Testicular tissue cryopreservation method based on single seminiferous tubule perfusion

A cryopreservation method and spermatogenesis technology, which is applied in the field of testicular tissue cryopreservation, can solve the problems of increasing osmotic damage of testicular tissue/cells, and it is difficult for liquid to reach the required concentration, so as to reduce the level of oxidative stress and chemical toxicity damage , the effect of reducing death

Active Publication Date: 2020-11-17
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The seminiferous tubule has a relatively closed lumen, and it is difficult to make the liquid in the lumen reach the required concentration by directly immersing the cryoprotectant. In addition, if the loading time of the cryoprotectant is prolonged, the amount of the cryoprotectant will increase. Penetrating damage to testicular tissue/cells

Method used

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  • Testicular tissue cryopreservation method based on single seminiferous tubule perfusion
  • Testicular tissue cryopreservation method based on single seminiferous tubule perfusion
  • Testicular tissue cryopreservation method based on single seminiferous tubule perfusion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 testicular tissue cryopreservation reagent combination

[0042] Including: cryoprotectant 1: composed of 10% DMSO, 10% FBS and 0.1M sucrose; and cryoprotectant 2: composed of 20% DMSO, 20% FBS and 0.5M sucrose; and resuscitation solution 1: composed of 10 Composed of % FBS and 1M sucrose; and Resuscitation Solution 2: composed of 10% FBS, 0.5M sucrose and DF12.

Embodiment 2

[0043]Example 2 Perfusion of cryoprotectant for single seminiferous tubules

[0044] The testicular tissue obtained from the microsperm extraction of patients with non-obstructive azoospermia who will be treated in Shanghai First People's Hospital is immediately placed in at least 20 times the volume of HTF solution and transported to the laboratory. During this period, 10ul cryoprotectant 2 was sucked into the fine glass needle for ICSI and connected to the microfluidic pump. After the sample is transported to the laboratory, put the sample into a small dish filled with HTF solution, use micro scissors and micro tweezers to gently separate the direct interstitium of the seminiferous tubules under the stereoscope, making it a single seminiferous tubule. seminiferous tubules. Use micro scissors to cut the seminiferous tubules longer than 1 cm to 1 cm. The seminiferous tubules were transferred to a small dish containing cryoprotectant 1, and soaked on a shaker for 10 minutes. ...

Embodiment 3

[0045] Embodiment 3 Cryopreservation and recovery of a single seminiferous tubule

[0046] Take a single seminiferous tubule out of the cryoprotectant 2, gently blot dry the cryoprotectant on the surface with absorbent paper, then place it on a Cryo-piece single sperm cryopreservation thin slice, and insert it into the Cryo-tubule Directly immerse in liquid nitrogen for rapid cooling. After the temperature stabilizes, move the frozen samples to the corresponding storage location in liquid nitrogen. Before resuscitation, heat the resuscitation solution to 37°C, take out the seminiferous tubules from liquid nitrogen, soak them in resuscitation solution 1 (10% FBS + 1M sucrose) for 1 min, and then place them in resuscitation solution 2 (10% FBS + 1M sucrose). Soak in 0.5M sucrose+DF123min) for 3min, and finally transfer to appropriate culture conditions (such as DF12 medium with 10% FBS).

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Abstract

The invention relates to a testicular tissue cryopreservation method based on single seminiferous tubule perfusion, which comprises the following steps: making testicular tissue into the single seminiferous tubule, soaking a single seminiferous tubule in a cryopreservation protective agent 1 for 10 minutes, and soaking and perfusing the seminiferous tubule by using a cryopreservation protective agent 2, so as to maximally reduce the permeation damage of the protective agent while achieve vitrification cooling, quickly transferring the poured spermatogenic tubule to a Cryo-genic single sperm cryopreservation slice, after sleeving of the Cryo-genic tubule, directly conducting immersing in liquid nitrogen to quickly achieve cooling, and quickly conducting soaking in a recovery solution aftercooling. Compared with a traditional testicular cell suspension cryopreservation method, the method disclosed by the invention has the advantages that (1) the complete structure of the seminiferous tubule is reserved; (2) the oxidative stress level of testicular cells is effectively reduced; (3) the death of a large number of spermatogonial cells and supportive cells after cryopreservation is effectively reduced; and (4) the recovery rate and activity of sperms in the spermatogenic tubules are high.

Description

technical field [0001] The invention relates to the technical field of cryopreservation of ball tissue, specifically, a method for cryopreservation of testicular tissue based on perfusion of a single seminiferous tubule, that is, a very small amount of testicular tissue is preserved by vitrification after perfusion of a cryopreservation solution in the seminiferous tubule. testicular tissue. Background technique [0002] At present, the fertility of people of childbearing age in the world is showing an overall downward trend. The WHO Human Reproduction Special Program reports that the infertility rate of the global population is at least 15%, and there are about 60-80 million infertile couples in the world. Cardiovascular disease is one of the three major diseases affecting human health today. According to the "Investigation Report on China's Infertility Status" published by the Population Association, the infertility rate in my country has reached 15%-20%, and there are ab...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 赵亮宇韩厦李铮洪艳李朋田汝辉
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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