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
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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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