Method for preparing male non-testicular-sourced protein-induced autologous-reproduction stem cells, kit, the stem cells and application

A reproductive stem cell and inducible technology, applied in the field of preparation of male non-testis-derived protein-induced autologous reproductive stem cells, can solve the problems of small number of reproductive stem cells and low safety

Inactive Publication Date: 2015-03-18
深圳百年干细胞技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems of low quantity and low safety of autologous reproductive stem cells obtained in males and male animals obtained in the prior

Method used

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  • Method for preparing male non-testicular-sourced protein-induced autologous-reproduction stem cells, kit, the stem cells and application
  • Method for preparing male non-testicular-sourced protein-induced autologous-reproduction stem cells, kit, the stem cells and application
  • Method for preparing male non-testicular-sourced protein-induced autologous-reproduction stem cells, kit, the stem cells and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0267] Example 1 Kit preparation

[0268] It is operated in a safe operation bench with a cleanliness level of 10-100, and is prepared under a low temperature of 4-10 degrees.

[0269] In 500ml of RPMI 640 culture medium, add:

[0270] 10 μM RHO kinase inhibitor Y-27632;

[0271] 10ng / ml stem cell factor;

[0272] Interleukin-3 at 10ng / ml;

[0273] Interleukin 6 at 10ng / ml;

[0274] Interleukin-11 at 10ng / ml;

[0275] 10ng / ml macrophage colony-stimulating factor (M-CSF);

[0276] 10ng / ml of granulocyte colony-stimulating factor (G-CSF);

[0277] 10μg / ml fucoidan;

[0278] Dextran sulfate at 10 μg / ml;

[0279] 10 nM AMD 3100 (1,1′-[1,4-phenylenebis(methylene)]-bis-1,4,8,11);

[0280] M-ALENDRONATE SODIUM TRIHYDRATE at 10 μM; and

[0281] 10 μM pamidronate disodium.

[0282] Fully dissolve, then filter and sterilize through a filter with a pore size of 0.22 microns, and prepare cell culture solution A.

[0283] In 500ml of DMEM culture medium, add:

[0284] 10 μ...

Embodiment 2

[0335] Example 2 Preparation of non-testis-derived protein-induced male autologous germ stem cells (M-PiGSC)

[0336] Such as figure 1 As shown, 50ml of male venous blood (human venous blood comes from the inventor himself and volunteers) was extracted, and conventional Ficoll centrifugal separation technology was used to centrifuge the male venous blood to obtain blood mononuclear cells. The obtained mononuclear cells were divided into 5x10 6 Density, with the cell culture solution A prepared in Example 1, at 37 ° C and 5% CO 2 Cultivate in the incubator for 3 days; Then use the cell culture solution B prepared in Example 1, continue to mononuclear cells with 5×10 6 The density was cultured for 6 days; then use the cell culture solution C prepared in Example 1, and continue to mononuclear cells with 5×10 6 density for 6 days. Gently pipet the somatic cells or mononuclear cells cultured in cell culture medium A, cell culture medium B and cell culture medium C to complete...

Embodiment 3

[0338] Example 3 The same method as in Example 2 was used to prepare non-testis-derived protein-induced male autologous reproductive stem cells. The only difference is that Balb / C male mice aged 3-5 months (purchased from the School of Radiology Medicine, Chinese Academy of Medical Sciences (Tianjin), animal qualification certificate number: SCXK (Jin) 2010-0002) were selected, and the peripheral blood was collected. and femoral blood, thereby replacing male venous blood.

[0339] The type identification and safety identification of the M-PiGSC prepared in Example 2 and Example 3 were carried out through the following tests.

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Abstract

The invention provides a method for preparing male non-testicular-sourced protein-induced autologous-reproduction stem cells. The method comprising following steps: (1) cultivating somatic cells in a cell culture fluid A for 1-3 days, cultivating the somatic cells in a cell culture fluid B for 1-6 days and then cultivating the somatic cells in a cell culture fluid C for 1-6 days; and (2) collecting the male non-testicular-sourced protein-induced autologous-reproduction stem cells. The invention also provides a kit used for preparing the male non-testicular-sourced protein-induced autologous-reproduction stem cells. The method and the kit are advantaged in yield, purity, and producing speed of the production of the protein-induced autologous hematopoietic stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing male non-testis-derived protein-induced autologous reproductive stem cells, a kit, stem cells and applications. Background technique [0002] Stem cells are a kind of pluripotent cells with self-replication ability, which can differentiate into various functional cells under certain conditions. Stem cells are divided into embryonic stem cells and adult stem cells according to their developmental stage. Embryonic stem cells are a type of early embryonic cells with unlimited self-renewal and differentiation potential. They are the cells of the inner cell pattern when fertilized eggs divide and develop into blastocysts. They have the ability to differentiate into cells of various germ layers. Adult cells refer to stem cells that exist in specific tissues or organs of humans and mammals (including bone marrow, nerves, muscles, epidermis, testes, or kidneys, etc.) ...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/076A01N1/02A61K35/52A61K35/545A61P15/00A61L27/38
Inventor 林雄斌林柳吟
Owner 深圳百年干细胞技术研究院有限公司
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