Dermalife basal medium

Residual gingival tissue from molar extractions (see above) was enzymatically digested with dispase II (4 mg/ml) for 30 min at 37 °C. After filtering through a 70 μm cell strainer and washing with PBS by centrifugation (400 × g for 5 min), the cell pellet is resuspended in complete DermaLife Basal medium supplemented with DermaLife K LifeFactors (Lifeline Cell Technology). Cell are grown on Collagen-A-coated (0,1 mg/mL Collagen A) tissue culture vessels under standard culture conditions.

Human skin melanoma (G361) cells and primary human melanocyte from Caucasian skin (PMC) were obtained from Japanese Collection of Research Bioresources (JCRB, Japan) and Kurabo Industries Ltd. (Osaka, Japan), respectively. G361 cells were cultured in McCoy's 5A medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum. PMC were cultured in Derma Life Basal Medium (Life Line Cell Technology, Carlsbad, CA). OAG (1-oleoyl-2-acetyl-glycerol) (Sigma, Japan) was dissolved in dimethylsulfoxide (DMSO) to obtain 10 mg/ml stock solution and added to the subconfluent (60-70% confluence) cells. After the initial dose response and toxicity assays for OAG, 30 μg/ml for G361 melanoma and 15 μg/ml for PMC were used. Cells were treated with TXC or other natural extracts (as indicated) at 60-70% confluency. Cell viability assay was performed using MTT (Life Technologies) and quantitative measurement of conversion of yellow MTT to purple formazan by mitochondrial dehydrogenases of viable cells. Statistical significance of the results was determined from 3-4 independent experiments including triplet or quadruplet sets in each experiment. Control and treated cells were observed under the microscope and photographed to record their morphology.

For co-culture of human dental pulp-derived cells and cells of epithelial origin (gingivium or skin derived) the condensates produced described by the method above are transferred at day 2 to 5 in a composite medium appropriate for both cell types (DMEM high glucose (with FCS)) and DermaLife Basal medium supplemented with DermaLife K LifeFactors (Lifeline Cell Technology); 1:1). A single cell suspension of epithelial cells in a ratio of 1:4 related to the initial cell number used for mesenchymal condensation was added and the resulting mixture was cultured under non-adherent conditions. To ensure constant culture conditions, medium was changed regularly every 1–3 days.