Blood Mesenchymal Stem Cell Culture From The Umbilical Cord With And Without Ficoll-paque Density Gradient Method [cultivo De Células Mesenquimais Do Sangue De Cordão Umbilical Com E Sem Uso Do Gradiente De Densidade Ficoll-paque]

摘要

Objectives: Implantation of cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood with and without using the Ficoll-Paque gradient density method (d=1.077g/ml). Methods: Ten samples of the umbilical cord blood obtained from full-term deliveries were submitted to two different procedures of mesenchymal stem cell culture: a) Method without the Ficoll-Paque density gradient, which concentrates all nucleated cells; b) Method with the Ficoll-Paque density gradient, which selects only low-density mononuclear cells. Cells were initially plated into 25 cm 2 cultures flasks at a density of 1×10 7 nucleated cells/cm 2 and 1×10 6 mononuclear cells/cm 2. Results: It was obtained 2-13x10 7 (median = 2.35×10 7) nucleated cells/cm 2 by the method without the Ficoll-Paque gradient density, and 3.7-15.7×10 6 (median = 7.2×10 6) mononuclear cells/cm 2 by the method with the Ficoll-Paque gradient density. In all cultures adherent cells were observed 24 hours after being cultured. Cells presented fibroblastoid and epithelioid morphology. In most of the cultures, cell proliferation occurred in the first week, but after the second week only some cultures - derived from the method without the Ficoll-Paque gradient density - maintained the growth rate reaching confluence. Those cultures were submitted to trypsinization with 0.25% trypsin/EDTA solution and cultured for two to three months. Conclusion: In the samples analyzed, cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood by the method without the Ficoll-Paque density gradient was more efficient than the method with the Ficoll-Paque density gradient.