FIG. 2.

Enzyme-assisted extraction of MSCs from two-dimensional (2D) and 3D cultures. (A) Nattokinase (NK) yielded a significantly higher percentage of MSCs extracted from 3D fibrin gels relative to the other proteolytic enzymes [trypsin (TR) and TrypLE (TRPLE)]. To quantify the percentage of cells recovered, MSC-seeded fibrin gels were dissolved 6 h after initial cell seeding (50K cells/gel). For 2.5 mg/mL fibrin gels, cells were efficiently retrieved after a 30-min incubation in the nattokinase-based fibrinolytic solution; 5 and 10 mg/mL gels were incubated for 60 min. Retrieved cells were pooled together from a total of three gels for each technical sample. *** indicates that all of the groups under the line are significantly different from those groups not under the line (p≤0.001). (B) After 14 days of preculture in 3D fibrin gels, nearly 100% of the MSCs extracted from nattokinase were viable, as quantified by propidium iodide staining and flow cytometry. (C) After 1, 7, or 14 days of preculture, MSCs were retrieved from 3D fibrin gels (using nattokinase) or harvested from 2D cultures (using trypsin). The number of cells retrieved from the cultures was comparable at all time points for both culture conditions.