Culture method for skin fibroblasts
A technology of fibroblasts and culture methods, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of low adherence rate, low cell crawling rate, cell mechanical damage, etc., and shorten the cell crawling. Slice time, simple operation effect
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Embodiment 1
[0033] Embodiment 1, the cultivation of mouse skin fibroblast
[0034] 1. Execute the mice by breaking the neck, select the abdominal skin, cut off the hair slightly with curved scissors, apply depilatory cream, let it stand for 5 minutes, smooth out the hair with clean gauze, and cut the skin with flat tweezers and curved scissors. into 1cm 2 Throw it into a beaker with PBS, wash it twice and put it into a clean bench.
[0035] 2. Transfer the skin to a clean 15cm glass dish, wash the skin 3 times with PBS containing 1% double antibody, and scrape off excess subcutaneous tissue and capillaries with tissue forceps and a scalpel.
[0036] 3. Transfer the skin to a 50mL centrifuge tube, add 10mL of 0.1% trypsin, and place it in a refrigerator at 4°C for overnight digestion (12-16 hours).
[0037] 4. Take out the skin tissue the next day, put it in a clean 15mL glass dish, pour an appropriate amount of PBS containing 1% double antibody, scrape off the epidermis with tissue forc...
Embodiment 2
[0043] Embodiment 2, cell morphology observation
[0044] Taking the existing tissue block adherence method as a comparative example, observe the cell morphology cultivated by the culture method described in Example 1, the results are shown in figure 1 .
[0045] The results show that after the culture method described in Example 1 was inoculated and cultured, it was found that most of the tissue pieces adhered to the 6-well plate after 2 days, and after 3 days, high-density round cells could be seen under the microscope (such as figure 1 a), after 7 days, it can be seen that there are more cells crawling out (such as figure 1 b). However, in the existing tissue block adhesion method, high-density round cells can be seen under the microscope after 5 days (such as figure 1 c), and less cells crawled out after 7 days (such as figure 1 d). It shows that the cell climbing out speed and the number of climbing out in the culture method of the present invention are better than t...
Embodiment 3
[0046] Embodiment 3, HE staining identification of dermal fibroblasts
[0047] The P2 cells cultured by the culture method described in Example 1 were digested with trypsin, terminated with complete medium, centrifuged at low speed and resuspended with fresh medium. Inoculate the suspension on a coverslip and place at 37°C, 5% CO 2 cultured in an incubator. After the cells were more than 60% confluent, the coverslips were taken out, rinsed with PBS three times, and then fixed with 95% ethanol for 30 min. Stain with hematoxylin staining solution for 5 min and rinse with tap water. Use alcohol containing 1% hydrochloric acid to separate the color for a few seconds, add eosin staining solution to stain for 1 min, and rinse with tap water. After drying the coverslips, mount the slides with neutral gum. Microscopic observation results see figure 2 .
[0048] Depend on figure 2 The results showed that most of the cells were of long-spindle type, a typical fibroblast shape, ...
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