Preparation method of artificial skin by taking VEGF165 gene modified hair follicle stem cells as seed cells
A hair follicle stem cell and artificial skin technology, applied in the field of artificial skin and its preparation, can solve the problems of low survival rate, easy necrosis of transplanted artificial skin, etc., and achieve the effects of abundant sources, restoration and regeneration of anatomical structures and physiological functions, and easy to obtain effects.
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Embodiment 1
[0045] Example 1 Isolation, culture and identification of rat hair follicle stem cells
[0046] 1.1) Select one-week-old SD rat tentacle skin, put 0.25% Dispase enzyme into it to digest at 37°C for 2 hours; use forceps and a disposable syringe needle to pull out the hair follicles from the end of the subcutaneous tissue, collect the hair follicles that are in good shape and in the growing phase, and look under the microscope. Cut the hair follicle into three equal parts, take the middle part, rinse with PBS, put it into a 50mL culture flask, add DMEM / F12 supplemented medium, and place it at 37°C, 5% CO. 2 In the incubator, the medium was changed every 2 days; the DMEM / F12 supplemented medium was composed of: 44ml DMEM / F12 medium, 5ml KSR serum substitute, 500μl penicillin-streptomycin mixture, 500μl L-glutamine, 500μl non- Essential amino acids, 20ng / ml recombinant human epidermal growth factor, 10ng / ml recombinant human basic fibroblast growth factor, 50 μl hydroxyethanol, 10...
Embodiment 2
[0053] Example 2 Preparation of gelatin sponge three-dimensional tissue scaffolds
[0054] 2.1) Preparation:
[0055] A. Take 5% gelatin and dissolve it in 10ml of distilled water at 25°C, add 0.05% chondroitin 6-sulfate sodium salt (C6S) and 0.2% hyaluronate sodium salt (HA) respectively;
[0056] B. After stirring with a magnetic stirrer for 60 minutes at room temperature, the crosslinker 0.5% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (EDC) and 0.25% N -Hydroxysuccinimide solution (N-Hydroxysuccinimide) was dropped into the solution and mixed for 5 minutes, then the solution was poured into the mold of the 12-well plate, and shaken horizontally evenly.
[0057] C. Freeze at -80°C for 2 hours
[0058] D. Then put on a freeze dryer, freeze-dried for 24 hours to obtain a porous sponge-like Gel-C6S-HA scaffold with a thickness of 2 mm.
[0059] 2.2) Observation: Take a small amount of stent samples, observe and record with scanning electron microscope...
Embodiment 3
[0060] Example 3 Preparation of hair follicle stem cells modified by vascular endothelial growth factor 165 gene (VEGF165)
[0061] 3.1 Packaging lentivirus: Take 293T cells in the logarithmic growth phase and inoculate them in a 100mm culture dish 24 hours in advance, and wait until the cells grow to 50%-70% the next day; the virus packaging is carried out by calcium transfer method: before transfection The cell culture medium was replaced with a medium without dual antibodies, including 10% FBS+DMEM high glucose; then, 50 ug of the target plasmid pLenti-IRES-VEGF165-EGFP and 5 ug of each of the three packaging plasmids VSVG, RSV-REV, and RRE were added to 50 ul of HBS solution Mix gently in medium, then add ddH 2 O to 500μl as B solution, prepare another 500μl CaCl 2 Solution A, then add solution B to solution A, blow out bubbles, and leave at room temperature for 2 minutes; add dropwise to the cell culture dish, shake horizontally for several times; after incubation for 10...
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