Mf medium

This study was reviewed and approved by the animal care and use committee of the Tohoku University Graduate school of Dentistry (2014DnA-36-3). All the animal experimental procedures performed based on approved guidelines of Regulations for Animal Experiments and Related Activities at Tohoku University. Eight healthy micro-mini pigs (Fuji Micra, Japan), aged 24–30 months and weighing 25–35 kg, were used and analyzed in the experiments. The greater omentum was resected from each micro-mini pig following general anesthesia. Briefly, the resected omentum was minced and digested at 37 °C for 90 minutes with 0.25% collagenase (Wako) in phosphate-buffered saline and centrifuged as described previously^{23 (link)}. The cell pellet was suspended in 2 ml of PBS and filtered through a 70 µm cell strainer. Density gradient centrifugation excluded the red blood cells with Histopaque (Sigma, UK), and the remaining cells were cultured in MF medium (Toyobo) until confluent. The cells were then washed with PBS and treated with 0.02% ethylenediaminetetraacetic acid (EDTA) (Nacalai tesque) for 2 minutes. Floating cells were collected and seeded on fibronectin (Wako)-coated dish in MF medium and maintained as ADMPC.

Upon euthanization, the maxillae of C57BL/6J mice were separated, and molars were extracted under a stereomicroscope. PDL was scraped from the surface of the mesial root of the first molar using micro-instruments, including a micro-curette (0.5 mm; Fine Science Tools, CA, USA). The scraped tissue was placed in a 6-well plate under a coverslip to prevent the tissue from floating and cultured in cell culture medium (α-Modification of Eagle’s Medium; FUJIFILM Wako Pure Chemical, Japan) containing 10% fetal bovine serum (Life Technologies, CA, USA), kanamycin (60 µg/mL; FUJIFILM Wako Pure Chemical), and FGF-2 (100 ng/mL; Kaken Pharmaceutical, Japan). The medium was incubated at 37℃ in 95% air and 5% CO₂. The cells that migrated and proliferated from the tissue were subcultured as murine PDL cells. Murine gingival fibroblasts (MGF) were established in this study. The dissected palatal gingiva of C57BL/6J mice was minced and placed in a 6-well plate with MF-start primary culture medium (Toyobo, Japan). The migrated cells from the tissue were subcultured with MF-medium (Toyobo) as MGF. Murine dental pulp-derived stromal cells (mDPSC) were also established from pulp explant of maxillary molars with the same protocol with MGF. Murine bone marrow and adipose tissue-derived undifferentiated stromal cells (mBMSC and mADSC) were purchased from Cyagen, USA.

hMSCs were purchased from Lonza (Basel, Switzerland) and cultured in MF-medium (TOYOBO, Tokyo, Japan). For the maintenance of hMSCs, the culture media were replenished every 2 days. hMSCs were detached and dissociated into single cell suspension by 4–5 min incubation with Trypsin solution [Trypsin/ethylenediaminetetraacetic acid (EDTA) for Mesenchymal Stem Cells, Lonza]. Live cell numbers were manually counted using hemocytometer.