RESUMEN
Mesenchymal stem cells (MSC) differentiate into different cell types and have immunomodulatory and paracrine effects. Cryopreservation of umbilical cord tissue as a source of MSC is very promising for regenerative medicine. We aim to evaluate a protocol for cryopreserving this tissue sectioned into small fragments with viable MSC. A total of 723 samples were frozen, thawed and cultured to obtain primary cultures of MSC. These were followed until 90-100% confluence and flow cytometric analysis were performed to confirm the mesenchymal phenotype. Samples in which protocol alterations at the collection of the samples were reported, were excluded for microbial contamination analysis leaving a total of 634 samples composed of 181 vaginal and 453 cesarean births. All cultures reach confluence with a media of 22.57 days and 97% in 28 or fewer days. Evaluated cultures showed low percentage of CD45+ and high of CD73 and CD90. Eight samples were subcultured 4 or 5 times and differentiated to chondrocytes and osteocytes to test differentiation potential with positive results. Umbilical cord tissue collections showed similar microbial profile and risk factors to those reported of umbilical cord blood collections, but with higher contamination frequencies. Cryopreserved tissue samples had viable cells that can be expanded without losing differentiation potential. Higher contamination frequencies compared to umbilical cord blood collection are not surprising, however, microbial load and survival of microorganisms to cryopreservation are expected to be lower.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Criopreservación/métodos , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Diferenciación Celular/genética , Proliferación Celular/genética , Condrocitos/citología , Sangre Fetal/citología , Humanos , Osteocitos/citología , Medicina Regenerativa/métodosRESUMEN
BACKGROUND AND OBJECTIVES: Umbilical cord blood is considered an alternative source of hematopoietic stem cells. Standard banking procedures use 50/55% DMSO in dextran 40 for cryopreservation and dextran-based solutions for thawing, however, due to the potential risk of crystallization of dextran, dextran 40 approved for clinical use has become limited or unavailable. This affects cryopreservation and thawing procedures. Carbohydrates, in particular sucrose, trehalose and glucose, have been shown to be effective in reducing cell damage during dehydration and have cryoprotective potential. We aim to study a 50/55% DMSO in 5% dextrose cryopreservation solution as an alternative to DMSO dextran. MATERIALS AND METHODS: Eighteen samples were divided into two aliquots and cryopreserved, one using standard solution and the other with DMSO dextrose experimental solution. Both aliquots were thawed and diluted with PBS or saline. Total nucleated cells counts, 7-AAD viability of CD45+ cells and recovery of CD34+ viable cells were assessed on thawed samples and compared between pair of aliquots. RESULTS: No differences were observed in the total nucleated cells recovery between cryopreservation solutions, however, higher viability and CD34+ viable cells recoveries were observed using the experimental solution. CONCLUSION: Results showed that DMSO dextrose cryopreservation solution had better results than the standard solution when thawed in an isotonic solution. This indicates that DMSO dextrose is probably a better alternative for direct infusion or when dextran thawing solutions are unavailable. Viability of CD45+ cells and recovery of CD34+ viable cells have positive correlation with engraftment, highlighting the relevance of the optimization of the cryopreservation and thawing process.