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

A germ stem cell and somatic cell technology, applied in the biomedical field, can solve the problems that limit the clinical application of iPS, the early stage of cell arrest, and the increased risk of potential abnormalities in cytogenetics and epigenetics

Pending Publication Date: 2020-08-04
林柳吟
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2009, Park et al. co-cultured human iPS and human fetal gonadal stromal cells in the first trimester, and found that iPS could differentiate toward germ stem cells, but the differentiated cells had no expression of meiosis-related genes and could not produce gametes
In 2010, Imamura et al. used BMP4 to successfully induce the iPS line expressing the germ stem cell marker Mvh-RFP into germ stem cells, express early germ cell markers, and induce differentiation into oocyte-like cells by trans-retinoic acid A, but These cells are stuck at an early stage and cannot mature
In addition, the low conversion efficiency of iPS and the increased risk of iPS reprogramming or potential abnormalities of cytogenetics and epigenetics during the subsequent cell culture process limit the clinical application of iPS
[0009] At present, how to obtain more and better non-ovary-derived female reproductive stem cells is a worldwide problem, and no satisfactory technical method has been obtained so far

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0306] The preparation of embodiment 1 kit

[0307] 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.

[0308] In 500ml of RPMI 640 culture medium, add:

[0309] 5% Serum Replacement (KSR);

[0310] EPO 10ng / ml;

[0311] M-GSF 10 ng / ml;

[0312] G-GSF 150ng / ml;

[0313] IL-3 10 ng / ml;

[0314] IL-6 10 ng / ml;

[0315] SCF 10 ng / ml;

[0316] bFGF 100 ng / ml;

[0317] Nanog 30ng / ml;

[0318] Oct4 30 ng / ml;

[0319] Sox2 20ng / ml;

[0320] c-myc 20 ng / ml.

[0321] Fully dissolve, then filter and sterilize through a filter with a pore size of 0.22 microns, and prepare cell culture solution 01.

[0322] In 500ml of M2 culture medium, add:

[0323] 5% Serum Replacement (KSR);

[0324] Nanog 30ng / ml;

[0325] Oct4 30 ng / ml;

[0326] Sox2 10 ng / ml;

[0327] c-myc 20 ng / ml;

[0328] DDX4(Mvh) 30 ng / ml;

[0329] Dazl 30ng / ml;

[0330] bFGF 100 ng / ml;

[0331] EGF 10 ng / ml;

[0332] IL-...

Embodiment 2

[0377] Example 2 Preparation of blood-derived female autologous reproductive stem cells

[0378] 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 divided into 5x10 6 Density, using the cell culture solution 01 prepared in Example 1, cultured in a 37°C and 5% CO2 incubator for 3 days; then change to the cell culture solution 02 prepared in Example 1, continue to mononuclear cells with 5x10 6 The density was cultured for 6-9 days to obtain blood-derived female autologous totipotent stem cells; 6 density for 6-9 days. Gently blow and blow the cells cultured in cell culture medium 01, cell culture medium 02 and cell culture medium 03 to completely suspend the cells, collect the cells in a centrifuge tube, perform centrifugation, and then use normal saline to dissolve the suspended cells After the was...

Embodiment 3

[0388] Example 3 Passage of blood-derived female autologous reproductive stem cells

[0389] When the blood-derived female autologous reproductive stem cells obtained in Example 2 covered 80%-90% of the bottom of the bottle, they were passaged at a ratio of 1:3. After adding an appropriate amount of PBS to wash twice, digest with 2ml of collagenase type IV for about 1-2min, observe under an inverted microscope during this period, if the cytoplasm shrinks and the intercellular space increases, immediately add serum-containing medium to stop the digestion. Collect the liquid, centrifuge at 1000rpm for 8min, discard the supernatant, add complete medium to resuspend, and inoculate and culture.

[0390] Figure 7 Passage diagram of female autologous germline stem cells is shown.

[0391] Figure 8 and Figure 9 The detection charts of markers of autologous reproductive stem cells of the first-generation and fifth-generation blood-derived females are shown respectively

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Abstract

The invention relates to a preparation method, a kit and application of hematogenous female autologous germline stem cells, in particular to a technology and a method for producing hematogenous femaleautologous germline stem cells through non-transgenosis by using female autologous blood cells as raw material cells. The preparation method of the hematogenous female autologous germline stem cellscomprises the following steps of: culturing female autologous blood cells in a cell culture solution O1 for 3 days; continuously culturing in a cell culture solution O2 for 6-9 days; continuously culturing in a cell culture solution O3 for 6-9 days; and collecting the hematogenous female autologous germline stem cells. The invention further provides a kit for preparing the hematogenous female autologous germline stem cells. The method and the kit disclosed by the invention have remarkable advantages in the aspects of sampling, production speed, yield and purity of the hematogenous female autologous germline stem cells.

Description

technical field [0001] The field of the present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a cell culture method for reversely differentiating female blood cells to produce blood-derived autologous reproductive stem cells, stem cells and applications. Background technique [0002] Nowadays, with the emergence of an aging society, the issue of aging is gradually being paid attention to by people. In women, the ovaries age significantly faster than other organs in the body. Ovarian aging affects women's health, triggering the onset of menopause, cancer and the occurrence of various chronic diseases. However, with the deterioration of the living environment, the increasing pressure of life and the damage to the ovarian function of women due to factors such as side effects of drug treatment, ovogenous infertility and pathological ovarian function decline have become major social health problems that plague modern wome...

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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