Endometrial stem cell preparation and preparation method for treating erectile dysfunction
A technology of erectile dysfunction and stem cell preparations, applied in the field of biomedicine, can solve problems such as ineffective treatment of erectile dysfunction and psychological burden, and achieve the effects of improving vascular microcirculation, effective treatment, and increasing blood supply speed and volume
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example 1
[0049] Example 1: Culture of endometrial stem cells:
[0050] Implant the menstrual cup on the second day of the menstrual period, continuously collect the menstrual blood for 12 hours, transfer the obtained menstrual blood into an anticoagulant, add penicillin / streptomycin and amphotericin B, and then add normal saline to make a suspension;
[0051] Take the lymphocyte separation medium, spread the suspension on the upper layer of the lymphocyte separation medium, the ratio of the lymphocyte separation medium to the suspension is 1:3, centrifuge at 800g for 20min, and obtain a white film between the lymphocyte separation medium and the suspension Floor;
[0052] Take the buffy coat, add normal saline, centrifuge at 2000rpm for 5min, discard the supernatant, then add normal saline, centrifuge at 2000rpm for 5min, discard the supernatant, suspend the cell pellet in the culture medium, and culture in a 37°C incubator; The culture medium was changed every 2 days, cultured for 16...
example 2
[0054] Get the endometrial stem cells prepared in Example 1, and carry out the following identification detection experiments:
[0055] ① Take the cultured endometrial stem cells, centrifuge and suspend them in 1×PBS, count them on a cell counter, and incubate the cells with CD45, CD34, CD90, CD105, and 7-AAD. All antibodies are directly labeled; after incubation, collect the cells , centrifuged and resuspended in 1×PBS, and detected by flow cytometry;
[0056] The channel setting conditions of the flow cytometer are: CD90FITC: 455V; CD45FITC: 455V; CD105PE: 730V; CD34PE: 730V; 7AAD: 876V. The emission wavelengths are: 488nm for FITC; 570nm for PE; 647nm for 7AAD. Tested separately by dosage group.
[0057] ②The cultured endometrial stem cells were taken, and the endotoxin was tested using Zhanjiang Anders Limulus reagent;
[0058] ③Take the cultured endometrial stem cells for fungal and bacterial detection;
[0059] The culture method was used for detection, and the cell ...
example 3
[0065] Example 3: Cryopreservation of endometrial stem cells:
[0066] Take the qualified endometrial stem cells prepared in Example 1, remove the culture medium, and wash the cells twice with PBS; add mild enzymes to digest the cells, shake the culture flask, put it back in the incubator for 1-2min, and observe the cells with an inverted microscope to ensure that all Cells are all detached and floating, tap the side of the flask to release residual adherent cells;
[0067] Then add PBS to dilute with mild enzyme to stop the digestion; take out 100μL for counting; centrifuge the remaining cells at 1000rpm for 5min, and put them in the cryopreservation solution at 1×10 6 Cells / mL density to resuspend the cells, pipette the cell suspension and transfer it to a cryopreservation tube, place it at -80°C overnight, and transfer it to a liquid nitrogen tank for storage.
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