Pftba

The preparation of PFTBA emulsion was performed as described in previous studies with modifications 20 (link), 22 (link). Briefly, egg yolk lecithin (190 mg, 99% purity, Sigma) was dissolved in Tyrode's solution (1 mL, Sigma) and sonicated. PFTBA (1 mL, 98% purity, Sigma) was then added with sonication (12 times at a temperature of 4 °C). For control groups, the emulsion with PBS (without PFTBA) was prepared. To determine the optimal composition and core-shell structure, the carboxyl-chitosan (5 % w/v, 7.5 % w/v, 10 % w/v, 12.5 % w/v, and 15 % w/v.; (Sigma) Figure 1A) was mixed with the PFTBA emulsion to reach the core solution. Thereafter, 20 % w/v. PCL (Sigma) was prepared as the shell solution in methyl alcohol and trichloromethane 1:4 v/v (Fuyu, China). To observe core-shell fiber formation, Rhodamine B (1 mg/mL, R6626, Sigma) was mixed into the core solution and 2 mg/mL of fluorescein isothiocyanate (FITC; YESE, China) was added to the shell solution.

Gas chromatograms were measured on a Perkin Elmer (Waltham, MA, USA) GC Clarus 590 connected to a Perkin Elmer MS Clarus SQ 8 S with an EI standard source, with a 4.0 mm Glass inlet liner with deactivated wool in split mode 1:50 on a Perkin Elmer Elite 5MS 30 m × 0.25 mm × 0.25 μm capillary column and a 1.0 mL/min flow rate with injection volume of 1 μL. The oven was programmed as follows: 100 °C for 2 min, then 10 °C/min heating rate to 300 °C, and afterwards hold for 5 min. Mass spectra were measured with electron ionization (EI) at 70 eV, transfer line at 250 °C, and a source temperature of 200 °C. The mass fragments were scanned between 50 and 500 m/z for all diesters with the exception of dimethyl oxalate, where fragments were scanned between 40 and 500 m/z. In all cases, a scan time of 0.3 s and inter-scan delay of 0.04 s were used. The Mass Spectrometer (MS) was calibrated and tuned with perfluoroterbutylamine (PFTBA, Sigma Aldrich). The samples were diluted in cyclohexane with a concentration of 0.1 mg/mL.

To assay the SCs in 3D cultures, a 3D matrix was generated using electrospun membranes and SCs in the fibrin gel ± PFTBA (Figure 3B) 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-Dye^TM 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 Table S1.