In vitro culture and identification of adipose tissue-derived mesenchymal stem cells in rats

Yuhong QIN; Guanghui CHEN; Suyan BIAN; Yanqin ZHANG; Tiande LI

ABSTRACT

BACKGROUND:

Adipose tissue-derived mesenchymal stem cells (ADMSCs) have the multilineage differentiation potential, and are relatively easier to be obtained, thus they have attracted more and more attention as a new seed cell for cell engineering.

OBJECTIVE:

To observe the in vitro culture conditions of ADMSCs isolated from rat's subcutaneous adipose tissue, and identify them using immunohistochemical staining.

DESIGN:

An animal experiment.SETTING; Department of Cardiology, the General Hospital of Chinese PLA.MATERIALS One healthy male Wistar rat of clean degree, 4 months old, weighing 200 g, was used. DMEM, fetal bovine serum were from GIBCO; Monoclone antibodies of rabbit-anti-rat CD13, CD34, CD44, CD45, CD105, D-related human leucocyte antigen (HLA-DR), factor-Ⅷ, vov Willebrand factor (VWF), Myosin, SABC kits and DAB staining kit from Wuhan Booster Biological Engineering, Co.,Ltd; Adeno-associated virus encoding green fluorescent protein from Vector Gene Technology Company Limited (Beijing).

METHODS:

The experiments were carried out in the Department of Internal Medicine, the General Hospital of Chinese PLA in October 2006. ① Cell isolation and culture 0.3 g adipose tissue was cut from subcutaneous adipose tissue of Wistar rat's groin under aseptic condition, then minced and digested before culture, DMEM was changed at 2-3 days after plenty of fusiform-shap ed attached cells were observed under microscope, and the cell growth was observed. The cell concentration was adjusted to 2×107 L-1, then seeded into 96-well plate, and 100 μL for each well. From the second day, 3 wells were randomly selected every day, the cells were released with tripsin, and counted with blood cell counting chamber under inverted microscope. ② Cell viability assay ADMSCs of passages 3 to 8 were added to DMSO freeze medium, and thawed after 2-4 weeks, and the cell viability was assessed by trypan blue dye exclusion. ③Immunohistochemical staining and identification 2 ×107 L -1 cells were seeded to culture plate, then the immunohistochemical (SABC method) identification and Oil red O staining were performed to determine the cell surface antibodies of CD13, CD34, CD44, CD45, CD105, HLA-DR, factor-Ⅷ, HLA-DR and VWF. ④Lineage-specific differentiation and identification The ADMSCs were plated on multi-well chamber and induced with lineage-specific media supplementation at least two weeks and identified by histologic/immunohistochemical assay of Oil red O for adipogenisis, alkaline phosphatase (ALP) stain for osteogenisis and Myosin monoclonal antibody for myogenisis. ⑤Transfected adenovirus carried green fluorescence protein (AD-GFP) medium The fourth generation of ADMSCs were seeded on 96-well plate, 3 000 cells for each well, serum-free DMEM was changed after 24 hours, and added by AD-GFP at the same time, then transfected with different multiplicity of infection (MOI) of 1∶50, 1∶100, 1∶150 and 1∶200respectively, and then the transfection was observed.MAIN OUTCOME

MEASURES:

① Results of cell isolation and culture; ② Cell viability after freezing and thawing; ③Results of immunohistochemical staining and identification; ④ Results of lineage-specific differentiation and identification;⑤ Results of transfected adenovirus carried AD-GFP.

RESULTS:

① About 3.6×105 attached cells were obtained from 0.3 g subcutaneous adipose tissue, and these cells could be subcultured for passages in vitro with stable population doubling time. ② The cells were thawed after freezing for 2-3 weeks, and the trypan blue staining showed that the cell viability was above 90%. ③ The immunocytochemical staining showed that CD13, CD44, CD105 were positive and CD45, factor-Ⅷ, HLA-DR and VWF negative in different generations. ④ From the second generation, a few Oil red O positively stained cells were observed, which were obviously increased after prolonging the refreshing. After lineage-specific differentiation, the cells were all positive by Oil red O staining, ALP staining and Myosin immunohistochemical staining. ⑤ 72 hours after transfection, it was observed under fluorescence microscope that most cells were green fluorescence when the MOI value was 1∶200, the transfection was successful, and it was generally determined that the transfection rate was above 90%.

CONCLUSION:

A large number of ADMSCs with multilineage differentiation potential can be easily obtained from rat adipose tissue, osteoblast, myoblasts, they can be expanded in large quantity and stored in vitro for long time, AD-GFP were also successfully transfected.