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

Abstract

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.

Effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cell sheets

“…To our gratification, the cryopreserved cell sheets did not show any noticeable disruption of the ECM structure or loss of viability and proliferative potential. Consistent with our findings, Kaku et al [16] and Vasconcelos et al [20] showed that long-term storage of periodontal ligament cells and cryopreserved periodontal ligament-derived undifferentiated mesenchymal cells in 10% (1.3 M) DMSO did not negatively affect their in vitro proliferative capacities or cell viability. These results demonstrated the feasibility of cryopreservation as a potential solution to the long-term storage of PDLSC sheets.…”

“…Recently, several groups demonstrated the feasibility of obtaining viable PDLSCs from frozen periodontal ligament tissues or intact whole teeth [20, 21]. However, to the best of our knowledge there has been no comprehensive study on the impact of cryopreservation on PDLSC sheets.…”

Potential for Stem Cell‐Based Periodontal Therapy

“…Two studies have shown that human PDLSCs can be recovered from cryopreserved PDLSCs and that cryopreservation does not affect the growth capacity of these cells (Seo et al, ; Vasconcelos et al, ). The cryopreserved PDLSCs maintained their stem cell characteristics such as expression of STRO‐1, multipotent differentiation capacity, and ability to form cementum/periodontal‐ligament‐like tissues (Seo et al, ; Vasconcelos et al, ). These data suggest that utilization of cryopreserved human PDLSCs for cell‐based therapy may be a valid clinical approach.…”

Proliferation of human periodontal ligament mesenchymal cells on polished and plasma nitriding titanium surfaces

“…Mesenchymal cells were isolated from periodontal ligament of impacted third molars, as previously described by Vasconcelos et al 9 (2012). Each tooth was immediately stored in a 50-ml Falcon tube containing alpha-MEM culture medium (Cultilab, Campinas, SP, Brazil), and then washed three times for 15 min each with alpha-MEM medium (Cultilab) supplemented with 10,000 IU/mL penicillin, 10,000 µg/mL streptomycin, 100 mg/mL gentamicin, and 250 µg/ mL amphotericin B (all antibiotics were purchased from Gibco, Grand Island, NY, USA).…”