Isolation and Expansion of Mandibular and Long Bone MSCs

Mandibular and long bones were collected to isolate cells independently. The attached soft tissues and teeth including incisors and molars were removed from the bones. All nucleated cells (ANCs) from mandibular bones were obtained by the digestion with 3 mg/mL collagenase type I (Worthington Biochem, Lakewood, NJ) and 4 mg/mL dispase II (Roche Diagnostic, Indianapolis, IN) for 60 min at 37°C. ANCs from long bones were obtained by flashing out from the bone marrow (Yamaza et al., 2008 (link)). Single cell suspension of ANCs from mandibular or long bones were obtained through 70 μm-cell strainers (BD Bioscience), and seeded at 1–1.5×10⁶ or 10–15×10⁶ on 100-mm dish (Corning, Corning, NY), respectively. MSCs were isolated and cultured following established protocol (Yamaza et al., 2008 (link)). The cells formed attached single colonies were recognized as passage 0 (P0) MSCs, and subsequently collected and passed to P1 expansion for subsequent experiments. There was no feeder cells were used in MSC culture. Multiple colony MSCs derived from several mice (n=3~5) were pooled and used in this study. Each test was repeated at least three times. To avoid hematopoietic cell contamination, we select multiple numbers of single colony clusters and pass to secondary culture.