CN108823150A - A kind of fallopian tubal buffering culture solution and preparation method thereof - Google Patents
A kind of fallopian tubal buffering culture solution and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of fallopian tubal buffering culture solutions and preparation method thereof, belong to Issues of Human Assisted Reproductive Technologies field.Fallopian tubal buffering culture solution of the present invention is that taurine, serine, threonine ingredient and chelating agent EDTA are added into salting liquid; sperm, ovum and the vigor of embryo can not only be effectively kept; and chelating agent EDTA can also chelate some non-essential heavy metal ion; reduce the toxicity of solution; the success rate for improving assisted reproductive technology, also provides strong guarantee for the safety of assisted reproductive technology.
Description
Technical field
The present invention relates to a kind of fallopian tubal buffering culture solutions and preparation method thereof, belong to Issues of Human Assisted Reproductive Technologies field.
Background technique
In Issues of Human Assisted Reproductive Technologies, fallopian tubal buffers culture solution for recovery of ova, washing sperm, micromanipulation, embryo
Tire transplanting flushing, gamete and embryo cryopreservation process etc., to keep sperm, ovum and the vigor of embryo, improve assisted reproductive technology
Success rate.Currently, the conventional fallopian tubal buffering culture solution of clinical use is only that the salt that HEPES or MOPS is buffering is molten
Liquid cannot effectively keep sperm, ovum and the vigor of embryo, the especially freezen protective to sperm and embryo.
Taurine (Taurine) is also known as β-aminoethanesulfonic acid, and earliest by separating in cow-bezoar, sterling is colourless or white
Ramp-like crystal, it is odorless.Taurine chemical property is stablized, and is a kind of non-protein amino of sulfur-bearing insoluble in organic solvents such as ether
Acid exists in vivo with free state, is not involved in the biosynthesis of vivo protein.Although taurine is not involved in protein synthesis,
But it is closely related with the metabolism of cystine, cysteine.The drug effect of taurine：1, strong liver cholagogic acts on：Cavy is real
It tests and shows that taurine can release cholestasia, be in choleretic effect；2, antipyretic and anti-inflammatory effect：By to maincenter 5-ht system or
The effect of catecholamine system reduces body temperature；3, antihypertensive effect；4, heart tonifying and antiarrhythmic effect；5, hypoglycemic effect；6,
The effect of relaxation skeletal muscle and antagonism myotonia.
Serine also known as β serine play work in the metabolism of fat and fatty acid and the growth of muscle
With because it facilitates the generation of immunoglobulin and antibody, maintain health immune system be also required to serine.In addition, silk
Propylhomoserin all plays effect in the synthesis of the sheath of the manufacture processing of cell membrane, musculature and encirclement nerve cell.
Threonine is that the important nutrition fortifier of one kind has alleviation human-body fatigue, enhancing development as tryptophan
Effect.Pharmaceutically, due to containing hydroxyl in the structure of threonine, there is water holding ability to human skin, in conjunction with oligonucleotide chain,
It plays an important role to protection cell membrane, phosphatide synthesis and fatty acid oxidation can be promoted in vivo.Its preparation, which has, promotes human body hair
Educate with the medicinal efficiency of anti-fatty liver, be compound amino acid infusion in an ingredient.Meanwhile threonine is that manufacture is a kind of efficiently again
The raw material of the antibiotic of low allergy --- monobactam.
Quaternary carboxylic acid ethylenediamine tetra-acetic acid (EDTA) is a kind of important artificial organic multicomponent acid complexing agent, most important
Chemical property be highly stable water soluble complex can be formed with various metal cations, therefore have strong complexing,
Precipitation by metallic ion can be prevented, prevents the catalytic action of metal ion, increases metal ion in solution availability, or from system
Middle removal metal ion.It is widely used in business and house detergent, weaving and paper technology bleaching process, metal plating mistake
Journey, food industry, medicine and agricultural.
Summary of the invention
The object of the present invention is to provide a kind of fallopian tubals that every Quality Control is up to standard to buffer culture solution, not only can be effective
Ground keeps the vigor of sperm, ovum and embryo, improves the success rate of assisted reproductive technology, is also the safety of assisted reproductive technology
Provide strong guarantee.
To solve the above problems, the technical scheme adopted by the invention is that：
A kind of fallopian tubal buffering culture solution, it is characterised in that：It is to dissolve taurine, serine, threonine and chelating agent
It is formulated in the salting liquid containing antibiotic and indicator.
Preferably：Fallopian tubal buffering culture solution be by taurine, serine, threonine, chelating agent point separately according to
The concentration of 0.20-0.25mmol/L, 0.05-0.15mmol/L, 0.20-0.40mmol/L, 0.01-0.05mmol/L, which are dissolved in, to be contained
It is formulated in the salting liquid for having antibiotic and indicator.
Preferably：The chelating agent is quaternary carboxylic acid ethylenediamine tetra-acetic acid.
Preferably：The antibiotic is gentamicin, and concentration is 5-12 μ g/ml.
Preferably：The indicator is phenol red, concentration 5.0-10.0 μ g/ml.
Preferably：The salting liquid be using HEPES or MOPS as buffer, based on sodium, potassium, magnesium, calcium ion, and
Using glucose, Sodium Pyruvate, sodium lactate as the compound culture solution of energy matter；Wherein include：Sodium chloride 97.00-
102.00mmol/L, potassium chloride 4.50-4.70mmol/L, magnesium sulfate 0.10-0.25mmol/L, calcium chloride 1.90-2.20mmol/
L, sodium bicarbonate 3.80-4.20mmol/L, HEPES or MOPS18.00-22.00mmol/L, glucose 2.60-2.88mmol/
L, Sodium Pyruvate 0.28-0.35mmol/L, sodium lactate 21.00-22.66mmol/L, potassium dihydrogen phosphate 0.35-0.39mmol/L.
Preferably：Energy matter in the salting liquid further includes albumin；The albumin is recombinant human serum albumin egg
It is white, it is to be dissolved in be prepared in saline solution using the recombination human serum albumin dry powder of medical grade, final concentration of 5mg/
ml。
The preparation method of the fallopian tubal buffering culture solution, includes the following steps：
1, good various components are weighed, it is spare；
2, salting liquid is prepared：In load weighted component, first by each group other than antibiotic, indicator, sodium bicarbonate
Divide and be dissolved in ultrapure injection stage water, the principle of liquid after first solid is followed in course of dissolution；The ultrapure injection stage is passed through with water
0.1-0.2 μM of membrane filtration, endotoxin < 0.015EU/ml；Then, load weighted antibiotic, indicator, carbonic acid are sequentially added
Salting liquid is made in hydrogen sodium；
3, the osmotic pressure and pH value of 2 gained salting liquid of detecting step, and record final osmotic pressure and pH value；The osmotic pressure
250-275mOsm/Kg is remained, the pH value remains 7.0-7.4；
4, by taurine, serine, threonine, chelating agent respectively according to 0.20-0.25mmol/L, 0.05-0.15mmol/
L, the concentration of 0.20-0.40mmol/L, 0.01-0.05mmol/L are dissolved in salting liquid, are stopped to abundant dissolution；
5, by step 4 acquired solution after 0.2 μm of membrane filtration sterilizes, sampling and testing；Test parameter：
A, pH value under the conditions of 20-25 DEG C：7.30-7.50；
B, osmotic pressure：265-285m Osm/Kg；
C, endotoxin：< 0.15EU/ml；
D, a cell Mouse embryos culture was by 96 hours：>=80% Blastocyst formation rate；
6, configured solution is subjected to aseptic subpackaged and label in hundred grades of workshops GMP.
Preferably：In the step 3, the osmotic pressure and pH value of step 2 gained salting liquid have been detected, and has recorded final infiltration
After pressure and pH value, according to the capacity of pre-configuration, recombination human serum albumin is added in salting liquid by final concentration of 5mg/ml.
Beneficial effect：Fallopian tubal buffering culture solution of the present invention is the addition taurine, serine, Soviet Union into salting liquid
Propylhomoserin nutritional ingredient and chelating agent EDTA can not only effectively keep sperm, ovum and the vigor of embryo, but also chelating agent
EDTA can also chelate some non-essential heavy metal ion, reduce the toxicity of solution, improve the success rate of assisted reproductive technology,
Also strong guarantee is provided for the safety of assisted reproductive technology.
Specific embodiment
Below with reference to specific implementation embodiment, the present invention will be further described.
Fallopian tubal of the present invention buffers culture solution, be by taurine, serine, threonine, chelating agent respectively according to
The concentration of 0.20-0.25mmol/L, 0.05-0.15mmol/L, 0.20-0.40mmol/L, 0.01-0.05mmol/L, which are dissolved in, to be contained
It is formulated in the salting liquid for having antibiotic and indicator.
The chelating agent is quaternary carboxylic acid ethylenediamine tetra-acetic acid；The antibiotic is gentamicin, and concentration is 5-12 μ g/
ml；The indicator is phenol red, concentration 5.0-10.0 μ g/ml.
The salting liquid is using HEPES or MOPS as buffer, based on sodium, potassium, magnesium, calcium ion, and with grape
Sugar, Sodium Pyruvate, the compound culture solution that sodium lactate is energy matter；Wherein include：Sodium chloride 97.00-102.00mmol/
L, potassium chloride 4.50-4.70mmol/L, magnesium sulfate 0.10-0.25mmol/L, calcium chloride 1.90-2.20mmol/L, sodium bicarbonate
3.80-4.20mmol/L, HEPES or MOPS18.00-22.00mmol/L, glucose 2.60-2.88mmol/L, Sodium Pyruvate
0.28-0.35mmol/L, sodium lactate 2l.00-22.66mmol/L, potassium dihydrogen phosphate 0.35-0.39mmol/L；
Energy matter in the salting liquid can also include albumin；The albumin is recombination human serum albumin,
It is to be dissolved in be prepared in saline solution using the recombination human serum albumin dry powder of medical grade, final concentration of 5mg/ml.
The preparation method of fallopian tubal buffering culture solution of the present invention, includes the following steps：
1, good various components are weighed, it is spare；
2, salting liquid is prepared：In load weighted component, first by each group other than antibiotic, indicator, sodium bicarbonate
Divide and be dissolved in ultrapure injection stage water, the principle of liquid after first solid is followed in course of dissolution；The ultrapure injection stage is passed through with water
0.1-0.2 μM of membrane filtration, endotoxin < 0.015EU/ml；Then, load weighted antibiotic, indicator, carbonic acid are sequentially added
Salting liquid is made in hydrogen sodium；
3, the osmotic pressure and pH value of 2 gained salting liquid of detecting step, and record final osmotic pressure and pH value；The osmotic pressure
250-275mOsm/Kg is remained, the pH value remains 7.0-7.4；
The osmotic pressure and pH value of step 2 gained salting liquid are detected, and after recording final osmotic pressure and pH value, it can be according to
The capacity of pre-configuration is added to recombination human serum albumin in salting liquid by final concentration of 5mg/ml；
4, by taurine, serine, threonine, chelating agent respectively according to 0.20-0.25mmol/L, 0.05-0.15mmol/
L, the concentration of 0.20-0.40mmol/L, 0.01-0.05mmol/L are dissolved in salting liquid, are stopped to abundant dissolution；
5, by step 4 acquired solution after 0.2 μm of membrane filtration sterilizes, sampling and testing；Test parameter：
A, pH value under the conditions of 20-25 DEG C：7.30-7.50；
B, osmotic pressure：265-285m Osm/Kg；
C, endotoxin：< 0.15EU/ml；
D, a cell Mouse embryos culture was by 96 hours：>=80% Blastocyst formation rate；
6, configured solution is subjected to aseptic subpackaged and label in hundred grades of workshops GMP.
【Direction memory and stability】
Storage：The fallopian tubal dispensed buffering culture solution is deposited in 2 DEG C -8 DEG C, liquid should be reduced as far as possible and be exposed to
CO2And air, to avoid 7.0 or lower pH level.It is not exposed to and is higher than at a temperature of 39 DEG C.
Stability：It can be until due date for being shown on label.The product of volume needed for being removed using sterile procedure, one
Denier removes, and the product of any amount is not returned to original container；Such as fruit product discoloration, muddiness have microbial contamination sign, ask
Do not use.
It to avoid pollution problem, is handled using asptic technique, and abandons in bottle or bottle and remain after completing operation
Remaining any rest products.
【Specifically used method】
Fallopian tubal buffering culture solution of the present invention is in no CO2The most reason tested in the atmospheric environment of incubator
The culture solution thought, can be used for obtaining egg mother cell, with during seed detergent, micromanipulation, embryo transfer flushing and embryo it is cold
Freeze etc..
Claims (9)
1. a kind of fallopian tubal buffers culture solution, it is characterised in that：It is to be dissolved in taurine, serine, threonine and chelating agent
It is formulated in salting liquid containing antibiotic and indicator.
2. fallopian tubal according to claim 1 buffers culture solution, it is characterised in that：It is by taurine, serine, Soviet Union's ammonia
Acid, chelating agent are respectively according to 0.20-0.25mmol/L, 0.05-0.15mmol/L, 0.20-0.40mmol/L, 0.01-
The concentration of 0.05mmol/L is dissolved in the salting liquid containing antibiotic and indicator and is formulated.
3. fallopian tubal according to claim 2 buffers culture solution, it is characterised in that：The chelating agent is quaternary carboxylic acid second
Ethylenediamine tetraacetic acid (EDTA).
4. fallopian tubal according to claim 3 buffers culture solution, it is characterised in that：The antibiotic is gentamicin, dense
Degree is 5-12 μ g/ml.
5. fallopian tubal according to claim 4 buffers culture solution, it is characterised in that：The indicator is phenol red, concentration
5.0-10.0μg/ml。
6. fallopian tubal according to claim 5 buffers culture solution, it is characterised in that：The salting liquid be with HEPES or
MOPS is buffer, based on sodium, potassium, magnesium, calcium ion, and using glucose, Sodium Pyruvate, sodium lactate as the change of energy matter
Close object culture solution；Wherein include：Sodium chloride 97.00-102.00mmol/L, potassium chloride 4.50-4.70mmol/L, magnesium sulfate
0.10-0.25mmol/L, calcium chloride 1.90-2.20mmol/L, sodium bicarbonate 3.80-4.20mmol/L, HEPES or
MOPS18.00-22.00mmol/L, glucose 2.60-2.88mmol/L, Sodium Pyruvate 0.28-0.35mmol/L, sodium lactate
21.00-22.66mmol/L, potassium dihydrogen phosphate 0.35-0.39mmol/L.
7. fallopian tubal according to claim 6 buffers culture solution, it is characterised in that：Energy matter in the salting liquid is also
Including albumin；The albumin is recombination human serum albumin, is molten using the recombination human serum albumin dry powder of medical grade
Xie Yu is prepared in saline solution, final concentration of 5mg/ml.
8. the preparation method of fallopian tubal buffering culture solution as claimed in claim 6, it is characterised in that：Include the following steps：
(1) good various components are weighed, it is spare；
(2) salting liquid is prepared：It is first that each component other than antibiotic, indicator, sodium bicarbonate is molten in load weighted component
In ultrapure injection stage water, the principle of liquid after first solid is followed in course of dissolution；The ultrapure injection stage water is through 0.1-
0.2 μM of membrane filtration, endotoxin < 0.015EU/ml；Then, load weighted antibiotic, indicator, sodium bicarbonate are sequentially added,
Salting liquid is made；
(3) osmotic pressure and pH value of salting liquid obtained by detecting step (2), and record final osmotic pressure and pH value；The osmotic pressure
250-275mOsm/Kg is remained, the pH value remains 7.0-7.4；
(4) by taurine, serine, threonine, chelating agent respectively according to 0.20-0.25mmol/L, 0.05-0.15mmol/L,
The concentration of 0.20-0.40mmol/L, 0.01-0.05mmol/L are dissolved in salting liquid, are stopped to abundant dissolution；
(5) by step (4) acquired solution after 0.2 μm of membrane filtration sterilizes, sampling and testing；Test parameter：
A, pH value under the conditions of 20-25 DEG C：7.30-7.50；
B, osmotic pressure：265-285m Osm/Kg；
C, endotoxin：< 0.15EU/ml；
D, a cell Mouse embryos culture was by 96 hours：>=80% Blastocyst formation rate；
(6) configured solution is subjected to aseptic subpackaged and label in hundred grades of workshops GMP.
9. the preparation method of fallopian tubal buffering culture solution according to claim 8, it is characterised in that：In the step (3), inspection
The osmotic pressure and pH value of salting liquid obtained by step (2) are surveyed, and after recording final osmotic pressure and pH value, according to the appearance of pre-configuration
Amount, recombination human serum albumin is added in salting liquid by final concentration of 5mg/ml.
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Cited By (2)
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| CN110100813A (en) * | 2019-06-06 | 2019-08-09 | 中国农业大学 | A kind of sheep embryo vitrifying freeze saves formula of liquid and freezing method |
| CN110669724A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Saccharum embryo culture solution and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110100813A (en) * | 2019-06-06 | 2019-08-09 | 中国农业大学 | A kind of sheep embryo vitrifying freeze saves formula of liquid and freezing method |
| CN110669724A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Saccharum embryo culture solution and preparation method thereof |
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