To assay the SCs in 3D cultures, a 3D matrix was generated using electrospun membranes and SCs in the fibrin gel ± PFTBA (Figure 3 B ) 20 (link). Briefly, fibrinogen in saline (80 mg/ml) was mixed with 1×106 SCs, and to achieve coagulation, the SC-fibrinogen mixture was added to thrombin (5 IU/ml)-PFTBA (10 wt. %; Sigma). SC-gel lacking PFTBA was also produced through cell-fibrinogen and thrombin-PBS mixing. SC-gel mixtures were then injected onto the membranes. The study groups included: Group 1: SCs without PFTBA injected onto the membrane in the absence of PFTBA (fibers + gel); Group 2: SCs without PFTBA injected onto the membrane with PFTBA (PFTBA fibers + gel); Group 3: SC-gel mixtures with PFTBA injected in the absence of PFTBA (PFTBA-gel); Group 4: SC-gel with PFTBA injected onto membrane with PFTBA (PFTBA fibers + PFTBA-gel).
After 48 h or 96 h of hypoxia, Live-Dead assays were performed through dual color staining (Live-Dead Cell Staining Kits, BioVision). Live-DyeTM fluoresces green (ex/em 488/518 nm) in both live and dead cells, while PI stains only dead cells (ex/em 488/615 nm). Following 3D culture, SCs were counted and total RNA was extracted using Trizol (Sigma) lysis in 3D cultures. RT-PCR was then performed. Primer sequences are shown inTable S1
After 48 h or 96 h of hypoxia, Live-Dead assays were performed through dual color staining (Live-Dead Cell Staining Kits, BioVision). Live-DyeTM fluoresces green (ex/em 488/518 nm) in both live and dead cells, while PI stains only dead cells (ex/em 488/615 nm). Following 3D culture, SCs were counted and total RNA was extracted using Trizol (Sigma) lysis in 3D cultures. RT-PCR was then performed. Primer sequences are shown in