Method for inducing and domesticating epidermal stem cells into adipocytes
A technology of epidermal stem cells and adipocytes, which is applied in the field of using epidermal hepatocytes to induce domestication into adipocytes, and can solve the problems of limited application range of epidermal stem cells
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Embodiment 1
[0033] Example 1 Preparation of Epidermal Stem Cells Before Induction and Domestication
[0034] 1. Preparation of epidermal stem cells:
[0035] Direct source and original source: With the consent of the patient, it was collected from healthy circumcision patients in the Surgical Outpatient Department of 301 Hospital.
[0036] (1) Take fresh adult foreskin (in vitro time is about 1-4 hours, stored at 4°C);
[0037] (2) soak the foreskin in dilute iodophor solution (containing iodophor 20%) for 1 min;
[0038] (3) Rinse with PBS / normal saline for 3 times to remove blood clots (to prevent affecting the digestion of enzymes);
[0039] (4) Trim the foreskin, remove the fat tissue, rinse with PBS / saline several times, and finally cut into 1cm 2 left and right organizational blocks;
[0040](5) Place the tissue block in a 6cm dish containing neutral protease (3mg / ml; D-hanks as solvent) solution and treat it at 4°C for 14-16h. If the treatment is not enough, it can also be plac...
Embodiment 2
[0111] Example 2 Induction and domestication of epidermal stem cells into adipocytes
[0112] 1. Culture medium composition
[0113] (1) The composition and dosage of the fat induction medium are: 90% IMDM (Bonat); 1uM Dexamethasone (sigma D1756); 0.2mM indomethacin (sigma.cat No.17378); 0.5mMinsulin (sigma cat No.19278); 0.5mM isobutyl-methylxanthine (sigma catNo.E9644); 10% FBS (Gibco, cat.no.10437010)
[0114] (2) The composition and dosage of dry maintenance medium are: 98% Epilife (Cascade Biologics, cat.no.M-EPI-500-CA), 1% PS double antibody (double antibody Invitrogen, cat.no.15070) and 1% HKGS supplement (HKGS, Cascade Biologics, cat. no. S-001-5).
[0115] 2. Induced domestication method:
[0116] (1) After the epidermal stem cells are cultured, take the epidermal stem cells with a density of 80%-95% for stable growth, wash the cells twice with PBS, replace with fresh Eplife medium, and replace it with 20% fat induction medium and 80% fat induction medium after 24...
Embodiment 3
[0120] Identification and result analysis of the target cell of embodiment 3
[0121] 1. Immunofluorescence identification of induced differentiation results
[0122] 1. Method:
[0123] (1) Take the cells at one, two and three weeks after induction, wash the cells twice with PBS, add 1ml of 4% paraformaldehyde to fix for 30min;
[0124] (2) Discard the fixative, wash the cells 3 times with PBS, 5 min each time;
[0125] (3) Add 1ml 0.05% permeate solution for 30min;
[0126] (4) discard the permeate, wash the cells with PBS 3 times, 5 min each time;
[0127] (5) Add 500ul sheep serum blocking solution, 1h. The primary antibody diluent dilutes the primary antibody according to the required ratio;
[0128] (6) Add the diluted SMA primary antibody, put it in a wet box, and refrigerate overnight at 4°C;
[0129] (7) Take out the cells incubated with the primary antibody the next day, wash once with PBS, and then wash 3 times with PBS, 10min each time
[0130] (8) Prepare t...
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