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Separation and culture method and application of testicle mesenchymal stem cells

A testicular interstitial, separation method technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of unclear cell types, low amount of cells obtained, lack of cell identification standards, etc.

Active Publication Date: 2014-12-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the transplantation of testicular mesenchymal stem cells for the treatment of LOH provides a good prospect for clinical application, the current method has many disadvantages such as the small amount of cells obtained, the type of cells is not clear, and the lack of cell identification standards, which obviously restricts clinical application and needs further in-depth study. Research
There are no research reports on the isolation and identification of testicular mesenchymal stem cells using CD51 as a marker

Method used

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  • Separation and culture method and application of testicle mesenchymal stem cells
  • Separation and culture method and application of testicle mesenchymal stem cells
  • Separation and culture method and application of testicle mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Isolation of CD51-positive cells in the interstitium of mouse adult testis

[0026]In this example, testicular mesenchymal stem cells were isolated from mouse adult testes.

[0027] The separation method is as follows:

[0028] a. Take out the male C57BL / 6 male mice with healthy reproductive system 7 days after birth, kill them by cervical dislocation, open the scrotum under aseptic conditions, take out the bilateral testes, remove the capsule and blood vessels under a dissecting microscope, and use Wash repeatedly with 1×PBS containing 1% double antibody. Then blow gently with a straw and a gun tip to disperse the interstitial tissue and the fine tubules as much as possible, and cut the tissue part with small scissors. Then put in 1mg / ml type IV collagenase solution containing a small amount of DNase enzyme, incubate at 37°C and 5% CO2 for 15 minutes, add a large amount of 1×PBS containing 0.5% BSA to terminate the digestion of collagenase, and centrifuge ...

Embodiment 2

[0031] Example 2: Identification of self-renewal and proliferation abilities of CD51-positive testicular mesenchymal stem cells

[0032] In this example, on the basis of Example 1, the self-renewal and proliferation abilities of the obtained CD51-positive testicular mesenchymal stem cells were identified.

[0033] a. Identification of self-renewal ability of CD51-positive testicular stromal stem cells:

[0034] 1) Single CD51-positive cells sorted from mouse adult testes were placed in each single well of a 6-well plate for culture. The components of the culture medium include ITS added with 1nM dexamethasone, 1ng / mlLIF, 5mg / literinsulin, 5mg / litertransferrin, 5ug / litersodiumselenite in DMEM-F12 medium, 5% chicken embryo extract, 0.1mMβ-mercaptoethanol, 1% non- Essential amino acids, 1% N2, 2% B27 (Gibco), 20 ng / ml bFGF, 20 ng / ml EGF, 20 ng / ml PDGFBB, 20 ng / ml OSM. Observe the formation of cell clones. When the size of cell clones reaches more than 50um and the density reach...

Embodiment 3

[0042] Example 3: Observing the role of CD51-positive testicular mesenchymal stem cells in tissue repair in vivo

[0043] Establishment of rat EDS model:

[0044] The ability of stem cells to regenerate damaged tissues in the body is an important attribute. Therefore, we investigated whether CD51-positive Leydig stem cells could promote Leydig cell recovery in a rat model of Leydig cell loss. Previous studies have shown that thecytotoxinethanedimethylenesulfonate (EDS) may deplete Leydig cells after 4 days of treatment. We selected three groups of adult rats, which were normal group, model group and cell group. Rats in model group and cell group were inoculated with EDS, 1x10 after 4 days 6 CD51-positive Leydig stem cells were injected into the cell group mice. Serum testosterone concentrations were measured on day 10 after transplantation. The result is as Figure 7 As shown, CD51-positive Leydig stem cells significantly promoted the secretion of testosterone in the tes...

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Abstract

The invention discloses a separation and culture method and application of testicle mesenchymal stem cells. The method comprises the following steps: separating a testicle from a 7-day or 2-month mouse, removing the envelope and vessels to expose a seminiferous tubule, digesting the seminiferous tubule with collagenase IV, and filtering the digested cell suspension; and on the basis of the cell specific marker of CD51, separating with a flow cytometry to obtain single cells, and collecting the cells for expressing CD51, thereby separating the CD51 positive testicle mesenchymal stem cells. The invention also discloses testicle mesenchymal stem cells separated according to the method, self-renewal and proliferation cells with multi-directional differentiation potential obtained in a culture medium, and application of the cells in preparing compositions for treating diseases related to low testosterone level.

Description

technical field [0001] The invention relates to the technical field of stem cells and tissue engineering, in particular to a method for separating and culturing testicular mesenchymal stem cells and its application. Background technique [0002] With the acceleration of global aging, various aging-related diseases have attracted increasing attention. Among them, the incidence rate of diseases such as delayed hypogonadism (LOH) and senile androgen deficiency caused by primary or secondary Leydig cell dysfunction is as high as more than 20% in men over 50 years old. It not only affects Quality of life can also easily lead to the occurrence of major diseases such as cardiovascular disease and mental disease. Testosterone replacement therapy is currently the main treatment. It is estimated that the market size of testosterone products in the United States was approximately US$49 million in 1997, and exceeded US$300 million in 2002. Statistics show that 20% of men over the age...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/48A61P5/26
Inventor 项鹏姜美花蔡炳臧志军汪建成
Owner SUN YAT SEN UNIV
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