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Small-molecular compound combination and method for inducing differentiated cells to prepare osteoblasts by using small-molecular compound combination

A small molecular compound and cell technology, applied in the field of cell biology, can solve the problems of induced transdifferentiation of cells and low feasibility of transdifferentiation of similar cells

Active Publication Date: 2018-05-22
深圳臻德济慈药品研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since there are about 25% genetic differences between humans and mice, it is not feasible to apply the above-mentioned successful patent application technical scheme in mouse cell experiments to human cells of the same kind; on the other hand, due to the induction of similar cells The specific theoretical basis and technical means of transdifferentiation to obtain different target cells are not the same. The applicant used the technical solutions reported above to repeat the experiments, but failed to successfully apply the transdifferentiation technical solutions applied to mouse cells to similar human cells. transdifferentiation, and failed to induce transdifferentiation of human differentiated cells into osteoblasts

Method used

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  • Small-molecular compound combination and method for inducing differentiated cells to prepare osteoblasts by using small-molecular compound combination
  • Small-molecular compound combination and method for inducing differentiated cells to prepare osteoblasts by using small-molecular compound combination
  • Small-molecular compound combination and method for inducing differentiated cells to prepare osteoblasts by using small-molecular compound combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] 1. Isolation of skin fibroblasts

[0091] 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.

[0092] 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.

[0093] 2. Reprogramming of skin fibroblasts: the first stage

[0094] After the treatment in the first step above, completely replace it with the induction medium of the first stage for cell culture. The culture time is 3-8 days, at 37°C, 5% CO 2 environment to grow ...

Embodiment 2

[0101] 1. Isolation of skin fibroblasts

[0102] 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.

[0103] 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.

[0104] 2. Activation of skin fibroblasts

[0105]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 ...

Embodiment 3

[0112] 1. Isolation of skin fibroblasts

[0113] 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.

[0114] 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.

[0115] 2. Activation of skin fibroblasts

[0116] 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...

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Abstract

The invention discloses a small-molecular compound combination and a method for inducing differentiated cells to prepare osteoblasts by using the small-molecular compound combination. The small-molecular compound combination includes a first-stage small-molecular compound and a second-stage small-molecular compound used in a staged manner according to time sequence, wherein the first-stage small-molecular compound includes TGF-beta receptor inhibitors, WNT / beta-catenin agonists and cAMP agonists; the second-stage small-molecular compound includes lysine deacetylases inhibitors, TGF-beta receptor inhibitors, PKC inhibitors, WNT / beta-catenin agonists and cAMP agonists. Through phased induction of the small-molecular compounds, the differentiated cells can be differentiated into the osteoblasts, precision quality control can be achieved in each step, and standardization operation and large-scale production are facilitated. The method provided by the invention has wide donor sources, a patient himself can be used as a donor, and the osteoblasts needed for basic research, clinical treatment, or tissue engineering production can be obtained in a relatively short period of time.

Description

technical field [0001] The invention relates to the fields of cell biology, tissue engineering and regenerative medicine, in particular to a combination of small molecule compounds and a method for preparing osteoblasts from differentiated cells induced by the combination of small molecule compounds. Background technique: [0002] The existing osteogenic seed cells for bone tissue engineering or regenerative medicine include late differentiated osteoblasts, osteoblast lines, bone marrow mixed cells or purified mesenchymal stem cells. However, the sources of such cells are limited, the acquisition process is complicated, and the donor is seriously injured, so the application is limited. [0003] A variety of differentiated cells such as skin fibroblasts have the advantages of abundant sources, easy acquisition, and easy long-term expansion and culture in vitro. At present, cell transdifferentiation technology can be used to directly induce differentiated cells such as skin f...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/071
CPCC12N5/0654C12N2500/38C12N2501/01C12N2501/15C12N2501/385C12N2501/415C12N2501/71C12N2501/73C12N2501/999C12N2506/09C12N2506/11C12N2506/13C12N2506/1307
Inventor 胡敏李燕皎王兆杰
Owner 深圳臻德济慈药品研发有限公司
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