| Stem cells are a group of cells with self-renewal and multi-directional differentiation potential. Now stem cells are the main source of seed cells. Stem cell are divided into embryonic stem cells and adult stem cells depend on source and differentiative stage. Although embryonic stem cells have the ability to differentiate into a variety of different tissues, ethical controversy greatly limits its application. Adult stem cells can derive from many sources, strong multiplication capacity, differentiate into variety mature cells and no limitations of the ethical and moral. It has become the first selection of ideal seed cells in tissue engineering at present.As a new kind of adult stem cells, ADSC get more and more attention because of rich source, drawing materials easily and power proliferation.In this study, we isolate and culture ADSC (adipose-derived stem cells) from the groin and epididymal adipose tissue in mice, and identified their biological function. We want to establish a perfect set of program of isolation and culture of ADSC of mice, which will provide experimental basis for study of mice ADSCMethods:Cut the adipose tissue from groin and epididymis in mice, digest the tissue using0.1%type I collagenase. The primary ADSC obtained from adipose tissue were cultured in DMEM/F12medium containing10%FBS, and purification the ADSC by the differential time of sticking wall. We observed the morphology of ADSC using the inverted microscope and transmission electron microscopy. The growth curve of ADSC were drawed through counting the number of cells. ADSC phenotype were identified by the flow cytometry and immunofluorescence cell biology. Finally, different cell inductor were used to induce ADSC to differentiate into fat cell and osteoplast. Compatibility between ADSC and collagen scaffold material were tested by scanning electron microscope.Results:1. The ADSC were long spindle-like or fibroblast-like. They grows crowdly and speed of proliferation accelerated after two days. At sixth day, the cells can reach more than90%integration. Subculture of cells grow slightly faster than the primary culture. Cells still maintain the long spindle-like in shape and arranged in scroll and fascicular. In vitro, ADSC could be passaged to9generation.2. The ADSC were large in size, round, oval or slightly irregular in shape. There were abundant microvilli on the cell surface. The nuclei locate in the center of cell, which were big in size, round or oval in shape and clear nuclear membrane. There were big and obvious nucleoli. There are nuclear notch in some nuclei. Some organelles were seen in cytoplasma, such as mitochondria, rough endoplasmic reticulum and so on. Crest of mitochondria were clear. There were many rough endoplasmic reticulum, which were moderate dilation.3. The ADSC can expres CD44and CD29, no expression of CD34.4. Many small lipid droplets were seen in the cytoplasm of ADSC after inducing by inductor. The small lipid droplets can be dyed red with oil red O.5. After induction of osteogenic inductor, the boundary line among ADSC were not clear and the structure of cells were fuzzy. There were many strong refractive granular material deposit at that field after dyeing with alizarin red.6. ADSC is spread on the collagen scaffold.Conclusion:1. The ADSC of mice were separated successful by using one digestive then many times collection method and different adherent time method.2. The ADSC were cultured and proliferated in medium containing DMEM/F12and10%FBS, which maintain the proliferative activity of ADSC and passage to9generation steadily.3. The ADSC expressed marker CD29and CD44, do not express marker CD34.4. The ADSC could differentiate into the fat cell and osteoblast after induction.5. There were good compatibility between ADSC and collagen scaffold material. |
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