Method for inducing differentiated cells to prepare mesenchymal stem cells and combination for regulating targets
A technology of mesenchymal stem cells and induction of differentiation, applied in the field of cell biology, can solve the problems of induced transdifferentiation of cells and the low feasibility of transdifferentiation of similar cells, and achieve high amplification efficiency, good multi-layered multi-directional differentiation potential, and convenient and accurate Operational and Systemic Quality Control Effects
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Embodiment 1
[0241] 1. Isolation of skin fibroblasts
[0242] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from the donor, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.
[0243] 1.2 Subsequent passage of cells to a large number of expansion, the number of cell generations between the 6th and 12th passages is used for the induction of transdifferentiation into mesenchymal stem cells. The day before initiation of differentiation (Day-1), inoculate cell density at 1-2.5×10 4 / cm 2 Cultured at 37°C, 5% CO 2 in the incubator.
[0244] 2. Activation of skin fibroblasts
[0245] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and cultivate the cells for 4-6 days. The first-stage culture medium refers to: 10% fetal ...
Embodiment 2
[0253] 1. The separation of skin fibroblasts is the same as in Example 1.
[0254] 2. When transdifferentiation is started (Day0), completely replace the basal culture medium with the following culture medium, and culture the cells for 4-6 days, and the culture time is 6-12 days, at 37°C, 5% CO 2 environment to grow cells. The second stage culture solution is: 10% fetal bovine serum (Hyclone)+100U / ml penicillin (Sigma)+100 μg / ml streptomycin (Sigma)+high sugar DMDM medium (Gibco)+forskolin (2μM~ ( 1~15μM)+Y-27632(3~15μM)+L-Ascorbin acid 2-phosphate(0.15~0.25mM), 10% fetal bovine serum in this culture system can also be replaced by serum substitute (invitrogen) with 10%- A concentration of 20% was substituted; 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.
[0255] 3. Then replace it with the following culture medium for 3-8 days, and culture the cells at 37°C and 5% CO2 environment. The medium for the feeding stage is: BMP4 (10-20ng / mL)+...
Embodiment 3
[0259] 1. The separation of skin fibroblasts is the same as in Example 1.
[0260] 2. Activation of skin fibroblasts
[0261] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and culture the cells for 4 to 6 days. The first-stage culture medium refers to: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin ( Sigma) + 100μg / ml streptomycin (Sigma) + high glucose DMDM medium (Gibco) + forskolin (2μM ~ 25μM) + Repsox (2 ~ 15uM) + CHIR99021 (1μM ~ 10μM) + VPA (0.5mM ~ 1.5mM ), 10% fetal bovine serum in this culture system can also be replaced by a serum substitute (invitrogen) at a concentration of 10% to 20%; 100U / ml penicillin (Sigma) and 100μg / ml streptomycin (Sigma) can not be used . at 37°C, 5% CO 2 environment to grow cells.
[0262] 3. Orientation induction of skin fibroblasts
[0263] After the treatment in the second step above, completely replace it with the second-stage culture mediu...
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