Primary Experimental Studies On BrdU Labeling Of Rabbit Adipose-derived Stem Cells

Objective:Investigating the effect of labeling rabbit adipose-derived stem cells (ADSCs) with 5-bromo-2'-deoxyuridine(BrdU),and making sure of the optimal method for BrdU labeling in intro to estimate the applicability of labeling ADSCs with BrdU.Method:Adipose tissue was dessected from the back of 6-months-old New Zealand rabbit neck,then the stem cells were isolated from the adipose tissue and cultured with collagenase digestion.The passaged cells were exposed to different defined mediums for adipocyte- and osteoblast-differentiation,and the biological characteristics of adipocyte and osteoblast were identified by using histochemistry. The third passage ADSCs were incubated with BrdU at different concentrations(5,10,15,20μmol/L) for different incubating time(24,48,72h).Immunohistochemistry was employed to identify the result of labeling,and the labeling rates of different labeling processes were calculated to make sure of the optimal labeling process.The security of the optimal labeling process was surveyed by trypan blue exclusion and calculating cell doubling time.Then the third passage ADSCs with the optimal labeling process were cultured in minimal essential medium without BrdU.Calculate the labeing rates of 5,7,9,11 passsages ADSCs in vitro to observe the attenuation of BrdU.At the third passage,ADSCs labeled with the optimal labeling process and ADSCs which were not labeled were seeded separately in PLGA scaffolds.We auto-transplanted the seeded scaffolds subcutaneously after grow them for 3 days in vitro.4 weeks after the operation,we observed the result of labeling ADSCs with BrdU in vivo through immunohistochemistry.Result:The stem cells isolated from the rabbit's adipose tissue demonstrated fibroblastlike morphology and could rapidly proliferate.We did not observe obviously change in cell morphology and spontaneous adipocyte-differentiation in serial passages.The ADSCs grown in adipogenic media demonstrated intracellular lipid vacuoles by Oil red-O staining at 12 days,and grown in osteogenic media demonstrated intense positive staining of calcium nodules with Von kossa at 14 days, so ADSCs had the ability to differentiate into adipocytes and osteoblast when treated with defined mediums.BrdU labeled ADSCs nuclei.Incubateing ADSCs with BrdU at 10μmol/L for 48h is the optimal labeling process.It makes no difference in the activity and proliferation of ADSCs whether they were incubated with the optimal labeling process or not.Labeing rates decreased the passages growing.It was about 95%in the preliminary survey,and 41%after 10 passage(about 4 weeks in vitro). BrdU positive staining stem cells can be detected 4 weeks after transplantation in vivo, which demonstrated transplanted stem cells could survive in vivo.Conclusion:ADSCs of rabbits,isolated easily in this trial,were characterized by quick self-proliferation and multi-directional differentiation in vitro.BrdU at 10μmol/L for 48h could label ADSCs effectively in intro and the process was easy to do.In addition,ADSCs labeled by BrdU can be traced in vivo one month after transplantation.BrdU could be used to label ADSCs.