Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro

A technology of osteoblast differentiation and bone marrow mesenchyme, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve problems affecting the source and application of tissue engineering cells, loss of differentiation potential, etc., and achieve simple acquisition methods Feasible, easy-to-source effects

Inactive Publication Date: 2014-03-26
HUZHOU CENT HOSPITAL
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Problems solved by technology

After the bone marrow mesenchymal stem cells isolated and purified in vitro have been passaged, the relevant biological characteristics have changed, and the appearance has not changed significantly, but they have gradually

Method used

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  • Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro
  • Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro
  • Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro

Examples

Experimental program
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Embodiment 1

[0026] Reagents used: fetal bovine serum (Hyclone), penicillin, streptomycin, DMEM medium, F12 medium (Gibco), dexamethasone, ascorbic acid, sodium glycerophosphate, 5'-azacytidine (Sigma).

[0027] 1. Take about 6-week-old ICR male adult mice (purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences), kill them by cervical dislocation, and take out the femur and tibia under aseptic conditions.

[0028] 2. Cut the epiphyses at both ends with sterile scissors, and use a syringe to draw complete medium to flush the bone marrow cavity. Complete medium components: 10% (v / v) fetal bovine serum, penicillin 100U / mL, streptomycin 100mg / L, The solvent is DMEM / F12 culture medium (that is, a mixture of DMEM and F12 with a volume ratio of 1:1).

[0029] 3. The obtained bone marrow cell suspension was filtered through a 200-mesh sieve, and inoculated in a 10 cm culture dish with an initial inoculation density of about 5×10 6 cells / mL, the cell culture medium i...

Embodiment 2

[0035] The bone marrow mesenchymal stem cells induced to differentiate in step 7 of this embodiment are cells that have been passed to the third passage through cell adherent culture and trypsinization, and other steps of this embodiment are the same as those of embodiment 1.

Embodiment 3

[0037] The composition of the induction medium in this example is that the complete cell medium is supplemented with the same concentrations of dexamethasone, ascorbic acid and sodium β-glycerophosphate as in Example 1.

[0038] Identification of osteoblast-induced differentiation of bone marrow mesenchymal stem cells:

[0039] After 21 days of osteogenic induction and differentiation of mouse bone marrow mesenchymal stem cells in Example 1 and Example 2, 3, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed 3 times with distilled water, and 0.2% Alizarin Red S Stain at room temperature for 30 minutes, then discard the staining solution, wash with distilled water three times, and observe the osteoblast differentiation results of mouse bone marrow mesenchymal stem cells under a microscope.

[0040] The result is shown in the attached picture, figure 1 Alizarin red S staining results for the osteogenic differentiated cells in Example 1 showed th...

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Abstract

The invention provides an inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro. The inducing culture medium is composed of 1*10<-8> mol/L of dexamethasone, 50 mu mol/L of ascorbic acid and 10 mmol/L of sodium beta-glycerophosphate; and solvent is a supernatant of a sclerite complete culture medium and comprises 10% of fetal calf serum, 100 U/mL of penicillin, 100 mg/L of streptomycin, a mixture of DMEM culture fluid and F12 culture fluid and multiple growth factors secreted by bone cells in the sclerite culture process. According to the invention, bone marrow mesenchymal stem cells of a mouse are purified by replacing the cell culture fluid through an adherent cell passage method, the obtained cells of the first generation are induced, and the supernatant of the sclerite complete culture medium cultured for 72-96 hours is used as the solvent of osteoblast differentiation inducer, thereby obviously improving the in vitro osteogenic differentiation efficiency of bone marrow mesenchymal stem cells.

Description

technical field [0001] The invention relates to an in vitro osteoblast induction and differentiation method of bone marrow mesenchymal stem cells and the used induction medium. Background technique [0002] Bone marrow mesenchymal stem cells can be differentiated into osteoblasts, chondrocytes, adipocytes and other cell types under the stimulation of appropriate conditions, and are easy to isolate and culture, and have strong self-renewal, differentiation ability and immunosuppressive advantages. It has important application prospects in the fields of tissue and organ defect diseases, tissue and organ degenerative diseases, cell therapy and tissue engineering. Recently, many domestic and foreign studies have explored the method of inducing the differentiation of bone marrow mesenchymal stem cells into osteoblasts, with a view to serving as the research basis of bone tissue engineering and the theoretical guidance of clinical biological treatment of bone-related diseases. Af...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 李静周国顺戴利成
Owner HUZHOU CENT HOSPITAL
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