“…Osmotic shock in distilled water was mainly applied prior to begin with decellularization 1 , 19 , 53 – 57 or, eventually, after a first freezing (heat shock). 58 For Villalba-Caloca et al, 18 Zhang et al, 19 Maughan et al, 59 and Sun et al 60 it was an intermediate phase; while, for Zhou et al, 56 immersion in deionized water was both the first step in decellularization and the last step of each cycle, specifically consisting in 4% SDC (25°C/4 h), 50 kU/mL of DNase-I in 1 M NaCl (37°C/3 h). Together with osmotic and temperature shock, other physical processes included constant agitation, 59 high-hydrostatic pressure, 61 ultrasonic bath, 18 and vacuum.…”