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Separation method for Leydig cell and use thereof

A cell and stem cell technology, applied in the field of separation of cells in the Leydig, can solve the problems of long duration, low acquisition rate, and difficulty in meeting the number and function of cells

Inactive Publication Date: 2008-06-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the classic methods to obtain high-purity Leydig cells are cell elutriation and Percoll density gradient centrifugation. The most prominent disadvantages are complicated separation techniques, many operating steps, and a long time.
After these two separation methods, the cells are very damaged, the acquisition rate is low, the survival rate is low, it is difficult to expand, and the long-term testosterone production function cannot be maintained in vitro, and it is difficult to meet the requirements of cell therapy and tissue engineering tissue construction. and the dual requirements of the function
Moreover, during these operations, a large amount of Leydig stem cells and precursor cells will be lost, resulting in the inability to maintain the number and function of Leydig cells for a long time

Method used

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  • Separation method for Leydig cell and use thereof
  • Separation method for Leydig cell and use thereof
  • Separation method for Leydig cell and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Acquisition, culture and detection of Leydig cells

[0105] 1. Obtaining Testicular Tissue

[0106] Male Wister rats, 30-45 days old, weighing 100-200 grams, were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences.

[0107] 2.5% pentobarbital anesthesia, bilateral inguinal canal medial oblique incision, cut the testicular sheath, carefully separate the connection between the testis and epididymis, obtain a complete testis after ligation of blood vessels, aseptically weigh the wet weight of each testis, That is, place in a sterile ice bath in phosphate buffered saline (PBS) without calcium and magnesium ions. The complete testis was obtained by repeated washing, and the outer layer of the testis and large blood vessels were peeled off on ice to keep the integrity of the testis shape and the continuity of the seminiferous tubules as much as possible. After removing the buffy coat and larger blood vessels visible to the naked eye, the wet wei...

Embodiment 2

[0140] Differential attachment time selection

[0141] According to the method of Example 1, cells in the testicular tissue and interstitial tissue of the testis were obtained, filtered and then inoculated and cultured.

[0142] The cultured cells were divided into four groups as follows: (1) 1-hour group; (2) 2-hour group; (3) 3-hour group and (4) 24-hour group.

[0143] The specific operation is as follows: After inoculation, after culturing for 1, 2, 3 and 24 hours according to the groups, the unattached suspension cells and culture fluid were collected respectively, and transferred to the corresponding new culture dishes of each group, with 5 % serum in DMEM / HAM'S F-12 (1:1) culture solution, after 24 hours, remove unattached cells and culture solution. At the same time, the adherent cells in the original culture dish of each group were added with serum-free DMEM / HAM'S F-12 (1:1) medium to continue culturing.

[0144] If the cells cultured after transfer do not re-attach t...

Embodiment 3

[0147] Optimization of Conditioned Medium for Leydig Cells

[0148] According to the method of Example 1, cells in the testicular tissue and interstitial tissue of the testis were obtained, filtered and then inoculated and cultured. The DMEM / HAM'S F-12 (1:1) culture medium for cell primary culture was divided into the following four groups: (1) serum-free group; (2) group added with 1% fetal bovine serum; (3) group added with 5% Fetal bovine serum group; and (4) adding 10% fetal bovine serum group. Then the cell morphology and growth characteristics were observed under an inverted fluorescent microscope and an electron microscope.

[0149] See Figure 5 .

[0150] The results show that the primary cells can maintain good cell growth morphology for more than 7 days when cultured under the condition of serum-free DMEM / HAM'S F-12 (1:1), and the DMEM / HAM'SF containing 1% fetal bovine serum -12 (1:1) culture medium can accelerate the spread and growth speed of early Leydig cell...

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Abstract

The invention discloses a method for obtaining Leydig cells and purposes of the method. The method disclosed by the invention has the advantages of quickness, simpleness and convenience, and capability of obtaining a large quantity of Leydig cells with high purity and good functions, and is used for establishing seed cells of tissue engineered androgen-secreting tissue and other purposes.

Description

technical field [0001] The invention relates to seed cells in tissue engineering and cell transplantation, in particular to a method for separating interstitial cells of the testis and uses thereof. Background technique [0002] Androgen deficiency is common in many related clinical departments such as andrology, urology, and plastic surgery. In recent years, with the accelerated pace of life, increased work pressure, obesity and population aging, the number of patients with androgen deficiency has increased year by year, and has become a refractory disease that seriously threatens men's health and quality of life worldwide. disease. At present, there is no long-term stable effective measure for the treatment of androgen deficiency clinically. The more commonly used method is exogenous testosterone supplementation therapy, but testosterone supplementation therapy requires long-term application, and cannot meet the physiological requirements of rhythmic testosterone secreti...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/071
Inventor 王晓云周广东邢新
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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