Cell isolation method,seeding number,and coating conditions for inducing and amplifying rat bone marrow mononuclear cells into endothelial progenitor cells
Objective To select the cell isolation method,seeding number,and coating conditions for culture flasks to induce and amplify rat bone marrow mononuclear cells(BMMNCs)into endothelial progenitor cells(BM-EPCs),aiming to establish an efficient,high-yield,and high-purity method for the separation,culture,induction,and amplification of BM-EPCs derived from bone marrow.Methods Two-week-old male SD rats were used and euthanized by cervical dislocation,then we isolated the tibia and femur bilaterally to collect bone marrow cell suspensions.We prepared Percoll cell separation solutions at concentrations of 30％,50％,60％,and 70％,and separated rat BMMNCs seed cells using the Percoll density gradient centrifugation method,and calculated the ratio of viable cells.The obtained BMMNCs were divided into six groups(1×105,5×105,1×106,2.5×106,5×106,and 1×107 cells)and were inoculated in the 25 cm2 sterile culture flasks.After 7 days of culture,we observed the number of cell colonies formed in each group under a microscope and calculated the number of colonies formed per 106 cells.Logistic fitting curves were plotted using GraphPad Prism 9.5 software to determine correla-tion based on the R2 coefficient,and the P-values were used to include statistically significant seeding numbers.We then used R language to define calculation functions and iteratively found the optimal BMMNCs seeding number based on the known total number of seed cells and the correlation function limitations.Fibronectin(FN)solutions at concentrations of 20 nmol/L,50 nmol/L,and 100 nmol/L were prepared,and culture flasks were coated for 2,6,12,and 24 h with blank solu-tions without FN as controls.We inoculated BMMNCs that did not adhere after 48 h into FN-coated culture flasks and cul-tured them for 3 days to calculate the number of colonies formed in each group,and determined the optimal FN concentration and coating time.BMMNCs that did not adhere after 48 h were inoculated in the 25 cm2 culture flasks,and were induced with EGM-2 complete medium,and we observed the colony formation and induction expansion process under a microscope.BM-EPCs cultured for 14 days were identified for purity using dual-positive staining and flow cytometry.Results The Per-coll separation method clearly separated BMMNCs into five layers,with the highest ratio of viable cells between the 30％and 50％Percoll layers.The optimal seeding number for BMMNCs was 2.5×106 cells.FN solutions at 50 nmol/L for 24 h or 100 nmol/L for 6 h effectively promoted colony formation.After 7 days of cell culture,well-formed cobblestone-like cells were observed,indicating that BMMNCs had been successfully induced into well-formed BM-EPCs.The percentage of Dil-Ac-LDL/FITC-UEA-1 dual-positive cells was 91.89％±5.77％,with CD31+KDR positive rate of 90.73％±0.61％,CD14 posi-tive rate of 0.53％±0.17％,and CD45 positive rate of 0.77％±0.34％,indicating that the obtained BM-EPCs were of high purity.Conclusions In the process of inducing and amplifying rat BMMNCs into BM-EPCs,Percoll density gradient cen-trifugation can be used to isolate BMMNCs.The optimal cell seeding number for BMMNCs is 2.5×106 cells,and the culture flask coating conditions are 50 nmol/L FN solution for 24 h or 100 nmol/L FN solution for 6 h.The obtained BM-EPCs through separation,culture,induction,and amplification methods are of good morphology and of high purity.
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