Stable glutamine

Sufficient cryopreserved ADMSC vials were thawed to provide the required dose for administration. The frozen ADMSCs were thawed and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% human serum, 1% penicillin-streptomycin solution, and 1% stable glutamine (Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C and 5% CO₂ incubator as described before [16 ]. Briefly, ADMSCs were recovered and resuspended in physiological saline solution and transferred to the patient with the temperature-controlled bag. The morphology of cell appearance and viability were assessed besides the identification of phenotypic characteristics of the cells and microbiological tests. Immunophenotyping characterization of ADMSC was performed by using flow cytometry analyses (Beckman Coulter, USA) with antibodies against CD45, CD73, CD90, CD105, CD11b, CD19, CD34, and CD44 (BD Stem Flow hMSC kit).

The in vitro biocompatibility testing of the materials was performed on two commercial cell lines, the L929 (mouse fibroblasts) and RAW 264.7 (mouse macrophages) cell lines, both obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The L929 cell line was taken for indirect culture system testing on fibroblasts, as it is the most commonly used model for testing dental materials on fibroblasts. The RAW 264.7 cell line is a macrophage line used for phagocytosis assay and immunomodulatory activity testing of materials. The cells of both cell lines were cultured in Dulbecco’s Modified Eagle Medium with 1 g⁄L glucose (DMEM) supplemented with 2 mM stable glutamine, 1% antibiotic-antimycotic solution, and 10% fetal bovine serum (complete DMEM), all purchased from Biological Industries (BioInd, Kibbutz Beit-Haemek, Israel), in standard cell culture conditions (humidified atmosphere, 5% CO₂, 37 °C).