Abstract
Bone marrow mesenchymal stem cells (BMSCs) have great potential in tissue engineering and clinical therapy, and various methods for isolation and cultivation of BMSCs have been reported. However, the best techniques are still uncertain. Therefore, we sought the most suitable among the four most common methods for BMSC separation from rabbits. BMSCs were obtained from untreated whole bone marrow (BM) adherent cultures, 3 volumes of red blood cells (RBC) lysed with ammonium chloride, 6 volumes of RBC lysed with ammonium chloride, and Ficoll density gradient centrifugation. Then, isolated BMSCs were evaluated with respect to primary cell yield, number of CFU-F colonies, proliferative capacity, cell phenotype, and chondrogenic differentiation potential. Our data show that BMSCs were successfully isolated by all four methods, and each method was similar with regard to cell morphology, phenotype, and differentiation potential. However, BMSCs from untreated whole BM adherent cultures had greater primary cell yields, larger colonies, and the shortest primary culture time (P<0.05). Moreover, the 4th generation of cultured cells had the strongest proliferative activity, the fastest growth rate and the most numerous cells compared with other cell passage generations (P<0.05). In conclusion, untreated whole BM adherent cultures are best for rabbit BMSC isolation and the 4th generation of cells has the strongest proliferation capacity.
Highlights
Stem cells have enormous utility in life science research
Through comparative analysis of different quantities of red blood cells (RBC) lysis buffer, we further evaluated the effects of erythrocytes and platelets on proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro
The whole bone marrow (BM) blood group had the most RBCs, followed by the group treated with 3 volumes of RBC lysis, the group treated with 6 volumes of RBC lysis, and the density gradient centrifugation group
Summary
Stem cells have enormous utility in life science research. bone marrow mesenchymal stem cells (BMSCs) which are adult stem cells derived from mesodermal cell lineages with self-renewable capacities and multi-directional differentiation potentials, are especially important [1]. Horn and colleagues [19] compared RBC lysis with Ficoll density fractionation and untreated whole BM adherent cultures, reporting that BMSCs can be efficiently isolated by RBC lysis. Their technique was faster and could be standardized more for clinical applications. Through comparative analysis of different quantities of RBC lysis buffer, we further evaluated the effects of erythrocytes and platelets on proliferation of rabbit BMSCs in vitro With this in mind, we compared untreated whole BM adherent cultures, 3 volumes of RBC lysis, 6 volumes of RBC lysis, and Ficoll density gradient centrifugation under the same conditions to find the best method for isolation and purification of rabbit BMSCs in vitro
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