USA; cat. no. A8955) for 30 min before being exposed to 0.2% O₂ + 15% CO₂. The treated cells from different groups were incubated in an air-tight chamber, in which the O₂ and CO₂ concentrations were controlled by a Carbon Dioxide and Oxygen Controller (ProOx C21, Biospherix, Lacona, NY, USA). A Blood Gas/Electrolyte Analyzer (Model 5700, IL, San Diego, CA, USA) was used to measure PO₂, PCO₂, and pH of supernatants.

Rat primary ventricular neonatal cardiomyocytes (PVNC) were isolated from 1–2-day old pups using the Pierce Primary Cardiomyocyte Isolation Kit (#88281), which includes a cardiomyocyte growth supplement to reduce fibroblast contamination. H9c2 cells (ATCC CRL-1446) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone), containing penicillin, streptomycin, and 10% fetal bovine serum (Hyclone). Media was supplemented with MEM Non-Essential Amino Acids Solution (Gibco) for MEFs. Cells were incubated at 37 °C and 5% CO2. Human induced pluripotent stem cell-derived cardiomyocytes (H-iPSC-CMs) were obtained from Cellular Dynamics (iCell Cardiomyocytes #01434). iCell Cardiomyocytes were cultured in maintenance medium as per the manufacturer’s protocol and differentiated for 72 h. Cell lines were transfected using JetPrime Polyplus reagent, as per the manufacturer’s protocol. For misoprostol treatments, 10 mM misoprostol (Sigma) in phosphate buffered saline (PBS; Hyclone) was diluted to 10 μM directly in the media and applied to cells for 24 h. To achieve hypoxia, cells were held in a Biospherix incubator sub-chamber with 1% O₂ (±1%), 5% CO₂, balanced with pure N₂ (regulated by a Biospherix ProOx C21 sub-chamber controller) at 37 °C for 24 h. BvO2, L161-982, L798-106, and H89 dihydrochloride (H89) were purchased from Sigma.