Target regulation system for dedifferentiation of somatic cells, kit and application of kit
A technology for regulating systems and somatic cells, applied in the field of cell biology, can solve the problems of difficult repetition of experiments, unclear components of the transdifferentiation system, and low efficiency.
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Embodiment 1
[0071] Example 1 Target Regulatory System Used to Prepare Osteoblasts
[0072] 1. Isolation of skin fibroblasts
[0073] 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.
[0074] 1.2 Subsequent passage of cells, a large number of expansion, the number of cell generation is between the 6th passage and the 12th passage, which is used to induce the transdifferentiation into osteoblasts. 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.
[0075] 2. Reprogramming of skin fibroblasts: the first stage
[0076] After the treatment in the first step above, completely replace it with the induction medium of the first stage for cell cult...
Embodiment 2
[0081] Example 2 Target Regulatory System Used to Prepare Osteoblasts
[0082] 1. Isolation of skin fibroblasts
[0083] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from the donor, separate the skin fibroblasts by the wall-adhesive method, and culture the isolated cells in the basal culture medium: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.
[0084]1.2 Subsequent passage of cells, a large number of expansion, the number of cell generation is between the 6th passage and the 12th passage, which is used to induce the transdifferentiation into osteoblasts. 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.
[0085] 2. Activation of skin fibroblasts
[0086] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and culture th...
Embodiment 3
[0092] Example 3 Target Regulatory System Used to Prepare Chondrocytes
[0093] 1. Isolation of skin fibroblasts
[0094] 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.
[0095] 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 chondrocytes. 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.
[0096] 2. Reprogramming of skin fibroblasts
[0097] After the treatment in the second step above, completely replace it with the second-stage culture medium for cell culture. The culture time is 6-12 day...
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