Collagenase type ι

Human exfoliated deciduous incisors were obtained as discarded biological samples from 6-to-8-year-old children in the Hospital of Stomatology, China Medical University, per the Institutional Review Board guidelines. Informed consent was obtained from the children's parents. The pulp was separated from the exfoliated teeth and digested in a solution of 4 mg/mL dispase (Boehringer Ingelheim, Mannheim, Germany) and 3 mg/mL collagenase type Ι (Worthington Biochemical Co., Lakewood, CO, USA) at 37°C for 30 min. Single-cell suspensions were obtained by passing the digest through a 70 μm strainer (Merck Millipore Ltd., Cork, Ireland). The cells were cultured in alpha modification of Eagle's medium (HyClone, Logan, UT, USA), 15% fetal bovine serum (ExCell Bio, Shanghai, China), 100 mmol/L L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and 100 U/mL penicillin-streptomycin (HyClone) at 37°C in the presence of 5% CO₂. SHED of passages 3–5 were used in the experiments. Serum-free culture medium was used as the cell suspension medium for transplantation.

Liver non-parenchymal cells were isolated as previously described.^{20 (link)} In brief, livers were harvested, perfused with calcium and magnesium-free Hank’s Balanced Salt Solution (HBSS; Beyotime Biotechnology), and digested with collagenase type Ι (Worthington, Lakewood, NJ, USA). The cell suspension was filtered through a 70 μm cell strainer (Sorfa, Zhejiang, China), centrifuged to eliminate hepatocytes, and then eliminated erythrocyte the Red Blood Cell Lysis Buffer (Beyotime Biotechnology) according to the manufacturer’s instructions. The non-parenchymal cells were incubated with different antibodies at 4°C for 30 min. Antibodies were used as follows: F4/80 (123108; BioLegend, San Diego, CA, USA); CD11b (101212; BioLegend); FITC Rat IgG2a κ (400505; BioLegend) and APC Rat IgG2b κ (400611; BioLegend).