Compound cell preparation, preparation method and application of preparation
A cell preparation, cell technology, applied in cell culture active agents, biochemical equipment and methods, animal cells, etc., to prolong amputation time and reduce limb pain and coldness.
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Embodiment 1
[0062] Example 1: Preparation of autologous ADSCs
[0063] 1.1 Fat collection
[0064] Before the collection, the donor should have a physical examination, no tumor history, no virus infection, no mycoplasma infection, and the fat collection should be collected in a professional medical collection institution; the specific method is abdominal subcutaneous liposuction 50mL. Immediately after collection, place them in a 125mL ice-bath sterile bottle (model 2019-0250, manufacturer Nalgene), which contains 50mL of preservation solution.
[0065] The preservation solution is DMEM / F12 with the following components added:
[0066] 50ug / mL gentamicin sulfate;
[0067] 10% sodium heparin anticoagulant.
[0068] 1.2 Isolation of fat cells
[0069] Remove small blood vessels and connective tissue, cut them into fluid state, and rarely see particles; resuspend in 2 times the volume of medical saline (containing 50ug / mL gentamicin sulfate), centrifuge at 500g for 10 minutes, and take t...
Embodiment 2
[0076] Example 2: Preparation of EPCs derived from ADSCs
[0077] Resuspend P2 generation ADSCs in ADSCs medium, adjust the cell density to 10000 / cm2, inoculate into 175 cm2 tissue culture flask, culture for 24 hours, change EPCs medium, continue to culture for 6 days, and harvest P0 generation EPCs.
[0078] The EPCs culture medium is DMEM / F12 containing the following components:
[0079] 5% animal-derived component-free serum replacement;
[0080] 10-8mol / L dexamethasone;
[0081] 20ng / mL recombinant human vascular endothelial growth factor;
[0082] 5ng / mL recombinant human basic fibroblast growth factor;
[0083] 5ng / mL insulin-like growth factor-1.
Embodiment 3
[0084] Embodiment three: PRP preparation
[0085]Clinically collect 100mL of the recipient’s own sodium citrate anticoagulated peripheral blood, centrifuge at 150g for 10 minutes, collect the supernatant and some red blood cells; take another 240g and centrifuge for 5 minutes, discard the upper 30% of the supernatant, and collect the middle layer and the upper part of the red blood cell layer For the buffy coat, about 40mL of PRP is reserved.
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