CN108728403A - A kind of spilting of an egg culture solution and preparation method thereof - Google Patents
A kind of spilting of an egg culture solution and preparation method thereof Download PDFInfo
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- CN108728403A CN108728403A CN201810610861.XA CN201810610861A CN108728403A CN 108728403 A CN108728403 A CN 108728403A CN 201810610861 A CN201810610861 A CN 201810610861A CN 108728403 A CN108728403 A CN 108728403A
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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
- C12N5/0603—Embryonic cells ; Embryoid bodies
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/32—Amino acids
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- 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
- C12N2501/90—Polysaccharides
- C12N2501/905—Hyaluronic acid
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Abstract
The present invention relates to a kind of spilting of an egg culture solutions and preparation method thereof, belong to Issues of Human Assisted Reproductive Technologies field.Spilting of an egg culture solution of the present invention is added into salting liquid by alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, proline, taurine, serine, threonine, tyrosine, methionine, lipoic acid, hyaluronic acid and chelating agent, vigor and the nutrition of embryo can not only be effectively kept, and, chelating agent can also chelate some non-essential heavy metal ion, reduce the toxicity of culture solution, it may advantageously facilitate the development of body early embryo, improve the success rate of auxiliary procreation technology, also strong guarantee is provided for the safety of auxiliary procreation technology.
Description
Technical field
The present invention relates to a kind of spilting of an egg culture solutions and preparation method thereof, belong to Issues of Human Assisted Reproductive Technologies field.
Background technology
In Issues of Human Assisted Reproductive Technologies, spilting of an egg culture solution is mainly used in the early stage culture flow of embryo.Currently, clinic makes
Conventional spilting of an egg culture solution be only using sodium bicarbonate as people's fallopian tubal salting liquid (HTF) of buffer, cannot effectively really
The development for protecting embryo, especially to the in vitro culture less effective of early embryonic development.
Invention content
The object of the present invention is to provide a kind of spilting of an egg culture solutions that every Quality Control is up to standard, can not only effectively keep embryo
The vigor of tire, improves the early embryonic development rate of auxiliary procreation technology, is also provided with and tries hard to keep for the safety of auxiliary procreation technology
Card.
To solve the above problems, the technical solution adopted in the present invention is：
A kind of spilting of an egg culture solution, it is characterised in that：It is by alanine, aspartic acid, asparagine, glutamic acid, glutamy
Amine, glycine, proline, taurine, serine, threonine, tyrosine, methionine, lipoic acid, hyaluronic acid and chelating agent
It is dissolved in the salting liquid containing antibiotic and indicator and is formulated.
Preferably：In the salting liquid, alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine,
Proline, taurine, serine, threonine, tyrosine, methionine concentration be 0.02-1.50mmol/L；Hyaluronic acid
A concentration of 0.01-0.50mg/mL；A concentration of 0.01-0.30mmol/L of lipoic acid；Appropriate chelating agent.
Preferably：The chelating agent is quaternary carboxylic acid ethylenediamine tetra-acetic acid.
Preferably：The antibiotic is gentamicin, a concentration of 4-12 μ g/mL.
Preferably：The indicator is phenol red, concentration 4.5-10.0 μ g/mL.
Preferably：The salting liquid is using sodium bicarbonate as buffer, based on sodium, potassium, magnesium, calcium ion, and with Portugal
Grape sugar, Sodium Pyruvate, sodium lactate, the compound solution that albumin is energy matter；Wherein include：Sodium chloride 89.00-
108.00mmol/L, potassium chloride 4.00-6.00mmol/L, magnesium sulfate 0.10-1.20mmol/L, potassium dihydrogen phosphate 0.3-
0.6mmol/L, sodium dihydrogen phosphate 0.10-0.50mmol/L, calcium chloride 1.60-2.50mmol/L, sodium bicarbonate 18.00-
26.00mmol/L, glucose 0.30-0.80mmol/L, Sodium Pyruvate 0.20-0.60mmol/L, sodium lactate 9.00-
23.00mmol/L, potassium dihydrogen phosphate 0.20-0.50mmol/L；
The albumin is recombination human serum albumin, is dissolved in using the recombination human serum albumin dry powder of medical grade
It is prepared in saline solution, final concentration of 0.0-6.0mg/mL.
The preparation method of spilting of an egg culture solution of the present invention, includes the following steps：
1, salting liquid is prepared：Various components first have been weighed according to each component dosage of salting liquid, it is spare；It again will be in addition to antibacterial
Each component other than element, indicator, sodium bicarbonate is dissolved in ultrapure injection stage water, and liquid after first solid is followed in course of dissolution
Principle；The ultrapure injection stage water is through 0.1-0.2 μM of membrane filtration, endotoxin < 0.015EU/mL；Then, it sequentially adds
Salting liquid is made in load weighted antibiotic, indicator, sodium bicarbonate；
2, the osmotic pressure and pH value of 1 gained salting liquid of detecting step, and record final osmotic pressure and pH value；The osmotic pressure
260-285mOsm/Kg is remained, the pH value remains 7.30-7.80；
3, according to the capacity of preformulation, it is molten that recombination human serum albumin by final concentration of 0.0-6.0mg/mL is added into salt
In liquid；
4, by alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, proline, taurine, silk
Propylhomoserin, threonine, tyrosine, methionine are dissolved in the concentration of 0.02-1.50mmol/L in salting liquid respectively；By hyalomitome
Acid is dissolved according to the concentration of 0.01-0.50mg/mL in salting liquid；Lipoic acid is dissolved with the concentration of 0.01-0.30mmol/L
In salting liquid；Appropriate chelating agent is dissolved in salting liquid；Stop to abundant dissolving；
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 20-30 DEG C：7.30-7.80；
B, osmotic pressure：260-285mOsm/Kg；
C, endotoxin：< 0.15EU/mL；
D, a cell Mouse embryos culture was by 96 hours：>=80% Blastocyst formation rate；
6, prepared solution is subjected to aseptic subpackaged and label in hundred grades of workshops GMP.
Advantageous effect：Spilting of an egg culture solution of the present invention is added alanine, aspartic acid, asparagus fern into salting liquid
Amide, glutamic acid, glutamine, glycine, proline, taurine, serine, threonine, tyrosine, methionine, sulphur are pungent
Acid, hyaluronic acid and chelating agent, can not only effectively keep vigor and the nutrition of embryo, moreover, chelating agent can also chelate
Some non-essential heavy metal ion, reduce the toxicity of culture solution, may advantageously facilitate the development of body early embryo, improve supplementary reproduction
The success rate of technology also provides strong guarantee for the safety of auxiliary procreation technology.
Specific implementation mode
With reference to specific implementation embodiment, the present invention will be further described.
Spilting of an egg culture solution of the present invention, be by alanine, aspartic acid, asparagine, glutamic acid, glutamine,
Glycine, proline, taurine, serine, threonine, tyrosine, methionine, lipoic acid, hyaluronic acid and chelating agent are molten
It is formulated in the salting liquid that Xie Yu contains antibiotic and indicator.In the salting liquid, alanine, aspartic acid, asparagus fern acyl
Amine, glutamic acid, glutamine, glycine, proline, taurine, serine, threonine, tyrosine, the concentration of methionine are equal
For 0.02-1.50mmol/L；A concentration of 0.01-0.50mg/mL of hyaluronic acid；A concentration of 0.01-0.30mmol/ of lipoic acid
L；The chelating agent is quaternary carboxylic acid ethylenediamine tetra-acetic acid, in right amount；The antibiotic is gentamicin, a concentration of 4-12 μ g/
mL；The indicator is phenol red, concentration 4.5-10.0 μ g/mL.
The salting liquid is using sodium bicarbonate as buffer, based on sodium, potassium, magnesium, calcium ion, and with glucose, third
Ketone acid sodium, sodium lactate, the compound solution that albumin is energy matter；Wherein include：Sodium chloride 89.00-108.00mmol/L,
Potassium chloride 4.00-6.00mmol/L, magnesium sulfate 0.10-1.20mmol/L, potassium dihydrogen phosphate 0.3-0.6mmol/L, biphosphate
Sodium 0.10-0.50mmol/L, calcium chloride 1.60-2.50mmol/L, sodium bicarbonate 18.00-26.00mmol/L, glucose 0.30-
0.80mmol/L, Sodium Pyruvate 0.20-0.60mmol/L, sodium lactate 9.00-23.00mmol/L, potassium dihydrogen phosphate 0.20-
0.50mmol/L；The albumin is recombination human serum albumin, is molten using the recombination human serum albumin dry powder of medical grade
Solution is prepared in saline solution, final concentration of 0.0-6.0mg/mL.
The preparation method of spilting of an egg culture solution of the present invention, includes the following steps：
1, salting liquid is prepared：Various components first have been weighed according to each component dosage of salting liquid, it is spare；It again will be in addition to antibacterial
Each component other than element, indicator, sodium bicarbonate is dissolved in ultrapure injection stage water, and liquid after first solid is followed in course of dissolution
Principle；The ultrapure injection stage water is through 0.1-0.2 μM of membrane filtration, endotoxin < 0.015EU/mL；Then, it sequentially adds
Salting liquid is made in load weighted antibiotic, indicator, sodium bicarbonate；
2, the osmotic pressure and pH value of 1 gained salting liquid of detecting step, and record final osmotic pressure and pH value；The osmotic pressure
260-285mOsm/Kg is remained, the pH value remains 7.30-7.80；
3, according to the capacity of preformulation, it is molten that recombination human serum albumin by final concentration of 0.0-6.0mg/mL is added into salt
In liquid；
4, by alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, proline, taurine, silk
Propylhomoserin, threonine, tyrosine, methionine are dissolved in the concentration of 0.02-1.50mmol/L in salting liquid respectively；By hyalomitome
Acid is dissolved according to the concentration of 0.01-0.50mg/mL in salting liquid；Lipoic acid is dissolved with the concentration of 0.01-0.30mmol/L
In salting liquid；Appropriate chelating agent is dissolved in salting liquid；Stop to abundant dissolving；
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 20-30 DEG C：7.30-7.80；
B, osmotic pressure：260-285mOsm/Kg；
C, endotoxin：< 0.15EU/mL；
D, a cell Mouse embryos culture was by 96 hours：>=80% Blastocyst formation rate；
6, prepared solution is subjected to aseptic subpackaged and label in hundred grades of workshops GMP.
【Direction memory and stability】
1, it stores：
The culture solution that do not uncap is deposited in 2-8 DEG C of refrigerator.
Before use, in incubator temperature (37 DEG C) and containing 5-6%CO2Atmospheric environment in rewarming.It should not be culture solution
In the environment of freezing or being exposed to more than 39 DEG C.
Opened bottle is put back to most handy containing 5-6%CO before refrigerator2Gas inflate again.Especially emphasize to measure training
PH value of nutrient solution under the conditions of experimental work can adjust use CO accordingly2Concentration, so as to obtain embryonic development need be most suitable for
PH value, spilting of an egg culture solution optimal pH is 7.30.
2, stability：Product is stable before the term of validity on label.
In use, according to correct sterile working method：
A. suitable product is taken out with sterile working method；
B. it should not be refunded in bottle remaining after taking out product；
C. it is in turbid shape as product changes colour, it is muddy or may have microbial contamination unusable.
【Specifically used method】
Spilting of an egg culture solution of the present invention is mainly used for the culture before after fertilization embryo's densification in the 1st day to the 3rd day.This hair
If the bright spilting of an egg culture solution has been added to recombination human serum albumin (rHSA), can use at any time.
Embryo Culture may be used in droplet, single hole ware or four orifice plates and carry out, and suggest covering paraffin oil, possess
CO2In vitro culture is carried out in the atmospheric environment of incubator.
Claims (7)
1. a kind of spilting of an egg culture solution, it is characterised in that：Be by alanine, aspartic acid, asparagine, glutamic acid, glutamine,
Glycine, proline, taurine, serine, threonine, tyrosine, methionine, lipoic acid, hyaluronic acid and chelating agent are molten
It is formulated in the salting liquid that Xie Yu contains antibiotic and indicator.
2. spilting of an egg culture solution according to claim 1, it is characterised in that：In the salting liquid, alanine, aspartic acid,
Asparagine, glutamic acid, glutamine, glycine, proline, taurine, serine, threonine, tyrosine, methionine
Concentration is 0.02-1.50mmol/L；A concentration of 0.01-0.50mg/mL of hyaluronic acid, a concentration of 0.01- of lipoic acid
0.30mmol/L；Appropriate chelating agent.
3. spilting of an egg culture solution according to claim 2, it is characterised in that：The chelating agent is quaternary carboxylic acid ethylenediamine tetraacetic
Acetic acid.
4. spilting of an egg culture solution according to claim 3, it is characterised in that：The antibiotic is gentamicin, a concentration of 4-
12μg/mL。
5. spilting of an egg culture solution according to claim 4, it is characterised in that：The indicator is phenol red, concentration 4.5-10.0 μ
g/mL。
6. spilting of an egg culture solution according to claim 5, it is characterised in that：It is buffering that the salting liquid, which is with sodium bicarbonate,
Agent, based on sodium, potassium, magnesium, calcium ion, and using glucose, Sodium Pyruvate, sodium lactate, albumin as the chemical combination of energy matter
Object solution；Wherein include：Sodium chloride 89.00-108.00mmol/L, potassium chloride 4.00-6.00mmol/L, magnesium sulfate 0.10-
1.20mmol/L, potassium dihydrogen phosphate 0.3-0.6mmol/L, sodium dihydrogen phosphate 0.10-0.50mmol/L, calcium chloride 1.60-
2.50mmol/L, sodium bicarbonate 18.00-26.00mmol/L, glucose 0.30-0.80mmol/L, Sodium Pyruvate 0.20-
0.60mmol/L, sodium lactate 9.00-23.00mmol/L, potassium dihydrogen phosphate 0.20-0.50mmol/L；
The albumin is recombination human serum albumin, is to be dissolved in brine using the recombination human serum albumin dry powder of medical grade
It is prepared in solution, final concentration of 0.0-6.0mg/mL.
7. the preparation method of spilting of an egg culture solution as claimed in claim 6, it is characterised in that：Include the following steps：
(1) salting liquid is prepared：Various components first have been weighed according to each component dosage of salting liquid, it is spare；Again will in addition to antibiotic,
Each component other than indicator, sodium bicarbonate is dissolved in ultrapure injection stage water, and liquid after first solid is followed in course of dissolution
Principle；The ultrapure injection stage water is through 0.1-0.2 μM of membrane filtration, endotoxin < 0.015EU/mL；Then, title is sequentially added
Salting liquid is made in measured antibiotic, indicator, sodium bicarbonate；
(2) osmotic pressure and pH value of salting liquid obtained by detecting step (1), and record final osmotic pressure and pH value；The osmotic pressure
260-285mOsm/Kg is remained, the pH value remains 7.30-7.80；
(3) according to the capacity of preformulation, recombination human serum albumin is added into salting liquid by final concentration of 0.0-6.0mg/mL
In；
(4) by alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, proline, taurine, silk ammonia
Acid, threonine, tyrosine, methionine are dissolved in the concentration of 0.02-1.50mmol/L in salting liquid respectively；By hyaluronic acid
It is dissolved in salting liquid according to the concentration of 0.01-0.50mg/mL；Lipoic acid is dissolved in the concentration of 0.01-0.30mmol/L
In salting liquid；Appropriate chelating agent is dissolved in salting liquid；Stop to abundant dissolving；
(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 20-30 DEG C：7.30-7.80；
B, osmotic pressure：260-285mOsm/Kg；
C, endotoxin：< 0.15EU/mL；
D, a cell Mouse embryos culture was by 96 hours：>=80% Blastocyst formation rate；
(6) prepared solution is subjected to aseptic subpackaged and label in hundred grades of workshops GMP.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109644984A (en) * | 2018-12-18 | 2019-04-19 | 陈子江 | A kind of cell glass freezing liquid and freezing method |
| CN110669725A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Cleavage embryo culture solution and preparation method thereof |
| CN111073848A (en) * | 2020-01-02 | 2020-04-28 | 广州裕康生物科技有限公司 | Cleavage stage embryo culture composition and cleavage stage embryo culture solution |
| CN111517551A (en) * | 2020-04-28 | 2020-08-11 | 南京优而生物科技发展有限公司 | Production water for liquid products for assisted reproduction technology |
| CN112608886A (en) * | 2021-01-05 | 2021-04-06 | 深圳英莱生命科学有限公司 | Ovum and embryo treating liquid and its preparing method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109644984A (en) * | 2018-12-18 | 2019-04-19 | 陈子江 | A kind of cell glass freezing liquid and freezing method |
| CN110669725A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Cleavage embryo culture solution and preparation method thereof |
| CN111073848A (en) * | 2020-01-02 | 2020-04-28 | 广州裕康生物科技有限公司 | Cleavage stage embryo culture composition and cleavage stage embryo culture solution |
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| CN111517551A (en) * | 2020-04-28 | 2020-08-11 | 南京优而生物科技发展有限公司 | Production water for liquid products for assisted reproduction technology |
| CN112608886A (en) * | 2021-01-05 | 2021-04-06 | 深圳英莱生命科学有限公司 | Ovum and embryo treating liquid and its preparing method |
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Application publication date: 20181102 |