A kind of human in vitro semen and preparation method thereof
A technology for semen and human body, applied in biochemical equipment and methods, embryonic cells, cell culture active agents, etc., can solve problems such as high prices, product performance decline, and patients' inability to achieve fertilization effects
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Embodiment 1
[0018] [Example 1] In vitro sperm formulation and preparation method:
[0019] A. In vitro semen formula:
[0020]
[0021] B. Preparation method:
[0022] a. Weigh all the solid components according to the formula and place them separately;
[0023] b. According to the principle of adding solid first, then liquid, and adding sodium bicarbonate last, add and dissolve all ingredients except gentamicin sulfate, phenol red, sodium bicarbonate and human serum albumin in ultrapure water;
[0024] c. Add gentamicin sulfate and phenol red to the solution in step b, and finally add sodium bicarbonate and dissolve to obtain a base solution;
[0025] d. Put the above base solution into an incubator with 5% carbon dioxide, 95% air and saturation temperature to balance for 7-12 hours;
[0026] e. Measure and adjust the pH value of the above-mentioned base solution at 7.3±0.1, and the osmotic pressure at 260-290mOsm / kg, and make a record;
[0027] f. For use in the isolator. Filter ...
Embodiment 2
[0028] [Example 2] Quality control detection of semen in vitro
[0029] 1. pH value detection
[0030] Take an appropriate amount of the sample to be tested and measure it three times with a pH meter, and take the average value of the three data obtained as the result, and it is considered qualified if it is 7.3±0.1.
[0031] Osmotic pressure test
[0032] After the osmometer is calibrated, take 500μl of the sample to be tested and put it into the test tube to start the test. When the value is stable, read the data, measure 3 data according to the above method, take the average as the result, and it is considered qualified if it is 260-290mOsm / kg .
[0033] Bacterial endotoxin detection
[0034] According to the requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China, the test is carried out by the limulus reagent gel method, and the result is ≤0.1EU / ml, which is considered qualified.
[0035] Cytotoxicity
[0036] Tested according to the p...
Embodiment 3
[0061] [Example 3] mouse embryology experiment
[0062] Experimental steps of in vitro fertilization in mice
[0063] 1. Experimental materials
[0064] (1) Mice: female C57, 4-5 weeks old, male C57, 10 weeks old. 12 light, temperature 20-25 ℃, free access to food and water;
[0065] (2) Consumables and equipment: 1ml syringe, ophthalmic scissors, ophthalmic forceps;
[0066] (3) Reagents: PMSG, hCG, HTF culture fluid, KSOM-AA embryo culture fluid, fertilization fluid of the experimental group.
[0067] 2. Petri dish preparation (prepared in the afternoon of the day before IVF and equilibrated in the incubator overnight)
[0068] (1) Fresh semen collection dish: 500μl HTF (35mm domestic), 500μl experimental group fertilized fluid (35mm domestic);
[0069] (2) Egg collection dish: 1ml M2 (35mm domestic) preheated to 37°C 1 hour in advance;
[0070] (3) In vitro fertilization dishes and petri dishes for cleaning: 60mm (HTF) dish--the upper part is used for fertilization drop...
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