Mycozap pr

HeLa, HaCaT, and HEK293T cell lines (ATCC) were maintained at 37°C in a humidified atmosphere of 5% CO₂ in fully supplemented media containing DMEM with 10% fetal bovine serum (FBS), nonessential amino acids, sodium pyruvate (Thermo Fisher Scientific), and Mycozap-CL (Lonza). Primary cardiac fibroblasts were isolated from neonatal mouse hearts as described (Zhang et al., 2014 (link)). Fibroblasts adhering to the dish during the pre-plating step were enriched for 1 week in DMEM:F12 supplemented with 3.5% FBS and 1x Mycozap-PR (Lonza). Primary glial cells were isolated from neonatal mouse brains as previously described (He et al., 2007 (link); Habas et al., 2013 (link)).

Human bone marrow MSCs (BMSCs) and adipose MSCs (ADSCs) purchased from Lonza (Basel, Switzerland) were cultured in mesenchymal stem cell basal medium (MSCBM, Lonza, Basel, Switzerland) or ADSC growth medium (ADSCBM, Lonza, Basel, Switzerland), respectively. Cells were cultured for 15 days following thawing from passage one and then after every seven days for successive passages. Cells were used to isolate EVs until passage six for BMSCs and seven for ADSCs and viability after starvation was established around 90% for both cell types (90% ± 2% for BMSC and 92% ± 3% for ADSC). For in vitro experiments, dermal microvascular endothelium cells (HMEC-1) were obtained by the ATCC (Manassas, VA, United States) and cultures following manufacturer’s instruction with MCDB131 medium (Thermo Fisher Scientific, Waltham, MA, USA). Normal Human Dermal Fibroblasts (NHDF) are primary adult fibroblasts (Lonza, Basel, Switzerland). NHDF were cultured in FGM-2 Growth Media (Lonza, Basel, Switzerland) supplemented with bullet kit and 1 mL Mycozap PR (Lonza, Basel, Switzerland). Normal Human Epidermal Keratinocytes (adult skin) (NHEK) were purchased from Lonza and cultured with KGMTM Gold Keratinocyte Growth Medium (Lonza, Basel, Switzerland).