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Separation and culture method of human testicular mesenchymal stem cells

A Leydig, separation method technology, applied in the field of separation and culture of human Leydig stem cells, can solve the problems of low purity of isolated human SLC, damage to spermatogenesis, increased cardiovascular and cerebrovascular events and prostate cancer risks, etc.

Active Publication Date: 2020-07-10
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some clinical symptoms of male hypogonadism can only be alleviated clinically by supplementing exogenous androgen (Testosterone Replacement Therapy, TRT), but there are the following obvious defects: 1. Long-term administration is required; 2. Loss of circadian rhythm; 3 .Increasing the risk of cardiovascular and cerebrovascular events and prostate cancer; 4. Impairing spermatogenesis and causing male infertility (N Engl J Med.2015; J Am CollCardiol.2016)
[0005] In summary, the isolated human SLC reported in these literatures or patents has low purity, so it may mislead our research and understanding of human SLC, especially restricting the clinical transformation of human SLC

Method used

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  • Separation and culture method of human testicular mesenchymal stem cells
  • Separation and culture method of human testicular mesenchymal stem cells
  • Separation and culture method of human testicular mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Protein CD248 is specifically expressed in human testicular mesenchymal stem cells (SLC) in human testis tissue

[0045] In this example, we chose to detect the expression of CD248 protein in human testicular tissue (derived from organ donors and deceased patients with prostate cancer), and found that CD248 was specifically expressed in SLC.

[0046] The specific method is as follows:

[0047] 1. Tissue acquisition and preparation:

[0048] Obtain clinically donated testicular tissue, place it in 4% paraformaldehyde solution, and fix it at 4°C for 12 hours; replace it with 30% sucrose solution, and dehydrate it at 4°C for 24 hours; put the tissue block into a frozen section In the embedding medium, place at -20°C for more than 30 minutes, and make sections in a cryostat for subsequent fluorescent staining.

[0049] 2. Immunofluorescence staining and detection:

[0050] The testis tissue slices were baked for 15 minutes, penetrated with 0.2% TritonX-100 for ...

Embodiment 2

[0053] Example 2 Isolation of human testicular mesenchymal stem cells

[0054] In this embodiment, human SLC is isolated from donated human testicular tissue (the source is the same as in Example 1).

[0055] The separation method is as follows:

[0056] 1. Tissue acquisition and digestion:

[0057] Obtain clinically donated testicular tissue specimens, put them into a centrifuge tube containing 5 μg / mL moxifloxacin DMEM / F12 medium, and place them on ice; wash the testicular tissue repeatedly with PBS 3-5 times in an ultra-clean bench. Then use micro scissors to cut the testicular tissue into fine tissue pieces (approximately 1 mm in size) 3 ), then put the shredded testicular tissue into DMEM / F12 digestion solution containing 1 mg / mL type IV collagenase and 200 μg / mL DNase I, and shake slowly on a shaker at 37 °C for 20-30 minutes (100 rpm / minutes), adding phosphate buffered saline (PBS) containing 10% bovine serum albumin (BSA) to terminate the digestion, filtered throug...

Embodiment 3

[0062] The cultivation of embodiment 3 human testicular mesenchymal stem cells

[0063] 1. The culture medium used for the proliferation and cultivation of human testicular mesenchymal stem cells is the formula of testicular mesenchymal stem cell serum-free culture medium for adding the following components to the DMEM / F12 basal medium:

[0064]

[0065]

[0066] 2. Culture method of human testicular mesenchymal stem cells:

[0067] The positive human testicular mesenchymal stem cells collected by the above sorting were incubated with the above culture medium at 37°C and 5% CO 2 Conditions were cultivated, and the culture medium was changed every 3 days. When the cell density reaches 80%, digest with Accutase for 1 min, add culture medium to stop digestion, and centrifuge at 1200 rpm for 4 min.

[0068] After the cell pellet was resuspended in culture medium, the positive human testicular mesenchymal stem cells were 5 Cells / mL were seeded in well plates for culture. ...

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Abstract

The invention discloses a separation and culture method of human testicular mesenchymal stem cells. The invention researches and provides a specific marker, namely CD248 protein, specifically expressed in the human testicular mesenchymal stem cells (SLC) in human testicular tissues so as to identify and separate high-purity human SLC from the human testicular tissues. Therefore, a method for separating and obtaining the high-purity SLC from the human testicular tissues is established; and the obtained SLC does not have other types of cells in a testis and has high purity. Meanwhile, a culturesystem of the human SLC is optimized and improved; a serum-free culture medium of the obtained testicular mesenchymal stem cells has clear components and is good for standardization; and an efficientand stable culture and amplification system of the testicular mesenchymal stem cells is established. According to the separation and culture method disclosed by the invention, the high-purity human SLC can be obtained from the human testicular tissues and high-quality seed cells are provided for clinical transformation of treating male hypogonadism by the testicular mesenchymal stem cells; and theseparation and culture method has important meanings on promoting characteristic researches of the human SLC, especially clinical transformation of the SLC.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to a method for isolating and culturing human testicular mesenchymal stem cells. Background technique [0002] More than 95% of testosterone in men is synthesized and secreted by Leydig cells (LC). When the ability of LC to synthesize testosterone is insufficient due to congenital or acquired diseases, male hypogonadism will occur, leading to sexual dysfunction and male infertility; and it is related to osteoporosis, muscle atrophy, central obesity, memory and cognitive function It is closely related to the occurrence and development of major diseases such as diabetes, cardiovascular and cerebrovascular diseases (N Engl J Med.2013; JAMA Intern Med.2017; JAMA.2017; Aging Male.2018 Deng Chunhua, etc.). At present, some clinical symptoms of male hypogonadism can only be alleviated clinically by supplementing exogenous androgen (Testosterone Replacement Therapy, TR...

Claims

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Application Information

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IPC IPC(8): C12N5/0775G01N33/569A61K35/28A61P15/08
CPCA61K35/28A61P15/08C12N5/0668C12N2500/25C12N2500/32C12N2500/44C12N2501/11C12N2501/115C12N2501/135C12N2501/235C12N2501/237C12N2501/39C12N2501/727C12N2509/00G01N33/56966G01N2333/7056G01N2469/10
Inventor 项鹏邓春华夏凯柯琼
Owner SUN YAT SEN UNIV
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