Objective To make a comparative study on the effects of whole bone marrow culture method and density gradient centrifugation method in isolating hBMSCs. Methods hBMSCs were obtained from healthy adult volunteers and isolated by whole bone marrow culture method and density gradient centrifugation method. Primary cell morphology was observed using inverted phase contrast microscope and the cells in the 2nd passage were stained with HE after being cultured for 7 days. And then, the generation time of the primary, 2nd and 3rd passage hBMSCs was comparedbetween two methods and the surface markers were detected by flow cytometer. In addition, the ALP expression inosteoinductive hBMSCs were evaluated by ALP activity kit at 3, 6 and 9 days and ALP staining was used for osteoinductivehBMSCs with Kaplow method at 9 days. Results Primary cells isolated with whole bone marrow culture method showedaggregation growth while cells isolated with density gradient centrifugation method showed diffusion growth. HE stainingshowed no significant difference in the morphology of the 2nd passage cells between these two methods. The generationtime of primary cells isolated by whole bone marrow culture method (15.36 ± 1.67) days was significantly shorter than that of cells isolated by density gradient centrifugation method [(18.57 ± 1.05) days] (P lt; 0.01), while the generation time of the 2nd and 3rd passage cells showed no statistically significant differences between these methods (P gt; 0.05). The concent of positive surface markers (CD29, CD44, CD71, CD105, CD166) and negative surface marker CD34 in the 2nd cells showed no significant difference between these two isolation methods (P gt; 0.05); however, negative markers CD14 and CD45 showed significant difference (P lt; 0.01). The ALP expression in osteoinductive cells showed no statistical significant (P gt; 0.05) at 3, 6 and 9 days; and the ALP staining positive cell ratio of whole bone marrow culture method was basically in accordance with that of density gradient centrifugation method at 9 days. Conclusion hBMSCs could be isolated by whole bone marrow culture method, and the cell isolation effects of whole bone marrow culture method are equivalent with density gradient centrifugation method.
Objective To explore the cytotoxicity, labeled time, marking rate, and effect on adhesion of quantum dot 655 (QD655) labeled rat bone marrow mesenchymal stem cells (BMSCs) in vitro, and to confirm its feasibil ity for stem cell label ing and tracer means for rat. Methods BMSCs were collected from the femur and tibia bone marrow cavity of a 2-week-old SD rat, cultured and identified. The 3rd passage of BMSCs were incubated with QD655 as the experimental groupaccording to the recommended concentration of the markers. The cells were not labeled by QD655 as control group. Thecell survival rate after QD655 label ing was detected by trypan-blue exclusion. The effect of QD655 on cell prol iferation was observed by MTT. The osteogenic differentiation potential was identified by Al izarin red staining, alkal ine phosphatase (ALP) staining, and real-time fluorogenic quantitative PCR. At immediately, 1, 2, 4, and 6 weeks, fluorescent microscopy was used to observe the labeled rate and scanning electron microscope was used to observe the cell adhesion to scaffold (bioglass/collagen composite). Results The cell survival rates were more than 90% in both experimental group and control group, showing no significant difference (P gt; 0.05). There was no significant difference in the cell prol iferation between 2 groups (P gt; 0.05). Al izarin red staining and ALP staining showed positive results. Real-time fluorogenic quantitative PCR result showed that the mRNA expression levels of osteopontin, osteocalcin, collagen type I, ALP, and BMP-2 in the experimental group was significantly higher than those in the control group. The labeled rates were 96.50% ± 1.59%, 93.30% ± 1.51%, 72.40% ± 2.90%, 40.10% ± 3.60%, and 10.00% ± 1.70% immediately, 1, 2, 4, and 6 weeks after label ing, respectively. The labeled rate in the control group was 0. Scanning electron microscope showed a good distribution of fusiform or polygonal cells in the pores of scaffold. Conclusion QD655 can be used as a label ing marker for BMSCs. Rat BMSCs labeled with QD655 is of high efficiency and safety.