In vitro comparative analysis of cryopreservation of undifferentiated mesenchymal cells derived from human periodontal ligament

Cryopreservation aims to cease all biological functions of living tissues in a reversible and controlled manner, i.e., to permit the recovery of cells by maintaining a high degree of their viability and functional integrity. The objective of this study was to evaluate in vitro the influence of cryopreservation on undifferentiated mesenchymal cells derived from the periodontal ligament of human third molars. Mesenchymal cells were isolated from six healthy teeth and cultured in α-MEM medium supplemented with antibiotics and 15% FBS in a humid atmosphere with 5% CO(2) at 37°C. The cells isolated from each tooth were divided into two groups: group I (fresh, non-cryopreserved cells) was immediately cultured, and group II was submitted to cryopreservation for 30 days. The rates of cell adhesion and proliferation were analyzed in the two groups by counting the cells adhered to the wells at 24, 48 and 72 h after plating. The number of cells per well was obtained by counting viable cells in a hemocytometer using the Trypan blue exclusion method. Differences between groups at each time point were evaluated by the Wilcoxon test. The Friedman test was used to determine differences between time points and, if detected, the Wilcoxon test with Bonferroni correction was applied. The results showed no significant difference in the in vitro growth capacity of undifferentiated mesenchymal cells between the two groups. In conclusion, cryopreservation for 30 days had no influence on periodontal ligament mesenchymal cells.