Pltgold human platelet lysate

PLTGold^®® Human Platelet Lysate (Sigma-Aldrich, St. Louis, MO, USA) is derived from normal human donor platelets collected at US blood centers. Multiple donor units are pooled in large batch sizes and manufactured to produce a consistent product [15 (link)].

Human MSCs were isolated from discarded lipoaspirate of 2 healthy volunteers (Sidra Medicine IRB#1609004389). Automated and clinical-grade fat digestion was performed through the Celution^® 800/CRS System (Cytori Therapeutics Inc., San Diego, USA). The isolated cells were seeded and cultured in flasks for expansion (2 × 10⁶ cells in T75 flask) with 5% PLTGold^® Human Platelet Lysate (Sigma-Aldrich), low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich), 1% 10,000 U/mL penicillin, and 10 mg/mL streptomycin (VWR), at 37 °C in a humidified 95:5 atmosphere of air/CO₂ (mol%). The AD-MSCs were adherent to plastic, displayed fibroblastoid morphology, and were compliant with the International Society of Cell Therapy criteria for MSCs^{97 (link)}. AD-MSCs were then cultured in adipo- (StemPro™ Adipogenesis Differentiation Kit (Gibco)) and osteo- (StemPro™ Osteogenesis Differentiation Kit (Gibco)) differentiating media, for 14 and 21 days respectively, as per manufacturer’s instructions, with the following conditions: AD-MSCs (control), AD-MSCs + RSG 1 µM (Cayman Chemical Company), AD-MSCs + DHA 10 µM (Cayman Chemical Company), AD-MSCs + RSG + DHA. Each condition was run in duplicate for RNA extraction and staining.

After 3 h of hypoxia, SV-HUC-1 cells were transferred back to the culture medium in 95% air and 5% CO₂ for reoxygenation for 24 h and treated with or without 2% PLTGold^® Human Platelet Lysate (Sigma-Aldrich, St. Louis, MO, USA).