Isolation and Culture of Gingival Mesenchymal Stem Cells

G-MSCs were obtained from the healthy gingival collars around partially impacted third molars at the Christian-Albrechts University of Kiel, Germany. The study was performed in accordance with Helsinki Declaration revised in 2008 and approved by the Ethics Committee of Christian-Albrechts University of Kiel, Germany. Cells' isolation and culture were done as formerly described [14 (link), 15 (link)]. Briefly, gingival soft tissue collars were detached, deepithelized, cut into pieces, and washed in a basic medium, consisting of Eagle's minimum essential medium alpha modification (Sigma-Aldrich GmbH, Hamburg, Germany), with 15% fetal calf serum (FCS, HyClone, Logan, UT, USA), 400 mmol/ml L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1% amphotericin (all from Biochrom, Berlin, Germany). The soft tissue parts were left to adhere for 30 min in dry tilted culture flasks, before the basic medium was slowly added to them. The flasks were incubated at 37°C with 5% CO₂ to let the cells grow and reach 80% confluence. The basic medium was replaced three times/week.
Immunomagnetic cell sorting employing anti-STRO-1 antibody (BioLegend, San Diego, CA, USA) and anti-IgM MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to isolate the G-MSCs as described before [14 (link)]. The obtained G-MSCs were cultured in a basic medium.