Culture medium for preparing endometrial stem cells as well as preparation method
A technology of endometrium and culture medium, applied in cell dissociation methods, cell culture active agents, non-embryonic pluripotent stem cells, etc., can solve the problems of limiting the application prospects of endometrial stem cells and the low survival rate of endometrial stem cells , to achieve the effect of promoting development and application and improving survival rate
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Embodiment 1
[0028] Embodiment 1: culture medium of the present invention
[0029] Based on Lonza serum-free medium, containing 2% (w / v) Pall serum replacement, 1% (w / v) penicillin / streptomycin, 1% (w / v) gentamicin sulfate, 2mM gluten Aminoamide, 2 mM MEM NEAA, 1 mM sodium pyruvate, 1 μg / ml EGF, 1 μg / ml bEGF and 1 μg / ml VEGF.
[0030] Among them, Lonza was purchased by the Swiss Lonza Group, batch number: 0000699907; Pall serum substitute was purchased by the American PALL Company, batch number: 259509 / R349-01; penicillin / streptomycin was purchased by Israel BI, batch number: 1807212; gentamicin sulfate Solarbio Cat. No. G8170; Glutamine sigma Cat. No. RNBG6180; MEM NEAA for Gibco Cat. No. 195809; Sodium Pyruvate for Sigma Cat. No. N1633-1
Embodiment 2
[0031] Embodiment 2: The method for preparing endometrial stem cells according to the present invention
[0032] Wash the obtained endometrial tissue material, add 10 times the volume of pre-cooled sodium chloride injection containing 2× double antibody and 0.1% fetal bovine serum, oscillate and shake well, absorb the cleaning solution; then add 10 times the volume of 75% alcohol, soak for no more than 30s, discard the alcohol; finally add tissue cleaning solution and wash twice;
[0033] Cut the washed endometrial tissue to 20-50mm 3, add an equal volume of mixed digestion solution, the digestion solution contains 4mg / ml collagenase I and 2mg / ml Dispase II, digest in a constant temperature shaker at 200R, 37℃ for 30-90min, and obtain a single cell suspension; the digested single cell Pass the cell suspension through a 100 μm disposable filter, discard tiny tissue pieces; centrifuge at 500 g for 10 min, resuspend the cell pellet with Lonza serum-free medium and inoculate it i...
Embodiment 3
[0035] Example 3: Comparative experiment on the survival rate of endometrial stem cells prepared by different techniques
[0036] Experimental group: embodiment 2 method:
[0037] Control group 1: the method is the same as in Example 2, the difference is that α-MEM is the basal medium, 2% Pall serum substitute, 1% (w / v) penicillin / streptomycin, 1% (w / v) cyanine sulfate Amamicin, 2mM glutamine, 2mM MEM NEAA, 1mM sodium pyruvate, 1μg / ml EGF, 1μg / ml PDGF-BB;
[0038] Control group 2: The method is the same as in Example 2, except that the mixed digestive juice contains 4 mg / ml collagenase I and 2 mg / ml collagenase IV;
[0039] Control group 3: the method is the same as in Example 2, the difference is that 4mg / ml collagenase I and 2mg / ml collagenase IV are contained in the mixed digestive juice; Bovine serum, 1% (w / v) penicillin / streptomycin, 1% (w / v) gentamicin sulfate;
[0040] Control group 4: the method is the same as in Example 2, the difference is that 4mg / ml collagenase ...
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