Isolation,culture and biological characterization of rat bone marrow-derived endothelial progenitor cells

Ming QIU; Mingzhao XIAO; Xin GOU; Yuxing YANG

ABSTRACT

BACKGROUND:

A steady method that can isolate endothelial progenitor cells(EPCs) need to be explored.Studies show that the EPCs riches in bone marrow with strong reproductive activity,which can be easily obtained.

OBJECTIVE:

To evaluate the isolating culture and biological characterization of EPCs derived from rat marrow.DESIGN,TIME AND

SETTING:

The in vitro cytology experiment was performed at the central laboratory of the First Affiliated Hospital of Chongqing Medical University from August to December 2008.MATERIALSTwenty SD rats,with two-week-old,were obtained from Experimental Animal Center of Chongqing Medical University.

METHODS:

femurs and tibias of rats were isolated under sterile conditions,and then the bone marrow cavity was washed by 4 ℃ 0.01 mol/L phosphate buffer(PBS).Mononuclear cells were collected from marrow suspension by density gradient centrifugation,followed by cultured with M199 culture medium containing 20% fetal bovine serum to obtained cell suspension.The cells were vaccinated into culture flask(coated with fibronectin) with the density of 1?109/L,and incubated at 37 ℃ in 5% CO2 atmosphere.MAIN OUTCOME

MEASURES:

Morphologic change of cells was observed by an invert microscope,the expression of CD133,CD34 as well as KDR was detected by immunocytochemistry,and the flow cytometry was used to analyze the growth curve.

RESULTS:

The adherent cells formed clusters that were found to be characterized by density packed round cells surrounded by spindle-likes cells.Adherent cells were identified positive for CD133,CD34 and KDR by immunocytochemistry on days 4,7,10 and 14.CD133-positive cells reduced after 10 days.Flow cytometry analysis indicated that the number of CD133/KDR double positive cells gradually increased to the maximum at day 10 and then declined.KDR-positive cells gradually increased up with the time.EPCs growth curve showed that cells proliferated fast on days 3-6.

CONCLUSION:

EPCs can be isolated and cultured by density gradient centrifugation from marrow suspension of SD rats.These cells exhibit the characterization of endothelial cells,and can differentiate into endothelial cells.