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Purifying method of buffalo spermatogonia stem cell like cells used for in vitro culture

A technology of spermatogonial stem cells and in vitro culture is applied in the field of purification of buffalo spermatogonial stem cells, which can solve the problems of increased cost, high cost and the like, and achieves the effects of simple operation, low cost and high repeatability.

Inactive Publication Date: 2019-02-26
卢克焕 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Higher concentration of spermatogonial stem cells can be obtained by immunomagnetic bead cell sorting, but this method not only needs to select a highly specific surface plateau for purification, but also requires special equipment and the purchase of disposable separation columns and antibody-labeled magnetic beads, which are expensive
A large number of relatively pure spermatogonial stem cells can also be obtained by separating and purifying spermatogonial stem cells by flow cytometry, but it is necessary to purchase a flow cytometer, which increases the cost

Method used

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  • Purifying method of buffalo spermatogonia stem cell like cells used for in vitro culture
  • Purifying method of buffalo spermatogonia stem cell like cells used for in vitro culture
  • Purifying method of buffalo spermatogonia stem cell like cells used for in vitro culture

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Embodiment 1

[0044] The method for purifying buffalo spermatogonial stem cell-like cells for in vitro culture of the present invention has the following specific steps:

[0045] (1) Transfer the 3-month-old buffalo testis tissue after removing the albuginea and epididymis to a petri dish, and add collagenase digestion solution with a collagenase concentration of 10mg / ml and DNA with a DNase concentration of 0.5mg / ml Enzyme digestion solution, place the petri dish in a 35℃, 4% carbon dioxide incubator for 20min; the addition amount of collagenase digestion solution is 0.5ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 150μl / g buffalo testis organization

[0046] (2) Centrifuge the materials in the petri dish in step (1), discard the supernatant, add PBS buffer with pH 2 to resuspend the pellet, centrifuge, centrifuge for 3 minutes and centrifuge at 3500 rpm; discard Clean, repeat the above operation twice to obtain a single fine tubule sediment;

[0047] (3) Add 0....

Embodiment 2

[0053] The method for purifying buffalo spermatogonial stem cell-like cells for in vitro culture of the present invention has the following specific steps:

[0054] (1) Transfer the 4-month-old buffalo testis tissue after removing the albuginea and epididymis to a petri dish, adding collagenase digestion solution with a collagenase concentration of 12mg / ml and DNA with a DNase concentration of 0.7mg / ml Enzyme digestion solution, place the petri dish in a 35.3℃, 4.3% carbon dioxide incubator for 18min; the addition amount of collagenase digestion solution is 0.7ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 210μl / g buffalo testis organization

[0055] (2) Centrifuge the materials in the petri dish in step (1), discard the supernatant, add PBS buffer with a pH of 2.4 to resuspend the pellet, centrifuge, and centrifuge for 3.6 min and 3150 rpm; discard; Supernatant, repeat the above operation twice to obtain a single fine tubule sediment;

[0056] (3) A...

Embodiment 3

[0062] The method for purifying buffalo spermatogonial stem cell-like cells for in vitro culture of the present invention has the following specific steps:

[0063] (1) Transfer the 5-month-old buffalo testis tissue after removing the albuginea and epididymis to a petri dish, adding collagenase digestion solution with a collagenase concentration of 15mg / ml and DNA with a DNase concentration of 0.9mg / ml Enzyme digestion solution, place the petri dish in a 35.6℃, 4.7% carbon dioxide incubator for 16min; the addition amount of collagenase digestion solution is 1ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 270μl / g buffalo testis tissue

[0064] (2) Centrifuge the materials in the petri dish in step (1), discard the supernatant, add PBS buffer with a pH of 2.8 to resuspend the pellet, and centrifuge with a centrifugal time of 4.2 min and a centrifugal speed of 2700 rpm; discard Supernatant, repeat the above operation twice to obtain a single fine tubule...

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Abstract

The invention discloses a purifying method of buffalo spermatogonia stem cell like cells used for in vitro culture. The purifying method of buffalo spermatogonia stem cell like cells used for in vitroculture comprises following steps: a culture dish I containing gelatin is inoculated with a buffalo testis single-cell suspension, a stem cell culture solution is added, and culturing is carried outfor 6 to 20h; suspending and not firmly attached cells in the culture dish I are collected, and are transformed into a culture dish II containing rat tail collagen for 2 to 6h of culturing, supernatant cells in the culture dish II are collected, and are transformed into a culture dish III containing laminin for 20 to 60min of culturing, and adherent cells in the culture dish III are collected so as to obtain the purified buffalo spermatogonia stem cell like cells. According to the purifying method, differential adherence method is adopted for purifying of buffalo spermatogonia stem cell like cells, the culture time of buffalo spermatogonia stem cell like cells is shortened greatly, cell activity is maintained, practice base is provided for obtaining of large scale high quality buffalo lines, and important meaning in treatment of mammal infertility and endangered species protection is achieved.

Description

Technical field [0001] The invention relates to the fields of genetic engineering, preservation of rare species and cell engineering, and more specifically to a method for purifying buffalo spermatogonial stem cell-like cells cultured in vitro. Background technique [0002] Spermatogonial stem cells (SSCs) are the precursor cells that form sperm. In male mammals, the proliferation and differentiation of spermatogonial stem cells provide a steady flow of power for the occurrence of sperm, while also ensuring that the genetic material is in the parent and child. Effective transfer between generations. Since the successful development of spermatogonial stem cell transplantation technology in 1994, the research of spermatogonial stem cells has become a hot spot, and in recent years, a method for in vitro culture of spermatogonial stem cells has been successfully established. In 2011, a serum-free and feeder-free culture system for mice was successfully carried out in vitro, bringing...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/32C12N2500/36C12N2500/44C12N2501/115C12N2501/13C12N2501/235C12N2501/998C12N2533/52C12N2533/54
Inventor 卢克焕李婷婷陆阳清杨小淦梁兴伟
Owner 卢克焕