CN112592888A - Sperm culture solution and preparation method thereof - Google Patents

Sperm culture solution and preparation method thereof Download PDF

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CN112592888A
CN112592888A CN202011626285.1A CN202011626285A CN112592888A CN 112592888 A CN112592888 A CN 112592888A CN 202011626285 A CN202011626285 A CN 202011626285A CN 112592888 A CN112592888 A CN 112592888A
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culture solution
sperm
mixture
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廖志杰
王远目
吴珏珏
刘丽娜
丁美露
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Shenzhen Yinglai Life Science Co ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Abstract

The invention relates to the technical field of assisted reproduction, in particular to a sperm culture solution and a preparation method thereof; the sperm culture solution is prepared by dissolving a basic culture solution and a compound modifier in a salt solution containing antibiotics and indicators at the concentration of 0.5-2 mmol/L and 150-300 mu mol/L respectively, wherein the compound modifier comprises succinate dehydrogenase, SOD, trehalose and free carnitine, and the molar ratio of the succinate dehydrogenase to the SOD to the trehalose to the free carnitine is as follows: 1-5: 30-50: 120-240: 4000-10000. The invention aims to provide a sperm culture solution and a preparation method thereof, the sperm culture solution can provide a stable culture environment for sperms, reduce the damage of active oxygen to the sperms, improve the sperm motility and contribute to improving the fertilization rate.

Description

Sperm culture solution and preparation method thereof
Technical Field
The invention relates to the technical field of assisted reproduction, in particular to a sperm culture solution and a preparation method thereof.
Background
At present, the number of couples who are infertile and infertile is increasing due to the influence of various aspects such as environment, food and life pressure. It is reported that the number of infertility patients in China exceeds 4000 ten thousand, and the rate of infertility of couples of childbearing age in China rises from 3% before 20 years to about 15% in recent years. Male infertility is mainly concentrated on azoospermia, oligospermia, asthenospermia and infertility due to normal sperm count. At present, assisted reproductive therapy can be used for helping patients with infertility, good sperms and ova need to be collected for in vitro fertilization in the assisted reproductive therapy, and the vitality of the sperms determines whether normal insemination can be performed. The purpose of the sperm cell culture solution is mainly to provide nutrition for sperm survival and improve sperm motility.
The sperm can generate excessive active oxygen during the in vitro culture process, and the sperm is more sensitive to the active oxygen and is more easily damaged by the active oxygen due to the specific cell structure of the sperm. The active oxygen is mainly harmful to destroying the sperm cell morphology and influencing the acrosome reaction in the fertilization process.
Therefore, a sperm culture solution and a preparation method thereof are provided.
Disclosure of Invention
The invention aims to provide a sperm culture solution and a preparation method thereof, the sperm culture solution can provide a stable culture environment for sperms, reduce the damage of active oxygen to the sperms, improve the sperm motility and contribute to improving the fertilization rate.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a sperm culture solution is prepared by dissolving a basic culture solution and a compound modifier in a salt solution containing antibiotics and indicators at the concentration of 0.5-2 mmol/L and 150-300 mu mol/L respectively, wherein the compound modifier comprises succinate dehydrogenase, SOD, trehalose and free carnitine, and the molar ratio of the succinate dehydrogenase to the SOD to the trehalose to the free carnitine is as follows: 1-5: 30-50: 120-240: 4000-10000.
Specifically, the basic culture solution comprises glutamic acid, cysteine, glycine, taurine, PUFA and a chelating agent, wherein the molar ratio of the glutamic acid to the cysteine to the glycine to the taurine to the PUFA to the chelating agent is as follows: 3-5: 0.5-1: 0.2-0.4: 4.5-6: 0.6-1.2: 0.08-0.16.
Specifically, the chelating agent is ethylenediamine tetraacetic acid, and the concentration of the ethylenediamine tetraacetic acid is 5-8 mug/mL.
Specifically, the antibiotic is penicillin, and the concentration of the penicillin is 6-12 mug/mL.
Specifically, the indicator is phenol red, and the concentration of the phenol red is 8-11 mug/mL.
Specifically, the salt solution comprises sodium chloride, potassium chloride, sodium bicarbonate, sodium pyruvate, trisodium citrate and glucose, wherein the molar ratio of the sodium chloride to the potassium chloride to the sodium bicarbonate to the sodium pyruvate to the trisodium citrate to the glucose is as follows: 100.5-120.6: 3.8-6.2: 10.9-12.4: 5.8-8.7: 20.1-23.5: 0.2 to 0.6.
A preparation method of a sperm culture solution comprises the following steps:
s1: weighing corresponding raw material components according to a ratio for later use;
s2: adding the preparation raw materials of the salt solution into an aqueous medium by taking medical-grade pure water as a medium, and uniformly mixing to obtain a mixture A;
s3: adding the preparation raw materials of the basic culture solution, the antibiotics and the indicator into the mixture A according to the corresponding proportion to obtain a mixture B;
s4: adding the composite modifier and the composite modifier into the mixture B according to a corresponding proportion to obtain a mixture C;
s5: performing sterile vacuum filtration on the mixture C, and introducing 2-4% by volume of N2And (5) gas is filled for 10-20 min, and sealed packaging is carried out to obtain the product.
Specifically, the temperature in the preparation method is 18-26 ℃, and the humidity is 40-60%.
Specifically, the filter membrane subjected to sterile vacuum filtration in the step S5 is a 0.2 μm filter membrane.
The invention has the beneficial effects that:
(1) according to the invention, succinate dehydrogenase, SOD, trehalose and free carnitine in a specific ratio are used as a composite modifier, the composite modifier is an antioxidant defense system, and the synergistic effect of the succinate dehydrogenase, the SOD, the trehalose and the free carnitine can effectively inactivate superoxide anion, active oxygen and H on the one hand2O2And the like, further protect cells from oxidative damage of free radicals, effectively reduce sperm lipid peroxidation, and prevent the generation of the sperm lipid peroxidationWhile preserving the integrity of the cell wall; on the other hand, can promote H2O2Decompose into oxygen molecules and water, thereby protecting cells from H2O2The damage of active oxygen to the sperms is reduced, and the sperm motility can be improved;
(2) the invention adopts the ethylenediamine tetraacetic acid as the chelating agent, can effectively chelate some unnecessary heavy metal ions, reduces the toxicity of the sperm culture solution, and further greatly improves the fertilization rate;
(3) the preparation process is simple and has strong practicability.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A sperm culture solution is prepared by dissolving a basic culture solution and a compound modifier in a salt solution containing antibiotics and indicators at the concentration of 2mmol/L and 300 mu mol/L respectively.
Specifically, the composite modifier comprises succinate dehydrogenase, SOD, trehalose and free carnitine, wherein the molar ratio of the succinate dehydrogenase to the SOD to the trehalose to the free carnitine is as follows: 5: 50: 240: 10000; the basic culture solution comprises glutamic acid, cysteine, glycine, taurine, PUFA and a chelating agent, wherein the molar ratio of the glutamic acid to the cysteine to the glycine to the taurine to the PUFA to the chelating agent is as follows: 5:1:0.4:6: 1.2: 0.16; the chelating agent is ethylenediamine tetraacetic acid, and the concentration of the ethylenediamine tetraacetic acid is 8 mug/mL; the antibiotic is penicillin, and the concentration of the penicillin is 12 mug/mL; the indicator is phenol red, and the concentration of the phenol red is 8-11 mug/mL; the salt solution comprises sodium chloride, potassium chloride, sodium bicarbonate, sodium pyruvate, trisodium citrate and glucose, wherein the molar ratio of the sodium chloride to the potassium chloride to the sodium bicarbonate to the sodium pyruvate to the trisodium citrate to the glucose is as follows: 120.6:6.2: 12.4: 8.7:23.5: 0.6.
a preparation method of a sperm culture solution comprises the following steps:
s1: weighing corresponding raw material components according to a ratio for later use;
s2: adding the preparation raw materials of the salt solution into an aqueous medium by taking medical-grade pure water as a medium, and uniformly mixing to obtain a mixture A;
s3: adding the preparation raw materials of the basic culture solution, the antibiotics and the indicator into the mixture A according to the corresponding proportion to obtain a mixture B;
s4: adding the composite modifier and the composite modifier into the mixture B according to a corresponding proportion to obtain a mixture C;
s5: performing sterile vacuum filtration on the mixture C, and introducing 2-4% by volume of N2And (5) gas is filled for 10-20 min, and sealed packaging is carried out to obtain the product.
Specifically, the temperature in the preparation method is 18-26 ℃, and the humidity is 40-60%.
Specifically, the filter membrane subjected to sterile vacuum filtration in the step S5 is a 0.2 μm filter membrane.
Example 2
The sperm culture solution of this embodiment is prepared by dissolving a basal culture solution and a complex modifier in a salt solution containing antibiotics and indicators at concentrations of 0.5mmol/L and 150 μmol/L, respectively, wherein the complex modifier comprises succinate dehydrogenase, SOD, trehalose and free carnitine, and the molar ratio of succinate dehydrogenase, SOD, trehalose and free carnitine is: 1: 30: 120: 4000.
specifically, the basic culture solution comprises glutamic acid, cysteine, glycine, taurine, PUFA and a chelating agent, wherein the molar ratio of the glutamic acid to the cysteine to the glycine to the taurine to the PUFA to the chelating agent is as follows: 3:0.5:0.2:4.5: 0.6: 0.08; the antibiotic is penicillin, and the concentration of the penicillin is 6 mug/mL; the indicator is phenol red, and the concentration of the phenol red is 8 mug/mL; the chelating agent is ethylenediamine tetraacetic acid, and the concentration of the ethylenediamine tetraacetic acid is 5 mug/mL; the salt solution comprises sodium chloride, potassium chloride, sodium bicarbonate, sodium pyruvate, trisodium citrate and glucose, wherein the molar ratio of the sodium chloride to the potassium chloride to the sodium bicarbonate to the sodium pyruvate to the trisodium citrate to the glucose is as follows: 100.5:3.8: 10.9: 5.8:20.1: 0.2.
example 3
The sperm culture solution of this embodiment is prepared by dissolving a basal culture solution and a complex modifier in a salt solution containing antibiotics and indicators at concentrations of 1.3mmol/L and 250 μmol/L, respectively, wherein the complex modifier comprises succinate dehydrogenase, SOD, trehalose and free carnitine, and the molar ratio of succinate dehydrogenase, SOD, trehalose and free carnitine is: 3: 40: 200: 7800.
specifically, the basic culture solution comprises glutamic acid, cysteine, glycine, taurine, PUFA and a chelating agent, wherein the molar ratio of the glutamic acid to the cysteine to the glycine to the taurine to the PUFA to the chelating agent is as follows: 4:0.8:0.3:5: 0.9: 0.11; the antibiotic is penicillin, and the concentration of the penicillin is 10 mug/mL; the indicator is phenol red, and the concentration of the phenol red is 9 mug/mL; the chelating agent is ethylenediamine tetraacetic acid, and the concentration of the ethylenediamine tetraacetic acid is 7 mug/mL; the salt solution comprises sodium chloride, potassium chloride, sodium bicarbonate, sodium pyruvate, trisodium citrate and glucose, wherein the molar ratio of the sodium chloride to the potassium chloride to the sodium bicarbonate to the sodium pyruvate to the trisodium citrate to the glucose is as follows: 118.5:5.4: 11.9: 6.3:22.7: 0.4.
comparative example 1
A sperm culture solution of this comparative example was prepared by dissolving the basal medium and the complex modifier in a salt solution containing an antibiotic and an indicator at concentrations of 0.4mmol/L and 120. mu. mol/L, respectively, and was prepared in the same manner as in example 1, and no further description is given herein.
Comparative example 2
A sperm culture solution of this comparative example was prepared by dissolving the basal medium and the complex modifier in a salt solution containing an antibiotic and an indicator at concentrations of 2.5mmol/L and 320. mu. mol/L, respectively, and was prepared in the same manner as in example 1, and no further description is given herein.
Comparative example 3
The preparation method of a sperm cell culture solution in this comparative example was the same as that of example 1, and no further description is given herein, except that the complex modifier was not incorporated in this comparative example.
Comparative example 4
The process for preparing a sperm culture solution of this comparative example was the same as that of example 1 and will not be described herein again, except that a sperm culture solution of this comparative example was prepared in which the molar ratio of succinate dehydrogenase, SOD, trehalose and free carnitine was: 0.5: 20: 100: 3800.
comparative example 5
A sperm culture solution of this comparative example was prepared in the same manner as in example 1, and no further description is given herein, except that a sperm culture solution of this comparative example was prepared in which the molar ratio of succinate dehydrogenase, SOD, trehalose and free carnitine was 6: 55: 250: 10040.
comparative example 6
One of the sperm culture solutions in this comparative example was a sperm culture solution commercially available from Kuck (China) medical trade company, Inc., and the type of the sperm culture solution was K-SISM-100.
Performing physical, chemical and biological index detection on the sperm culture solutions of the examples 1-3 and the comparative examples 1-6, and sequentially performing pH value detection, osmotic pressure detection and bacterial endotoxin detection, wherein the pH value detection comprises the following steps: taking a certain amount of sperm culture solution, and detecting by using a blood gas analyzer; osmotic pressure, 20 μ L of liquid is dropped into a 0.5ml EP tube and put into a probe of an osmometer (freezing point osmometer) for detection; and (3) detecting bacterial endotoxin, namely detecting by adopting a limulus reagent gel method in the detection method of endotoxin in Chinese pharmacopoeia, and judging that the endotoxin is qualified when the endotoxin is less than 1 EU/mL.
The results of the measurements are shown in Table 1 below.
TABLE 1 results of physicochemical and biological index measurements of culture solutions of examples 1 to 3 and comparative examples 1 to 6
Numbering PH value (Room temperature) Osmotic pressure (mOsm/Kg) Botulinum toxin (LAL)
Example 1 7.2～7.4 275～285 ＜0.15EU/m
Example 2 7.2～7.4 275～285 ＜0.15EU/m
Example 3 7.2～7.4 275～285 ＜0.15EU/m
Comparative example 1 7.3～7.5 280～295 ＜0.5EU/m
Comparative example 2 7.3～7.5 280～295 ＜0.5EU/m
Comparative example 3 7.3～7.5 280～295 ＜0.5EU/m
Comparative example 4 7.2～7.4 275～285 ＜0.3EU/m
Comparative example 5 7.2～7.4 275～285 ＜0.3EU/m
Comparative example 6 7.0～7.5 270～285 ＜0.8EU/m
The culture solutions of examples 1 to 3 and comparative examples 1 to 6 were tested for the effect of culturing sperm:
1. sperm preparation, when fresh semen is adopted in the test, the sperm concentration and the motility are checked after the semen is liquefied, the concentration should be more than 200 ten thousand/ml, and the motility should be more than 50%, and the checking method is as follows: taking 1 microliter of semen, placing the semen in a warm (30-35 ℃) Makler counter, covering a cover glass, and inspecting under a microscope with a 20-time objective lens and a 10-time eyepiece; there are 100 squares in the Makler counter, each counting 10 squares; counting motile and non-motile sperm separately; after the counter is checked, the sperm concentration and the sperm motility are calculated. (sperm concentration is the average number of motile and non-motile sperm in the counter multiplied by one million/ml sperm motility is the percentage of motile sperm divided by the average number of sperm)
When frozen semen was used for the test, the thawing method was as follows: preparing water at 35 ℃, adding about 100 ml of heated sterile water (35 ℃) into a 200 ml beaker, taking 1 tube of semen sample (1 ml per tube) from a liquid nitrogen storage tank, and putting the semen sample into the water at 35 ℃ for 10-20 minutes; after thawing, sperm concentration and motility were checked in the same manner as described above. The concentration should be greater than 20 ten thousand/ml and the sperm motility should be greater than 40%.
2. Test method
Adding the semen (fresh or unfrozen) into a centrifugal tube, sorting the semen from the semen by adopting a density gradient centrifugation method, placing Percoll separating medium (45% -90% of each 2ml, 90% of each lower part and 45% of each upper part) with a density gradient of 45% -90% (v/v) into a 10ml centrifugal tube, slowly adding 1ml of the semen on the gradient liquid, centrifuging for 20 minutes at 200g, discarding the supernatant, re-suspending the semen by using spermrinse, centrifuging for 10 minutes at 100g, discarding the supernatant, and re-suspending the sperm by using spermrinse to obtain a sperm suspension; selection of 5X 10 in sperm suspension7Adding sperm of about a certain number into the above sperm culture solution and commercial HTF-S culture solution, and standing at 35 deg.C and 3% N2Was cultured in an incubator for 2 hours, and then a fertilized oocyst embryogenesis test was performed, the test results of which are shown in table 2 below.
TABLE 2 results of sperm motility, fertilization rate and blastocyst formation rate
Numbering Sperm motility (%) Fertilization Rate (%) Blastocyst formation Rate (%)
Example 1 98.32 98.27 ≥95
Example 2 96.05 97.36 ≥90
Example 3 97.33 97.95 ≥93
Comparative example 1 91.76 93.42 ≥88
Comparative example 2 90.55 92.73 ≥85
Comparative example 3 78.34 83.39 ≥80
Comparative example 4 92.35 91.82 ≥87
Comparative example 5 91.18 92.85 ≥86
Comparative example 6 85.55 88.25 ≥82
The above results show that the components of the sperm culture solution of the present invention are matched with each other and complement each other. The change or the lack of any raw material in the formula of the sperm culture solution can influence the culture effect of the sperm culture solution on the sperm, and reduce the sperm motility and the sperm quality.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (9)

1. A sperm culture solution is characterized in that the sperm culture solution is prepared by dissolving a basic culture solution and a compound modifier in a salt solution containing antibiotics and indicators at the concentration of 0.5-2 mmol/L and 150-300 mu mol/L respectively, wherein the compound modifier comprises succinate dehydrogenase, SOD, trehalose and free carnitine, and the molar ratio of the succinate dehydrogenase to the SOD to the trehalose to the free carnitine is as follows: 1-5: 30-50: 120-240: 4000-10000.
2. A sperm cell culture solution according to claim 1, wherein the basal medium comprises glutamic acid, cysteine, glycine, taurine, PUFA and a chelating agent in a molar ratio of: 3-5: 0.5-1: 0.2-0.4: 4.5-6: 0.6-1.2: 0.08-0.16.
3. A sperm cell culture solution according to claim 2, wherein said chelating agent is ethylenediaminetetraacetic acid, wherein the concentration of ethylenediaminetetraacetic acid is 5-8 μ g/mL.
4. A sperm cell culture solution according to claim 1, wherein said antibiotic is penicillin, and the concentration of said penicillin is 6-12 μ g/mL.
5. A sperm cell culture solution according to claim 1, wherein said indicator is phenol red, said phenol red being present in a concentration of 8-11 μ g/mL.
6. A sperm cell broth according to claim 1 wherein said saline solution comprises sodium chloride, potassium chloride, sodium bicarbonate, sodium pyruvate, trisodium citrate and glucose in a molar ratio of sodium chloride, potassium chloride, sodium bicarbonate, sodium pyruvate, trisodium citrate and glucose: 100.5-120.6: 3.8-6.2: 10.9-12.4: 5.8-8.7: 20.1-23.5: 0.2 to 0.6.
7. A process for the preparation of a sperm cell culture solution according to any one of claims 1 to 6, comprising the steps of:
s1: weighing corresponding raw material components according to a ratio for later use;
s2: adding the preparation raw materials of the salt solution into an aqueous medium by taking medical-grade pure water as a medium, and uniformly mixing to obtain a mixture A;
s3: adding the preparation raw materials of the basic culture solution, the antibiotics and the indicator into the mixture A according to the corresponding proportion to obtain a mixture B;
s4: adding the composite modifier and the composite modifier into the mixture B according to a corresponding proportion to obtain a mixture C;
s5: performing sterile vacuum filtration on the mixture C, and introducing 2-4% by volume of N2And (5) gas is filled for 10-20 min, and sealed packaging is carried out to obtain the product.
8. A process according to claim 7, wherein the temperature is 18-26 ℃ and the humidity is 40-60%.
9. The method of claim 7, wherein the membrane filter used in the step S5 is a 0.2 μm membrane filter.
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