GMSCs collection adhered to the Declaration of Helsinki and associated regulations. Consent forms were signed by all volunteers, with ethical approval granted by the Ethics Committee of the Stomatological Hospital of Southern Medical University (Approval No.: EC-CT-[2022]41).
The isolation and culture of GMSCs followed a modified protocol from Zhang et al. (Zhang et al., 2009 (link)). Gingival tissues, acquired from healthy individuals aged 23–30 years, were carefully segmented into 1–2 mm3 pieces and thoroughly rinsed with sterile PBS. Subsequently, the tissues were incubated with Dispase II (Solarbio, Beijing, China) overnight at 4 °C to separate the epithelial layer from the underlying connective tissue. Collagenase Type IV (Solarbio) digestion was then carried out at 37 °C for 1 h. The centrifuged pellet was then resuspended in Dulbecco’s Modified Eagle Medium supplemented (DMEM; Gibco, MA, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P/S; Gibco). The cells were cultured in T25 flasks at 37 °C with 5% CO2. Passages were performed using 0.25% trypsin when the cell confluence reached 70%–80%. Experiments were conducted using cells at passages 3-6.
The isolation and culture of GMSCs followed a modified protocol from Zhang et al. (Zhang et al., 2009 (link)). Gingival tissues, acquired from healthy individuals aged 23–30 years, were carefully segmented into 1–2 mm3 pieces and thoroughly rinsed with sterile PBS. Subsequently, the tissues were incubated with Dispase II (Solarbio, Beijing, China) overnight at 4 °C to separate the epithelial layer from the underlying connective tissue. Collagenase Type IV (Solarbio) digestion was then carried out at 37 °C for 1 h. The centrifuged pellet was then resuspended in Dulbecco’s Modified Eagle Medium supplemented (DMEM; Gibco, MA, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P/S; Gibco). The cells were cultured in T25 flasks at 37 °C with 5% CO2. Passages were performed using 0.25% trypsin when the cell confluence reached 70%–80%. Experiments were conducted using cells at passages 3-6.
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