Stem cell composition for preventing and treating diabetic foot and its use, and a stem cell preparation
A stem cell preparation and stem cell technology are applied in the field of stem cells to achieve the effects of simple separation, less damage to the donor site, and improved microenvironment
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Embodiment 1
[0043] Embodiment 1 The cultivation and collection of umbilical cord mesenchymal stem cells
[0044] (1) Under sterile conditions, take fresh umbilical cords that have been away from the mother for no more than 2 hours, and screen whether the mother is carrying infectious disease pathogens. Negative) fresh umbilical cords were placed in PBS containing double antibodies and transported back to the laboratory in a 4°C incubator;
[0045] (2) Clean the fresh umbilical cord with sterile PBS, then disinfect it with 75% ethanol, and then clean it with PBS. After static and arterial, cut the umbilical cord to 20mm 3 Evenly inoculate the pieces with the size of 0.5 ~ 1cm in the petri dish, slowly add 50 ~ 100μl / cm 2 complete medium at 37°C, CO 2 Static culture in an incubator with a concentration of 5%;
[0046] (3) After the cells adhered to the wall and fused, they were digested with trypsin, centrifuged, and divided into multiple culture dishes for subculture.
Embodiment 2
[0047] Example 2 Culture and Collection of Bone Marrow Mesenchymal Stem Cells
[0048] (1) Under sterile conditions, transfer the patient's bone marrow tissue extracted by bone marrow puncture into a 50ml centrifuge tube, add DMEM-LG medium to dilute and mix, and then centrifuge at 500rpm / min for 10min. After centrifugation, absorb the fat in the upper layer and discard the supernatant;
[0049] (2) Add DMEM-LG medium to the remaining precipitate to dilute and mix it evenly, and add it along the tube wall to a test tube containing Ficoll lymphatic separation solution equal to the volume of the remaining precipitate, and centrifuge at 2200rpm / min for 30min. Collect the mononuclear cells in the mist layer after centrifugation, transfer to a new 50mL centrifuge tube, add PBS to wash the cells, discard the supernatant after centrifugation, and adjust the cell density to 2×10 5 cell / ml inoculated in a petri dish, placed at 37°C, CO 2 Static culture in an incubator with a concentrat...
Embodiment 3
[0051] Example 3 Culture and collection of adipose-derived mesenchymal stem cells
[0052] (1) Aliquot the adipose tissue extracted under aseptic conditions into 50ml centrifuge tubes, 20ml in each tube. Add an equal volume of 0.5% type Ⅰ collagenase (final concentration: 0.25%) to each tube, mix thoroughly, seal, transfer to a constant temperature air shaker, and digest for 1 hour at 37°C and 100rpm. Add 4ml FBS to each tube to stop digestion and mix well. Centrifuge at 1500rpm / min for 5min. Discard the upper two layers of liquid, add 40ml PBS to each tube to resuspend the cells, count manually and collect by centrifugation. Discard the supernatant and divide the cells at 1 x 10 5 cells / ml inoculated in a petri dish, placed at 37°C, CO 2 The concentration was 5% in the incubator for static culture.
[0053] (2) Change the medium for the first time after 24 hours, discard the suspended cells, and then change the medium every three days. When the cell confluency reached 8...
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