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Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro.

Zhang W, Zhang F, Shi H, Tan R, Han S, Ye G, Pan S, Sun F, Liu X - PLoS ONE (2014)

Bottom Line: 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 4(th) 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 4(th) generation of cells has the strongest proliferation capacity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, College of Clinical Medicine, Yangzhou University, Yangzhou, Jiangsu Province, China ; The Research Center for Translational Medicine, Yangzhou University, Yangzhou, Jiangsu Province, China.

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 4(th) 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 4(th) generation of cells has the strongest proliferation capacity.

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Flow cytometry.CD44 positive BMSCs were 98.12, 98.85, 93.32, and 94.32%, and CD34 positive cells were 3.25, 2.37, 5.31, and 3.50%, as shown by flow cytometry (autofluorescence is marked as a white filled histograms). There were no obvious differences among the four groups (p<0.05). Cells that expressed CD44 markers, but not CD34, correspond to BMSCs.
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pone-0088794-g004: Flow cytometry.CD44 positive BMSCs were 98.12, 98.85, 93.32, and 94.32%, and CD34 positive cells were 3.25, 2.37, 5.31, and 3.50%, as shown by flow cytometry (autofluorescence is marked as a white filled histograms). There were no obvious differences among the four groups (p<0.05). Cells that expressed CD44 markers, but not CD34, correspond to BMSCs.

Mentions: Immunofluorescent staining indicated that the CD44-PE cell marker fluoresced green on the cell surface under a fluorescent microscope. CD34-PE was fluorescent brown (not green). So, cultured cells were positive for CD44, but negative for CD34 (Figure 3). Flow cytometry was used to quantify BMSCs percentages, and CD44-positive cells were 98.12, 98.85, 93.32, and 94.32%, and the CD34-positive cells were 3.25, 2.37, 5.31, and 3.50%. These data confirmed that cells obtained from all four methods were BMSCs. There were no obvious differences among the four groups of BMSCs (Figure 4).


Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro.

Zhang W, Zhang F, Shi H, Tan R, Han S, Ye G, Pan S, Sun F, Liu X - PLoS ONE (2014)

Flow cytometry.CD44 positive BMSCs were 98.12, 98.85, 93.32, and 94.32%, and CD34 positive cells were 3.25, 2.37, 5.31, and 3.50%, as shown by flow cytometry (autofluorescence is marked as a white filled histograms). There were no obvious differences among the four groups (p<0.05). Cells that expressed CD44 markers, but not CD34, correspond to BMSCs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928292&req=5

pone-0088794-g004: Flow cytometry.CD44 positive BMSCs were 98.12, 98.85, 93.32, and 94.32%, and CD34 positive cells were 3.25, 2.37, 5.31, and 3.50%, as shown by flow cytometry (autofluorescence is marked as a white filled histograms). There were no obvious differences among the four groups (p<0.05). Cells that expressed CD44 markers, but not CD34, correspond to BMSCs.
Mentions: Immunofluorescent staining indicated that the CD44-PE cell marker fluoresced green on the cell surface under a fluorescent microscope. CD34-PE was fluorescent brown (not green). So, cultured cells were positive for CD44, but negative for CD34 (Figure 3). Flow cytometry was used to quantify BMSCs percentages, and CD44-positive cells were 98.12, 98.85, 93.32, and 94.32%, and the CD34-positive cells were 3.25, 2.37, 5.31, and 3.50%. These data confirmed that cells obtained from all four methods were BMSCs. There were no obvious differences among the four groups of BMSCs (Figure 4).

Bottom Line: 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 4(th) 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 4(th) generation of cells has the strongest proliferation capacity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, College of Clinical Medicine, Yangzhou University, Yangzhou, Jiangsu Province, China ; The Research Center for Translational Medicine, Yangzhou University, Yangzhou, Jiangsu Province, China.

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 4(th) 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 4(th) generation of cells has the strongest proliferation capacity.

Show MeSH
Related in: MedlinePlus