A preparation method of human corneal epithelial cells, its conditioned medium and its preparation method
A corneal epithelial cell and conditioned medium technology is applied in the field of preparation of human corneal epithelial cells, which can solve the problems of limited source of LSCs donors, the inability of oral mucosal epithelial cells to provide stable cell phenotype, and immune rejection of allogeneic limbal transplantation.
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Embodiment 1
[0082] Primary culture, subculture and purification of embodiment 1 hAMSCs
[0083] Peel off the amniotic membranes of the placental fetal surface under aseptic conditions, transfer to the laboratory at 4°C, and use blue chain double antibody (penicillin 1×10 5 U / L, streptomycin 1×10 5 U / L) sterile DPBS solution was repeatedly washed twice, the surface of the amniotic membrane was repeatedly scraped with a cell scraper, the connective tissue of the membrane was removed, and blood stains were washed with DPBS solution.
[0084] Cut the placental amniotic membrane with scissors, and cut it into about 1cm strips, squeeze it with tweezers, and wash it with normal saline repeatedly until it becomes clear. Heavy. Then add an appropriate amount of normal saline, and use a hand-held electric homogenizer to process it into fine particles (about the size of rice grains), rinse it again and again with normal saline until it is clear, and then continue to use the homogenizer to process ...
Embodiment 2C
[0093] Embodiment 2 CCK-8 method detects hAMSCs proliferative activity
[0094] Take the 3rd generation hAMSCs cells with a confluence rate of 85%, digest and make a density of 1×10 4 cells / mL cell suspension, take 100 μL and add it to a 96-well plate. Add 100 μL PBS solution to the quadrilateral wells (total 36 wells) of the 96-well plate, and place the cell culture plate at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, carefully add 10 μL-CCK-8 solution to 5 auxiliary wells, measure once every 24 hours, and incubate the culture plate in the incubator for 3 hours. The absorbance value at a wavelength of 450nm was measured by a microplate reader, and the continuous measurement was repeated for 7 days. Data were recorded daily and proliferation curves were drawn.
[0095] Plot the result as Figure 5 shown. The results of CCK-8 test showed that the subcultured hAMSCs grew slowly on the 1st day, and their proliferative ability was weak, but the proliferative ...
Embodiment 3
[0096] Embodiment 3 flow cytometry identification hAMSCs phenotype
[0097] Take the third generation with a fusion rate of more than 85%, discard the culture medium, wash twice with PBS, add a concentration of 0.125% trypsin-0.02% EDTA digestion solution, put it in a 37°C incubator for digestion for 3 minutes, and add an equal volume to stop solution to terminate the digestion, blow and mix the digested cells, centrifuge at 1500r / min for 6min, discard the supernatant, resuspend the cells by blowing and blowing with PBS, and centrifuge at 1500r / min for 6min, repeat twice to adjust the cell concentration to 2×10 9 L-1, put into a flow tube, add 100 μL of cell suspension to each tube, add mouse anti-human monoclonal antibodies CD44-PE, CD90-FITC, CD105-PerCP-Cy, CD73-APC and Negative Control mixture CD34-PE, CD19-PE, CD45-PE, CD11b-PE, HLA-DR, incubate at room temperature in the dark for 30min, add 2m LPBS to each flow tube, shake fully, centrifuge at 1500r / min for 6min, discar...
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