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Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells

A spermatogonial stem cell and blood-derived technology, applied in the field of biomedicine, can solve problems such as safety concerns, time-consuming, and complicated processes

Active Publication Date: 2020-06-30
深圳百年干细胞技术研究院有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the technical source of male autologous spermatogonial stem cells obtained in the prior art is difficult, the process is complicated, time-consuming, the quantity is small, and the problems of safety concerns, the present invention provides a male blood-derived autologous spermatogonial stem cells (blood-derived autologous spermatogonial stem cells) Germ stem cells) preparation method, the method is simple, fast, safe, large yield

Method used

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  • Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells
  • Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells
  • Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 A kit for preparing male blood-derived autologous spermatogonial stem cells

[0045] The kit is prepared in a safe operating bench with a cleanliness level of 10-100 and prepared at a low temperature of 4-10°C.

[0046] The kit includes cell culture medium S1, cell culture medium S2 and cell culture medium S3.

[0047] Preparation of cell culture medium S1: In 500 mL of RPMI 1640 culture medium, add: 5% Knockout serum substitute, 10 ng / mL erythropoietin, 10 ng / mL macrophage colony-stimulating factor, 150 ng / mL granulocyte colony-stimulating factor, 10ng / mL interleukin-3, 10ng / mL interleukin-6, 10ng / mL stem cell factor, 100ng / mL basic fibroblast growth factor, 30ng / mL Nanog protein, 30ng / mL Oct4 protein, 20ng / mL Sox2 protein, 20ng / mL C-myc protein; fully dissolved, then filtered and sterilized through a filter with a pore size of 0.22 microns, and prepared into cell culture medium S1.

[0048] Preparation of cell culture medium S2: In 500mL of M2 medium, add:...

Embodiment 2

[0051] Example 2 Preparation method of male blood-derived autologous spermatogonial stem cells

[0052] (1) Take 50mL of male venous blood (from volunteers), and use the conventional Ficoll centrifugal separation technology to centrifuge the venous blood to obtain blood mononuclear cells. The view under the microscope is as follows figure 1 shown;

[0053] (2) The obtained mononuclear cells were divided into 5x10 6 density, with the cell culture solution S1 prepared in Example 1, at 37 ° C and 5% CO 2 Cultivate in the incubator for 3 days to obtain cell 1; then use the cell culture medium S2 prepared in Example 1, and use 5×10 6 Density at 37°C and 5% CO 2 The incubator was cultivated for 3-6 days to obtain cell 2; 6 Density at 37°C and 5% CO 2 Culture in an incubator for 3-6 days to obtain cell 3;

[0054] (3) Gently blow and blow the cells 3 obtained after being cultured in cell culture medium S1, cell culture medium S2 and cell culture medium S3 successively, after th...

experiment example 1

[0056] Experimental example 1 Detection of blood-derived autologous spermatogonial stem cells

[0057] (1) Apply the male blood-derived autologous spermatogonia stem cells prepared in Example 2 on a poly-D-lysine glass slide, and use a cell slide centrifuge to rotate and throw the slide at 1800RCF for 2 minutes to make a cell slide And make a mark, dry the cell slides and store them in a -20°C refrigerator;

[0058] (2) Take out the cell slide from the -20°C refrigerator and dry it at room temperature, and use a marker pen to delineate the area on the reverse side; then fix it with 4% paraformaldehyde, and punch holes with 0.1% Triton X-100 for 5 minutes and after washing with phosphate buffered saline (PBS, pH 7.4), block with 10% normal goat serum at room temperature for 1 hour to obtain the sample;

[0059] (3) Add the primary antibody reagent to the sample, the primary antibody used is rabbit anti-human DAZL polyclonal antibody (diluted 1:100), and react the antigen in th...

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Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method for producing blood-derived autologous spermatogonial stem cells through reverse differentiation of male blood cells, and the blood-derived autologous spermatogonial stem cells and applications thereof. According to the preparation method, autologous cells are used as raw material cells, and are cultured sequentially with a cell culture liquid S1, a cell culture liquid S2 and a cell culture liquid S3 to obtain the blood-derived autologous spermatogonial stem cells. According to the present invention, the human autologous blood cells are used as the autologous raw material cells for conveniently and simply produce the autologous spermatogonial stem cells through the reverse differentiation; by using the formula of the cell culture liquid comprising the small molecule substances, in the case of no change of the human somatic chromosome DNA sequence and no insertion of any foreign genes or DNA fragments, the human autologous blood cells are subjected to rapid reverse differentiation so as to produce the blood-derived autologous spermatogonial stem cells; and the production rate, the yield and product purity of the male blood-derived autologous spermatogonial stem cell of the present invention are significantly superior to the production rate, the yield and product purity of the male blood-derived autologous spermatogonial stem cell in the prior art.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method for reversely differentiating male blood cells to produce blood-derived autologous spermatogonial stem cells, stem cells and applications thereof. Background technique [0002] Stem cells are a kind of pluripotent cells with self-replication ability. Under certain conditions, it can differentiate into a variety of functional cells. Stem cells are divided into embryonic stem cells (Embryonic StemCell, ES cells) and adult stem cells (Somatic Stem Cell) according to their developmental stages. Stem cells are divided into three categories according to their developmental potential: totipotent stem cells (Totipotent Stem Cells, TSCs), pluripotent stem cells (Pluripotent Stem Cells) and unipotent stem cells (Unipotent Stem Cells). Stem cells are not yet fully differentiated and immature cells, which have the potential to regenerate various tissues...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/30C12N2500/33C12N2501/105C12N2501/11C12N2501/115C12N2501/125C12N2501/13C12N2501/14C12N2501/155C12N2501/165C12N2501/22C12N2501/2303C12N2501/2306C12N2501/305C12N2501/31C12N2501/602C12N2501/603C12N2501/605C12N2501/606C12N2501/727C12N2501/998C12N2506/11
Inventor 林雄斌汤世坤任海英廖唤昭徐胜美黄卫红吴铭
Owner 深圳百年干细胞技术研究院有限公司
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