Isolation and Culture of Human Gingival MSCs

Gingival tissue biopsies were obtained from five healthy adult volunteers with no gingival inflammation. The gingival specimens were completely de-epithelialized with a scalpel, for the exclusion of the most of the keratinocytes resident in the gingival. In brief, the connective tissues were grinded and then washed several time with PBS (LiStarFish, Milan, Italy) and subsequently cultured using TheraPEAK™MSCGM-CD™ BulletKit serum free, chemically defined (MSCGM-CD) medium for the growth of human MSCs (Lonza, Basel, Switzerland). The medium was changed twice a week, and cells spontaneously migrating from the explant fragments after reaching about 80% of confluence, were trypsinized using Triple Select (LiStar Fish) (Diomede et al., 2014 (link)). On day 6, colonies of 50 or more cells were scored as colony-forming unit fibroblasts (CFU).