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Preparation method and kit to prepare hematogenous female autologous germline stem cells and application of kit

A technology of reproductive stem cells and somatic cells, applied in the field of biomedicine, can solve the problems of time-consuming isolation and culture, and the scarcity of acquisition.

Inactive Publication Date: 2019-08-16
林柳吟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Scholars at home and abroad have invented some methods for the preparation of reproductive stem cells, such as the in vitro differentiation of natural embryonic stem cells to produce reproductive stem cells, or the in vitro differentiation of iPS pluripotent stem cells (transgenic stem cells) to produce reproductive stem cells, or the isolation and culture of ovarian tissue to prepare reproductive stem cells. Stem cells, isolation and culture are time-consuming and difficult, and the number of acquisitions is extremely rare

Method used

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  • Preparation method and kit to prepare hematogenous female autologous germline stem cells and application of kit
  • Preparation method and kit to prepare hematogenous female autologous germline stem cells and application of kit
  • Preparation method and kit to prepare hematogenous female autologous germline stem cells and application of kit

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Experimental program
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Effect test

Embodiment 1

[0293] The preparation of embodiment 1 kit

[0294] 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.

[0295] In 500ml of RPMI 640 culture medium, add:

[0296] 5% Serum Replacement (KSR);

[0297] EPO 10ng / ml;

[0298] M-GSF 10 ng / ml;

[0299] G-GSF 150ng / ml;

[0300] IL-3 10 ng / ml;

[0301] IL-6 10 ng / ml;

[0302] SCF 10 ng / ml;

[0303] bFGF 100 ng / ml;

[0304] Nanog 30ng / ml;

[0305] Oct4 30 ng / ml;

[0306] Sox2 20ng / ml;

[0307] c-myc 20 ng / ml.

[0308] Fully dissolve, then filter and sterilize through a filter with a pore size of 0.22 microns, and prepare cell culture solution O1.

[0309] In 500ml of M2 culture medium, add:

[0310] 5% Serum Replacement (KSR);

[0311] Nanog 30ng / ml;

[0312] Oct4 30 ng / ml;

[0313] Sox2 10 ng / ml;

[0314] c-myc 20 ng / ml;

[0315] DDX4(Mvh) 30 ng / ml;

[0316] Dazl 30ng / ml;

[0317] bFGF 100 ng / ml;

[0318] EGF 10 ng / ml;

[0319] IL-...

Embodiment 2

[0364] Example 2 Preparation of blood-derived female autologous reproductive stem cells

[0365] 50ml of female venous blood (from volunteers) was extracted, and the venous blood was centrifuged using conventional Ficoll centrifugal separation technology to obtain blood mononuclear cells. The obtained mononuclear cells were cultured at a density of 5x106 with the cell culture solution O1 prepared in Example 1 in an incubator at 37°C and 5% CO2 for 3 days; then replaced with the cell culture solution prepared in Example 1 O2, continue to treat mononuclear cells with 5x10 6 The density is cultivated for 6-9 days; then use the cell culture solution O3 prepared in Example 1, and continue to mononuclear cells with 5×10 6 density for 6-9 days. Gently pipette the cells cultured in cell culture medium O1, cell culture medium O2 and cell culture medium O3 to completely suspend the cells, collect the cells in a centrifuge tube, perform centrifugation, and then use normal saline to dis...

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Abstract

The invention relates to a preparation method and kit to prepare hematogenous female autologous germline stem cells and application of the kit. The hematogenous female autologous germline stem cells are prepared from female autologous blood cells as raw cells through a non-transgenic technique and method of producing hematogenous female autologous germline stem cells. The preparation method of thehematogenous female autologous germline stem cells comprises: culturing female autologous blood cells in a cell medium for 3 days; continuing to culture in cell medium O2 for 6-9 days; continuing toculture in a cell medium O3 for 6-9 days; collecting the hematogenous female autologous germline stem cells. The invention also provides a kit to prepare the hematogenous female autologous germline stem cells. The method and kit herein have significant advantages in terms of material acquiring, production speed, yield and purity for hematogenous female autologous germline stem cells.

Description

technical field [0001] The invention relates to a preparation method, kit and application of blood-derived female autologous reproductive stem cells, and belongs to the field of biomedical technology. Specifically, the invention relates to a cell that reversely differentiates female blood cells to produce blood-derived autologous reproductive stem cells Culture methods, stem cells and applications. Background technique [0002] In female mammals, the germline stem cells in the gonads are derived from a rare type of germline stem cell inherited from the embryo. In the process of embryogenesis and ovary formation, reproductive stem cells settle in the ovary, leading the differentiation, development and maturation of the ovary. Germ stem cells develop sequentially to form oogonia ► primary follicles ► secondary follicles ► mature follicles ► eggs. Germ stem cells have Mvh + and Dazl + Positive specific cell phenotype. The reproductive stem cells of the ovary are a slow con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/078C12N5/073C12N5/071C12N5/077A61K35/545A61P15/00A61P15/08C40B40/00
CPCA61K35/545A61P15/00A61P15/08C12N5/0611C12N2500/38C12N2501/105C12N2501/11C12N2501/115C12N2501/117C12N2501/125C12N2501/13C12N2501/14C12N2501/155C12N2501/16C12N2501/165C12N2501/22C12N2501/2303C12N2501/2306C12N2501/305C12N2501/31C12N2501/392C12N2501/602C12N2501/603C12N2501/605C12N2501/606C12N2502/025C12N2502/09C12N2502/11C12N2502/1305C40B40/00
Inventor 林雄斌
Owner 林柳吟
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