Sb 10

A diffusion method of chemical decellularization, adapted from previously published methods (30 (link), 31 (link)), was employed. Embryonic hearts were placed in 2 mL low retention tubes (Fisher Scientific 02-681-321), submerged with detergent and/or buffer, and placed on a rotator at 15 rpm (Thermo Fisher) for the following durations. Hearts were washed with ddH₂O for 8 h, sulfobetaine-10 (SB-10, Sigma Aldrich D4266) for 4 h, 100 mM Na/50 mM phosphate-buffered saline (PBS) for 15 min, 5% sodium deoxycholate (SD, Sigma Aldrich D6750)/sulfobetaine-16 (SB-16, Sigma Aldrich H6883) for 4 h, 100 mM Na/50 mM PBS for three 15-min periods, SB-10 for 1.75 h, 100 mM Na/50 mM PBS for 15 min, 5% SD/SB-16 for 3 h, 50 mM Na/10 mM PBS for two 15-min periods, deoxyribonuclease I (DNase I, Sigma-Aldrich D4527) solution for 12 h (with no rotation), and 50 mM Na/10 mM PBS for 15 min. Decellularized hearts, in fresh 50 mM Na/10 mM PBS solution, were stored at −80°C until further use.

The NPC-collagen microspheres were decellularized using a protocol optimized previously, involving a combination of the anionic detergent, Triton X-200, and the zwitterionic detergents, sulfobetaine-10 and -16 (i.e., SB-10 and SB-16; both from Sigma)^{12 (link)}. In brief, the samples were washed with PBS and immersed in 10 mM phosphate-buffered 50 mM sodium solution containing 50 mM SB-10 for 30 min. After rinsing with 50 mM phosphate-buffered 100 mM sodium solution, the samples were then immersed in 10 mM phosphate-buffered 50 mM sodium solution containing 0.6 mM SB-16 and 0.14% Triton X-200 for 30 min. After washing with PBS three times, the above steps were repeated, but the incubation times with detergent were reduced to 15 min. All the steps were carried out with gentle shaking at room temperature.