Isolation of rabbit bone marrow mesenchymal stem cells using density gradient centrifugation and adherence screening methods

Cailong ZHANG; Changsuo XIA; Zhengyao JIANG

ABSTRACT

BACKGROUND:

Under special conditions, bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and chondroblasts. However, MSCs are few in bone marrow. How to harvest, purity and rapidly proliferate in vitro is a foundation of application in tissue engineering technique.

OBJECTIVE:

To optimize, collect, purity, assess rabbit BMSCs and to observe the biological character of BMSCs. DESIGN, TIME AND

SETTING:

The observational study was performed at the Animal Experimental Center of Tongji Medical College from September 2005 to July 2006. MATERIALS One female New Zealand rabbits aged 2 months were used for MSC collection and primary culture.

METHODS:

Bone marrow solution was purified by density gradient centrifugation and adherence screening method. Culture solution was obtained. BMSCs were incubated in phosphate buffered solution (PBS), supplemented with 2.5 g/L trypsin (3.0 mL), and placed in an incubator at 37 ℃ for two or three minutes. Cell morphology was observed using an inverted microscope. The digestion was stopped when cytoplasm recovery, long and thin cells with large intercellular space, and few round cells appeared. Subsequently, BMSCs were incubated in serum-free L-DMEM, and placed in a plastic culture flask at 1.0×108/L. MAIN OUTCOME

MEASURES:

MSC morphology, ultrastructura and surface marker; Proliferation of the first, third, fifth, eighth and tenth passages of BMSCs; Cell growth curve was drawn.

RESULTS:

BMSCs was pure following density gradient centrifugation and adherence screening method. The third and fifth passage of cells had typical whirlpool-shape. Transmission electron microscope demonstrated that round or oval MSCs possessed large nuclei, big nucleus proportion, a few cellular organ. These were low-differentiated cells. Growth curve of cultured MSCs was "S" shape. The first, third and fifth passage cells had strong reproductive capability. The eighth and tenth passage of cells had significantly reduced proliferation. Cells isolated were positive for CD44 and CD90, but negative for CD34. These were low-differentiated cells under the electron microscope.

CONCLUSION:

Isolated cells are MSCs, with the property of stem cells. The third and fifth passage cells are pure, with strong reproductive capability.