CN111607559A - Sperm separating liquid and preparation method thereof - Google Patents
Sperm separating liquid and preparation method thereof Download PDFInfo
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- CN111607559A CN111607559A CN202010472919.6A CN202010472919A CN111607559A CN 111607559 A CN111607559 A CN 111607559A CN 202010472919 A CN202010472919 A CN 202010472919A CN 111607559 A CN111607559 A CN 111607559A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention provides a sperm separating medium which is characterized by comprising the following components: 13. + -. 1mM NaHCO3，0.36±0.02mM KH2PO4，0.32±0.02mM C3H3O3Na，17±1mM C3H5O3Na，0.5±0.1mM C6H12O620 + -0.5 mM HEPES, 0.1 + -0.05 mM glutathione, 0.51 + -0.06 mM vitamin C, 0.21 + -0.02 mM vitamin E, 0.07 + -0.02 mM β -carotene, 0.82 + -0.09 mM catechol, 100 + -5 mM NaCl, 4.6 + -0.2 mM KCl, 0.2 + -0.01 mM MgSO4·7H2O and 2.0. + -. 0.1mM CaCl2·2H2O, 5mg/L gentamicin, 5g/L human serum albumin, and 80% by volume or 40% by volume of a silane-coated colloidal silica solution, dissolved in water for injection. According to the invention, by adding antioxidants such as glutathione, vitamin C and vitamin E into the sperm separation solution, the phenomenon that oxidation stress reaction is generated on the sperm by oxidation substances in the semen and the separation solution can be avoided, and the activity of the sperm is ensured.
Description
Technical Field
The invention relates to the field of sperm separation, in particular to a sperm separating medium and a preparation method thereof.
Background
The most common cause of infertile men with normal semen index is abnormal sperm DNA, and the fertility depends not only on the absolute number of sperm, but also on sperm function. The motility of sperm is a reliable indicator of sperm fertilisation, and in Assisted Reproduction (ART), the number of motile sperm separable in the semen directly affects the treatment regime for infertility. The separation operation process and the separation reagent can cause the oxidation damage of the sperms, and the oxidation damage influences the movement capability and the function of the sperms and further influences the outcome of the subsequent assisted reproduction. Sperm density gradient separation is a commonly used assisted genital extracorporeal operation technology, and can effectively remove leukocytes, epithelial cells, dead sperm, malformed or immature sperm and cell debris in semen, but can also cause oxidative damage to the sperm.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides the sperm separating medium with clinical application prospect, and ensures the vitality and DNA integrity of the sperm.
In order to solve the technical problems, the invention adopts the technical scheme that: a sperm cell separation solution comprising the following components: 13. + -. 1mM NaHCO3，0.36±0.02mM KH2PO4，0.32±0.02mM C3H3O3Na，17±1mM C3H5O3Na，0.5±0.1mM C6H12O620. + -. 0.5mM HEPES, 0.1. + -. 0.05mM glutathione, 0.51. + -. 0.06mM vitamin C, 0.21. + -. 0.02mM vitamin E, 0.07. + -. 0.02mM β -carotene, 0.82. + -. 0.09mM catechol, dissolved in water for injection.
Further, the sperm separating solution also comprises 100 mM NaCl, 4.6 mM KCl, 0.2mM MgSO 0.01mM4·7H2O and 2.0. + -. 0.1mM CaCl2·2H2O。
Further, the sperm separating solution also comprises 5mg/L gentamicin, 5g/L human serum albumin and colloidal silicon dioxide solution which is wrapped by 80% by volume or 40% by volume of silane.
The invention adopts another technical scheme that: a method for preparing a sperm separating solution, comprising the steps of:
s1, weighing 100 + -5 mM NaCl, 4.6 + -0.2 mM KCl and 0.2 + -0.01 mM MgSO4·7H2O and 2.0. + -. 0.1mM CaCl2·2H2O，13±1mM NaHCO3，0.36±0.02mM KH2PO4，0.32±0.02mM C3H3O3Na，0.5±0.1mM C6H12O620 +/-0.5 mM HEPES dissolved in 150ml of water for injection and filtered through a 0.22 mu m filter membrane;
s2, adding 0.1 +/-0.05 mM glutathione, 0.51 +/-0.06 mM vitamin C, 0.21 +/-0.02 mM vitamin E, 0.07 +/-0.02 mM beta-carotene and 0.82 +/-0.09 mM catechol into the mixed solution obtained in the S1, and uniformly stirring;
s3, adding 17. + -. 1mM C to the mixture obtained in S23H5O3Na, 5mg/L gentamicin and 5g/L human serum albumin, and the volume is adjusted to 200mL by using water for injection after the materials are uniformly stirred.
S4, equally dividing the mixed solution obtained in the S3 into two parts, adding 400mL of silane-coated colloidal silica solution into one part, fixing the volume to 500mL, and preparing into 80% v/v sperm separating solution; 200mL of silane-coated colloidal silica solution was added to the other portion, and water for injection was added to make a volume of 500mL, thereby preparing a 40% v/v sperm separating solution.
Further, the osmotic pressure of the solution prepared in S4 is 280-320mOsm/Kg, and the pH value is 7.0-7.5.
Further, the sperm separating medium is used for separating human sperm.
Compared with the prior art, the invention has the beneficial effects that: by adding antioxidants such as glutathione, vitamin C, vitamin E and the like into the sperm separation solution, the oxidative damage to the sperm in the separation operation process can be avoided, and the activity and DNA integrity of the sperm are ensured.
Detailed Description
It is easily understood that according to the technical solution of the present invention, a person skilled in the art can propose various alternative structures and implementation ways without changing the spirit of the present invention. Therefore, the following detailed description is merely illustrative of the technical solutions of the present invention, and should not be construed as being all of the present invention or limiting or restricting the technical solutions of the present invention.
Glutathione itself is easily oxidized by some substances, so that sulfhydryl groups in molecules such as a plurality of proteins and enzymes can be protected from being oxidized by harmful substances, thereby ensuring the normal exertion of molecular physiological functions such as the proteins and the enzymes.
The vitamin C has stronger reducibility, so that the vitamin C is strived to have oxidation-reduction reaction with oxygen in the air and is added into the sperm separation liquid, thereby effectively reducing the influence of oxides on the sperm and ensuring the sperm motility.
Vitamin E is a fat-soluble vitamin whose hydrolysate is tocopherol, one of the most important antioxidants, which promotes sex hormone secretion and increases sperm motility and number in men.
The antioxidant properties of beta-carotene are mainly manifested by its ability to scavenge free radicals. The beta-carotene molecule contains a plurality of double bonds, and is easy to be oxidized in the presence of light, heat, oxygen and free radical ions with strong activity, thereby protecting the organism from being damaged. Lipid peroxidation and free radical reaction exist in organisms in large quantities, so that the functions of cells are reduced, the bodies are aged and diseases occur, and the lipid peroxidation can be reduced by the existence of beta-carotene.
The following description will be made with reference to specific examples, based on 10L of sperm cell separation solution:
example 1
55.52g NaCl, 3.28g KCl, 0.47g MgSO were weighed4·7H2O and 2.79g CaCl2·2H2O，10.08gNaHCO3，0.46g KH2PO4，0.33g C3H3O3Na，0.72g C6H12O646.47g HEPES dissolved in 1.5L of water for injection and filtered through a 0.22 μm filter membrane, 0.15g glutathione, 0.9g vitamin C, 0.9g vitamin E, 0.9g β -carotene and 0.9g catechol were added to the above mixture and stirred well, and 17.93g C g HEPES was added to the resulting mixture3H5O3Na is uniformly stirred and then the volume is determined to be 2L by using water for injection; equally dividing the obtained solution into two parts, adding 4L of silane-coated colloidal silica solution into one part, metering the volume to 5L, and preparing a sperm separating solution with the volume of 80% v/v; and adding 2L of silane-coated colloidal silica solution into the other part, adding injection water to the volume of 5L, and preparing the sperm separating solution with the volume of 40% v/v.
Example 2
58.44g NaCl, 3.43g KCl, 0.49g MgSO were weighed out4·7H2O and 2.94g CaCl2·2H2O，10.92gNaHCO3，0.48g KH2PO4，0.35g C3H3O3Na，0.9g C6H12O647.66g HEPES dissolved in 1.5L water for injection and filtered through 0.22 μm filter membrane, 0.31g glutathione, 1g vitamin C, 1g vitamin E, 1g β -carotene and 1g catechol were added to the mixture and stirred uniformly, and 19.05g C was added to the mixture3H5O3Na, 0.5g of gentamicin and 50g of human serum albumin, stirring uniformly, and then fixing the volume to 2L by using water for injection; equally dividing the obtained solution into two parts, adding 4L of silane-coated colloidal silica solution into one part, metering the volume to 5L, and preparing a sperm separating solution with the volume of 80% v/v; and adding 2L of silane-coated colloidal silica solution into the other part, adding injection water to the volume of 5L, and preparing the sperm separating solution with the volume of 40% v/v.
Example 3
61.36g NaCl, 3.58g KCl, 0.52g MgSO were weighed out4·7H2O and 3.09g CaCl2·2H2O，11.76gNaHCO3，0.52g KH2PO4，0.37g C3H3O3Na，1.08g C6H12O648.85g HEPES dissolved in 1.5L water for injection and filtered through a 0.22 μm filter membrane, 0.46g glutathione, 1.1g vitamin C, 1.1g vitamin E, 1.1g β -carotene and 1.1g catechol were added to the mixture and stirred well, and 20.17g 20.17g C was added to the mixture3H5O3Na is stirred uniformly and then the volume is determined to be 2L by using water for injection; equally dividing the obtained solution into two parts, adding 4L of silane-coated colloidal silica solution into one part, metering the volume to 5L, and preparing a sperm separating solution with the volume of 80% v/v; and adding 2L of silane-coated colloidal silica solution into the other part, adding injection water to the volume of 5L, and preparing the sperm separating solution with the volume of 40% v/v.
Filtering the separated liquid obtained in the above embodiment with 0.22 μm filter membrane again, taking a certain sample for test experiment, repeating the operation three times at room temperature of 30 deg.C, extracting 20mL each time for measurement, wherein the pH value is 7.30, 7.42, 7.28, the average value is 7.33, and is in the range of 7.2-7.4; the osmotic pressure was 290mOms/Kg, 285mOms/Kg, 295mOms/Kg, with the three-time average value of 290 mOms/Kg.
In vitro cytotoxicity assay
In vitro cytotoxicity test was performed according to GB/T16886.5, 1mL of sperm isolate was aspirated, injected into an equal amount of cell suspension, the dish was gently rotated to distribute cells evenly on the dish surface, 1mL of each of ZDBC and ZDEC was aspirated as negative and positive controls, added to the other dishes, and the dishes were placed in a carbon dioxide incubator until colonies appeared. And observing the colony morphology under a microscope, observing that 10 percent of cells are loose and attached to the wall, evaluating that the reaction degree is slight, the grade is 1, and the toxicity test of the density separating solution is qualified.
Skin sensitization detection
Sensitization test was performed according to GB/T16886.10, 3 mice were harvested, the hair on both sides of the spine of the mice was removed, 0.5mL of sperm isolation solution was dropped onto each piece of gauze, the gauze was applied to the site of the mice where the hair was removed, the dressing was removed after 4 hours of application, the contact site was marked with permanent ink, the residual test material was removed, and the contact area was washed with warm water. The contact sites were recorded (1. + -. 0.1), 24. + -. 2, 48. + -. 2, 72. + -.2) h after removal of the dressing. The contact parts have no erythema and edema, the average mark is 0.3, the reaction type is extremely light, and the skin sensitization of the sperm separating solution is qualified.
Bacterial endotoxin detection
The detection is carried out by adopting a gel method, the sensitivity of the limulus reagent is 0.25EU/mL, the test is divided into four groups, the four groups are respectively, water for endotoxin detection is used as a negative control, an endotoxin standard substance is used as a positive control, an endotoxin standard substance and a sperm separating medium are used as a sample positive control group, the sperm separating medium is used as a sample group, each group is repeated for two times, after the preparation is finished, the test tube is placed into a (37 +/-1) DEG C test tube thermostat for reaction for 1h, then the test tube is inverted for observation, the result is that the negative control and the sample are both negative, and the positive control and the sample positive control are. The content of endotoxin in the sperm separating medium is qualified.
Sperm motility test
3 parts of semen are prepared and liquefied at 37 ℃. The sperm-separated solution was added to the test tube, and the lower layer was 1mL of 80% v/v separated solution and the upper layer was 1mL of 40% v/v separated solution. After the semen is liquefied, 1mL of semen is carefully added above the separation solution, 400g of the solution is centrifuged for 20min at 300-. The pellet was resuspended in 2mL of 37 deg.C pre-warmed medium and centrifuged at 200g for 5 min. The supernatant was removed, 2mL of the culture was added to resuspend the pellet, and 200g was centrifuged for 5 min. 1mL of a culture solution preheated at 37 ℃ was added thereto, and the mixture was cultured at 37 ℃ for 24 hours. The sperm quality analyzer detects the sperm motility, the sperm motility is respectively 86.4%, 89.4% and 88.3%, the average value is 88.03%, and the standard is far over 75%.
The technical scope of the present invention is not limited to the above description, and those skilled in the art can make various changes and modifications to the above-described embodiments without departing from the technical spirit of the present invention, and such changes and modifications should fall within the protective scope of the present invention.
Claims (6)
1. A sperm cell separation solution comprising the following components: 13. + -. 1mM NaHCO3，0.36±0.02mMKH2PO4，0.32±0.02mM C3H3O3Na，17±1mM C3H5O3Na，0.5±0.1mM C6H12O620. + -. 0.5mM HEPES, 0.1. + -. 0.05mM glutathione, 0.51. + -. 0.06mM vitamin C, 0.21. + -. 0.02mM vitamin E, 0.07. + -. 0.02mM β -carotene, 0.82. + -. 0.09mM catechol, dissolved in water for injection.
2. The sperm separator of claim 1 further comprising 100 ± 5mM naci, 4.6 ± 0.2mM KCl, 0.2 ± 0.01mM MgSO 04·7H2O and 2.0. + -. 0.1mM CaCl2·2H2O。
3. The sperm cell separation solution of claim 2, further comprising 5mg/L gentamicin, 5g/L human serum albumin, and an 80% by volume or 40% by volume silane encapsulated colloidal silica solution.
4. A method for preparing a sperm separating solution, which is characterized by comprising the following steps:
s1, weighing 100 + -5 mM NaCl, 4.6 + -0.2 mM KCl and 0.2 + -0.01 mM MgSO4·7H2O and 2.0. + -. 0.1mM CaCl2·2H2O，13±1mM NaHCO3，0.36±0.02mM KH2PO4，0.32±0.02mM C3H3O3Na，0.5±0.1mM C6H12O620 +/-0.5 mM HEPES dissolved in 150mL of water for injection and filtered through a 0.22 mu m filter membrane;
s2, adding 0.1 +/-0.05 mM glutathione, 0.51 +/-0.06 mM vitamin C, 0.21 +/-0.02 mM vitamin E, 0.07 +/-0.02 mM beta-carotene and 0.82 +/-0.09 mM catechol into the mixed solution obtained in the S1, and uniformly stirring;
s3, adding 17. + -. 1mM C to the mixture obtained in S23H5O3Na, 5mg/L gentamicin and 5g/L human serum albumin, stirring uniformly, and then fixing the volume to 200mL by using water for injection;
s4, equally dividing the mixed solution obtained in the S3 into two parts, adding 400mL of silane-coated colloidal silica solution into one part, fixing the volume to 500mL, and preparing into 80% v/v sperm separating solution; 200mL of silane-coated colloidal silica solution was added to the other portion, and water for injection was added to make a volume of 500mL, thereby preparing a 40% v/v sperm separating solution.
5. The method of claim 4, wherein the solution prepared in S4 has an osmotic pressure of 280-320mOsm/Kg, a pH value of 7.0-7.5, and an endotoxin value of <0.25 EU/mL.
6. The sperm cell separation solution of claim 3, wherein said sperm cell separation solution is used to separate human sperm cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112391339A (en) * | 2020-11-13 | 2021-02-23 | 深圳韦拓生物科技有限公司 | Sperm gradient separating medium and preparation method thereof |
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