Human Dental Pulp Mesenchymal Stem Cells Cryopreservation

There are challenges related to quality and safety in the cryopreservation of human dental pulp stem cells. It is important to establish a method of cryopreservation of human dental pulp stem cells (hDPSCs) for future therapeutic uses. Objectives: In this study we evaluated the effect of two cryopreservation methods in three different times on the viability and phenotype of hDPSCs. Methods: An in vitro study of 48 healthy dental pulp taken from subjects between 14 and 31 years of age who signed an informed consent approved by the Institutional Review Board. The samples were mechanically and enzymatically dissociated, then CD105 + cells were magnetically isolated by Miltenyi MiniMACS ™, then cryopreserved by 2 different methods in 3 different times: 1, 7 and 30 days, evaluating their viability and cell phenotype postcryopreservation by flow cytometry. The data was described using percentages with their respective standard deviation, and for the statistical analysis the Student t test and Mann Whitney U test were used. Results: For method 1 after day 30 yielded an average of 59.5% cell viability while method 2 for day 1 and day 7 yielded an averaged of 65.5% and 56% for cell viability respectively. The phenotype showed increased expression for the CD105+/CD34- markers at day 1 and day 7 in method 1; CD105+/CD45- has a greater expression for the three different times of cryopreservation by method 1; CD34-/CD45- markers have increased expression for the three different times by method 2. CD73+ marker yielded better results at day 1 and day 7 by method 1. Conclusion: The cryopreservation method employed would modify the markers that characterize the phenotype of hDPSCs. Cryopreservation could modify cellular proteins affecting its detection by flow cytometry or may compromise the stage of cell differentiation.