Combination of small molecular compounds and method for preparing osteoblasts from cells induced by the combination of small molecular compounds

A technology of small molecular compounds and osteoblasts, applied in the field of cell biology, can solve the problems of low feasibility of transdifferentiation of similar cells, induced transdifferentiation of cells, etc. Effect

Active Publication Date: 2021-05-28
深圳臻德济慈药品研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Combination of small molecular compounds and method for preparing osteoblasts from cells induced by the combination of small molecular compounds
  • Combination of small molecular compounds and method for preparing osteoblasts from cells induced by the combination of small molecular compounds
  • Combination of small molecular compounds and method for preparing osteoblasts from cells induced by the combination of small molecular compounds

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 combination of small molecule compounds and a method for preparing osteoblasts from differentiated cells induced by the combination of small molecule compounds. The combination of small molecule compounds includes the first-stage small-molecule compound and the second-stage small-molecule compound used in stages according to time series, and the first-stage small-molecule compound includes TGF-β receptor inhibitors, WNT / β-catenin agonists and cAMP agonists; the second-stage small molecule compounds include lysine deacetylase inhibitors, TGF-β receptor inhibitors, PKC inhibitors, WNT / β-catenin agonists and cAMP agonists. The present invention can transform differentiated cells into osteoblasts through stage-by-stage induction by small molecular compounds, each step can realize precise quality control, and is convenient for standardized operation and large-scale production. The method provided by the invention has a wide range of sources of donors, and patients themselves can be used as donors, and osteoblasts required 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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Patent Type & Authority Patents(China)
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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