Isolation,culture and multi-directional differentiation of rat bone marrow mesenchymal stem cells by using the whole bone marrow adherence method

Peng CAI; Shaoxing ZHU; Yiming SU; Shiyong HUANG

ABSTRACT

BACKGROUND:

There is few mesenchymal stem cells(MSCs) in the bone marrow,about one BMSC in 1?104-1?105 monocytes.Following in vitro isolation,purification and amplification,it is satisfactory for in vivo requirement.

OBJECTIVE:

To observe biological characteristics and potentiality of multi-directional differentiation of BMSCs in vitro.DESIGN,TIME AND

SETTING:

The cytological in vitro study was performed at the Institute of Urinary Surgery,Union Hospital Affiliated to Fujian Medical University from October 2007 to December 2008.MATERIALSA total of 30 clean male Sprague Dawley rats of inbreeding line were supplied by Silaike,Shanghai,China.

METHODS:

BMSCs from rats were isolated and cultured in vitro by using the whole bone marrow adherence method.When 80%-90% confluency,BMSCs were digested by trypsin.At the third passage,BMSCs were induced to differentiate into osteoblasts and adipocytes.MAIN OUTCOME

MEASURES:

Cell morphology was observed under the inverted phase contrast microscope.BMSCs surface markers were detected using flow cytometry.Osteogenic ability was examined by alizarin red staining.Adipogenic ability was measured by Oil red O staining.

RESULTS:

The primary cells and the passage cells were mostly fusiform in shape,to be similar to fibroblasts.Cell still kept a high potential of growth and amplification following over 10 subcultures.At the third passage,BMSCs were positive for CD44,CD90,CD106,but negative for CD34,CD45,CD11b.Following 21 days of osteogenic induction,cell alizarin red staining showed that alizarin red was positive in osteoblasts.Following 2 weeks of adipogenic induction,oil red O staining showed that red lipid droplet existed in adipocytes.

CONCLUSION:

Whole bone marrow adherence method can isolate,purify and amplify BMSCs in vitro.The obtained cells have general biological characteristics of MSCs,and also have potentiality of multi-directional differentiation.