| The skin is the body's largest organ, it has a complex organiz- ational structure and the multiple physiological functions. When external factors such as injury or disease caused skin defect, the skin's repair and reconstruction become thorny problems of clinical. Epidermal stem cells have self-renewal and strong proliferative potential, to be counted as the ideal seeded cells of tissue- engineered skin. Epidermal stem cells are a variety of epidermal cells of the progenitor cells, derived from embryonic ectoderm, with bi-directional differentiation. On the one hand, the downward migrat- ion may be differentiated into epidermal basal layer, thereby to generate hair follicles; On the other hand may be upward migration, and eventually differentiate into a variety of epidermal cells. Rheinwald cultured the first human epidermal cells in vitro successly in 1975, Green and O'Connor in vitro cultured autologous epidermal cells for wound coverage, and created the new method of tissue-engineering technique in the treatment of skin defects. However, the next research and application found that epidermal cells in vitro are easy-aging, they have the limited of proliferation ability. It have long-cycle to application, and the lack of skin attachment constrain application and development of skin tissue-engineering. With the development of molecular biology,cell biology,tissue engineering and bio-engineering ,epidermal stem cells gain more and more attention in gene therapy,cell therapy by virtue of its unique biological advantages, successfully isolation and culture of epidermal stem cells play an important role in clinical research of wound healing. However, there are few epidermal stem cells, basal cells of the total number is only 1%-10%, so isolation and culture of epidermal stem cells successfully play an important role.We use dispaseⅡ- trypsin- typeⅣcollagen attachment method in this experiment to isolate cells and to culture in serum-free medium. After 20 minutes, we can observe that the adherent cells are small and round with strong light refraction. With 1 day culture, the cells become big and flat with irregular polygon, a larger and round nucleus in the central cytoplasm; two days later, the number of cells increase, are cluster-like growth; after3-4 days, the cells are integration into a chip, cell morphology are uniform, arrange like paving stones. When 80%-90% cells are mixed, the cells are subcultured. Because the 24-well plates have been coated, the adherent cells are difficult to be digest, so we apply to digest for two steps. First of all, add about 300 L of 0.25% trypsin containing 0.02% EDTA in each hole and place at 37℃in an atmosphere containing 5% CO2-air and saturated humidity for 3 minutes, then under the inverted microscope we can find that some cells become round, cytoplasm retraction with the gap of cells become larger, add medium containing the serum to quit digestion, beat the cells again and make the cells off the wall, add them to 15 ml centrifuge tube. The next step is as the same as above. Centrifugate 5 minutes and 1000r/min, discard the supernatant. According to the degree of cell density we apply the ratio of 1:2 or 1:3 passage to continue to culture. And then identification. Confocal microscope to observe the immunofluorescence staining results of CK10, CK19 and CK15 antibody, the cytoplasm shows the green fluorescence (nuclei stained red by PI fluorescence); the cultured cells were positive for CK19 and CK15, so the cultured cells have high-purity. CK10 is a negative marker, so they can't be observed under the confocal microscope. Further illustration that in this experiment the cells have high-purity.
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