Comparison of separation methods on biological characteristics of rabbit bone marrow mesenchymal stem cells

Abstract

Objective By comparing the different separation methods of rabbit bone marrow mesenchymal stem cells (BMSCs),to find suitable BMSCs culture ways.Methods BMSCs were obtained by whole bone marrow adherent culture,half red blood cell lysis,whole red blood cell lysis and Ficoll density gradient centrifugation.Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the growth of BMSCs at 4th,7th,10th,and 13th day.The growth curves of BMSCs at generation of2nd,3rd,4th and 5th were drawn by MTT.Inverted phase contrast microscope was used to observe the morphological changes of cells.CD34 and CD44 antigens of BMSCs were identified by flow cytometry and immunofluorescence.Results BMSCs could be separated by each method.The adherent cells showed shuttle or multiple angle shapes,with rich cytoplasm,and the positive rate of BMSCs labeled by CD44 antigen was 95.57％,and that by CD34 antigen was 0.99％.The more cell number,larger colonies and shortest primary culture time were presented in whole bone marrow adherent culture group (P ＜ 0.05).The 4th-generation BMSCs showed more excellent proliferative activity,higher cell growth rate and more count of cells obtained than other generations (P ＜ 0.05).Conclusion Whole bone marrow adherent culture is the best suitable cultured ways of rabbit BMSCs.The 4th-generation BMSCs exerted the strongest proliferation capacity. Key words: Bone marrow mesenchymal stem cells; Cell culture