Dispase

SHED and SHED-CM were prepared as previously described^{26 (link)}. The pulp was gently removed and digested for 1 h at 37 °C (SDminiN, Taitec, Koshigaya, Japan) in a solution containing collagenase type I (3 mg/ml) (Fujifilm Wako, Tokyo, Japan) and dispase (4 mg/ml) (Fujifilm Wako). Single-cell suspensions were plated on culture dishes in DMEM (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and then incubated at 37 °C in 5% CO₂ (Forma 310 Direct-Heat CO₂ Incubator, Thermo Fisher Scientific). The human skin fibroblast line, derived from a 36-year-old individual, was obtained at passage 12 from the Health Science Research Resources Bank (Osaka, Japan). SHED (passage 9), and Fibro (passage 13) for CM preparation were seeded at 1 × 10⁵ cells per dish. Cells at 70–80% confluence were washed with PBS and serum-free DMEM, followed by replacement with serum-free DMEM. Media were incubated for 48 h at 37 °C in a humidified atmosphere of 5% CO₂ (Forma 310 Direct-Heat CO₂ Incubator), then collected and centrifuged for 10 min at 2000 × g at 4 °C (AX-511, Tomy Seiko, Tokyo, Japan). Since the precipitates contained cell debris, we carefully collected the conditioned medium without nearing the precipitates. We adjusted the protein concentration of each CM to 3 µg/ml in serum-free DMEM.

HEK293 cells (ECACC 85120602) were cultured in DMEM (Sigma) supplemented with 10% FBS (Biowest), Fao cells (ECACC 89042701) in RPMI 1640 (GIBCO) supplemented with 10% FBS, and NIN/3T3 cells (ECACC 93061524) in DMEM supplemented with 10% calf serum (Biowest). Embryonic fibroblasts were isolated from 13.5-dpc homozygous embryos by treatment with trypsin (GIBCO), and resuspended and cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate (GIBCO), and MEM Non-Essential Amino Acids Solution (GIBCO)^{46 (link)}. To isolate primary hepatocytes, the liver was perfused by cannulation in the portal vein with HBSS containing collagenase (Nitta Gelatin) and dispase (WAKO), and the isolated cells were resuspended and cultured in WME (GIBCO) supplemented with 10% FBS, 100 nM dexamethasone, and 1 nM human insulin^{47 (link)}.

For the isolation of primary mouse keratinocytes, skin was removed from neonatal mice. The skin was floated on 1000 PU/mL dispase (Wako) dermis-side down, and kept at 4 °C overnight. The epidermis was peeled off from the dermis using a tweezers and floated on accutase basal-side down for 7 min at room temperature. The epidermis sheet was rubbed with a tweezers to separate the cells and filter them through a 100 µm cell strainer. Cell suspensions were centrifuged and resuspended in CnT-Prime Medium (CellnTec). The number of live cells was counted by trypan blue staining and 3 × 10⁵ cells were seeded in each well of collagen-coated 12-well plates. Primary keratinocytes were incubated in 37 °C for 3 h and observed by microscopy.

Mouse POBs were isolated according to the published methods^{2 (link),32 (link)}. POBs were obtained from the calvariae of neonatal C57BL/6J Jc1 mice (Clea Japan, Japan). A mixture of 0.1% collagenase (FUJIFILM Wako, Japan) and 0.2% dispase was used to dissociate cells from the bone fragments. POBs were then seeded onto either a 6-well plate or an 8-well chamber slide II at a density of 2.0 × 10⁶ or 3.2 × 10⁵ cells/well, respectively. The cells were cultured in αMEM (Thermo Fisher Scientific) with 10% FCS supplemented with 50 U/ml streptomycin (Thermo Fisher Scientific) and 50 μg/ml penicillin (Thermo Fisher Scientific). The medium was changed every 48 h for 1~2 weeks until bone-like nodules were formed.

Synovium was digested with 2 mg/mL collagenase (Wako), 3 mg/mL dispase (Wako), and 25 μg/mL deoxyribonuclease I (Sigma-Aldrich) prepared in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies) with shaking at 37°C for 1 hour. Bone fragments were crushed with a pestle, after which the crushed bones were washed gently once in phosphate buffered saline (PBS) (for remove the marrow cells). Bone and bone fragments were incubated for 1 hour at 37°C in DMEM in the presence of 2 mg/mL collagenase (Wako Chemicals) and 25 μg/mL deoxyribonuclease I (Sigma-Aldrich). The cell suspensions (synovium and bone fragments) were filtered through a cell strainer (Falcon, 70 μm) to remove debris. After lyse the red blood cells, these cells were re-suspended in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS) (Gibco) supplemented with 2% FBS, 10 mM HEPES and 1% penicillin/streptomycin.