CN108251354A - Single step embryo medium and preparation method thereof - Google Patents
Single step embryo medium and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to supplementary reproduction fields, and in particular to a kind of single step embryo medium and preparation method thereof.The composition of single step embryo medium is used to prepare in the present invention, the composition is made of (±) alpha lipoic acid, acetyl L carnitines or acetyl L carnitine hydrochlorides, N acetyl group L cysteines；(±) alpha lipoic acid, acetyl L carnitines or acetyl L carnitine hydrochlorides, N acetyl group L cysteines molar ratio be 0.055 0.098：0.044‑0.078：0.702‑0.858.The single step embryo medium prepared using the composition remains the active ingredient that embryo is secreted into culture solution; it can play the role of protecting cell and inhibiting to decompose utilizing noxious products, avoid the problem that the mechanical damage that may be subject to during embryo transfer and Blastocyst formation rate caused by stress reaction caused by culture environment difference be not high.
Description
Technical field
The invention belongs to supplementary reproduction fields, and in particular to a kind of single step embryo medium and preparation method thereof.
Background technology
Vitro fertilization-embryo implanting In Vitro Fertilization-Embryo Transfer abbreviation IVF-ET are
Refer to after respectively taking out ovum and sperm, it is in vitro fertilization, then by embryonic precursors --- zygote transplation, which is returned in maternal uterine, develops
Into fetus.Test-tube baby is that ovum and sperm is allowed to be fertilized in vitro and carry out early embryonic development by artificial means, is then transplanted
The baby for developing and being born in maternal uterine.
Since first " test tube " baby in the world on July 25th, 1978 after Britain is born, on July 1st, 2012
Have 5,000,000 " test tube " babies to be born.IVF is more than to account for 50,000,000 Infertile Couples to bring Gospel for the whole world.However, 30
Clinical practice for many years is not significantly increased the success rate of IVF.2011, gestation association of the U.S. count 35 years old patient IVF into
Power (pregnancy rate) in 25-30% or so, and 1997 also 30% or so, and babies' birthrate is lower.One of them is important
The reason of be IVF embryo mediums quality, this is one of most important deciding factor of IVF embryo qualities.
IVF cultures are generally divided into two kinds of flows i.e. two kinds of culture solution series：Whole (list/single step culture solution series) and divide
Journey (double culture solution series).Embryo's early and late stagess are provided using double culture solution series flows different culture solution into
Part, but the important effective ingredient being secreted by embryo in culture solution is also lost, such as the embryotrophy factor.Whole culture solution system
Row flow remains these important effective ingredients, but also remains the decomposition product being harmful in culture solution.
The hypotrophy allocated by sperm used at present or its physically or chemically index excess enthalpy is wide, similarly tested
Journey easily makes in incubation sperm with batch sperm or egg cell is dead or capacitation deficiency vigor is relatively low or ovum is thin
Born of the same parents' existence is affected, and such as in sperm motility experiment, the ratio that sperm maintains vigour is relatively low or its ratio is near or below conjunction
Case marker standard (maintain vigour rate >=75%).Some are higher by the consistency of sperm, are not done containing oxious component and inflammatory cell removal
Only, the growing of fertilized eggs is seriously affected.
In addition, general culture solution cannot intuitively judge the quality of validity before use, if after it is rotten still after
Continuous use will cause irreversible result to experiment or clinical manipulation.Therefore it needs to develop a kind of nothing and needs to change culture solution, contains
There is the single step embryo medium that important active ingredient can simultaneously inhibit noxious products.
Invention content
In view of this, the purpose of the present invention is to provide a kind of composition for being used to prepare single step embryo medium and one
Step formula embryo medium, single step embryo medium of the invention remain the active ingredient that embryo is secreted into culture solution, energy
Play the role of protecting cell and inhibiting to decompose utilizing noxious products.
To achieve the above object, the technical scheme is that：
The composition of single step embryo medium is used to prepare, the composition is by (±)-alpha-lipoic acid, acetyl L-carnitine
Or acetyl L-carnitine hydrochloride, n-acetyl-L-cysteine composition；(±)-alpha-lipoic acid, acetyl L-carnitine or acetyl
L-carnitine hydrochloride, n-acetyl-L-cysteine molar ratio be 0.055-0.098：0.044-0.078：0.702-
0.858。
Critical component A：(±)-alpha-lipoic acid
Alpha-lipoic acid (LA) two kinds of structure mixtures in the form of closed loop disulfide form and open chain reduction exist, both
Form recycles mutually conversion by oxidationreduction, and as biotin, lipoic acid is not in fact usually free to be existed, but with
ε-the NH of lysine residue in the same enzyme molecule of its carboxyl (such as dihydrolipoic acid transacetylase)2Base is with amido bond covalent bond
(quite similar with biocytin in structure).Lipoic acid enjoys the title of " universal antioxidant ", is widely present in vivo, is
A kind of disulfide with extensive biological activity has very strong antioxidant activity, and it is free that LA can remove internal oxygen
Base, chelated metal ions regenerate other internal antioxidants to play antioxidation, and protection mitochondrial function adjusts energy
Metabolism.
Wherein active oxygen (reactive oxidative species, ROS) oxidative stress caused by/oxygen radical is to man
One of the reason of damage of property testis and sperm may be male sterility.LA can be converted into reduced form in vivo
DHLA, the two collective effect can almost remove ROS and free radical all in reproductive system, such as singlet oxygen, hydroxyl free
Base, hydrogen peroxide, peroxynitrite, nitric oxide and hypochlorous acid etc..For slight oxidative damage, vitamin C
(vitamin C, VitC), vitamin E (vitamin E, VitE) and LA can be repaired, but the oxidative damage of severe only ties up life
Plain E and LA are effective, especially LA, can not only repair heavy oxidation damage, and can resist long-time, the oxidation damage of duration
Wound.
The Antioxidative Defense System of body is mainly anti-oxidant by LA, VitC, VitE, Co-Q10,5 kinds of glutathione (GSH)
Agent is formed.LA is that antioxidant all in the system can uniquely be made to include LA oneself (including DHLA), is regenerated as restoring
Type antioxidant so as to activate the metabolic cycles of other antioxidant in organism, forms unique antioxidant regeneration cycle net
Network, it is common to play biological antioxidant effect, maintain the normal oxidation antioxidation dynamic equilibrium of body.
Chelated metal ions testis is one of important target organ of heavy metal, and poisoning maximum heavy metal to human body is mainly
Lead, mercury, chromium, arsenic, 5 kinds of cadmium, cadmium are the known poisonous substances for being easiest to accumulate in vivo, and the velocity of discharge is slow, and chronic accumulation can be direct
It destroys testicular cell DNA structure and inhibits its reparation, be catalyzed a large amount of free radicals of generation, easily cause testis and spermatoblast film fat
The serious Reproductive Damage such as matter peroxidating.LA and DHLA can chelate contents of many kinds of heavy metal ion in vivo and in vitro, as copper, zinc, lead,
The metal ions such as arsenic, cadmium.
Mitochondria is the control centre of cell activities, is intracellular energy converting system and energy supply center, and participate in
The regulation and control of the important physiology course of general anti-oxidative defense and Apoptosis etc..Energy needed for sperm motility comes from glycolysis and line grain
The ATP generated in body respiratory metabolism, so structure of mitochondria caused by a variety of causes and function change can lead to mitochondria energy
Dyssynthesis is measured, sperm motility reduces.LA is referred to as chondriosome nutrient, you can protection cyclophorase reduces mitochondria ROS's
It generates or removes excessive ROS, induction II phase enzyme enhancing cellular anti-oxidant capacity, repair mitochondria.
Critical component B：Acetyl L-carnitine or acetyl L-carnitine hydrochloride
It is the carboxylate of l-cn, its white powder is soluble easily in water, insoluble in acetone.Acetyl L-carnitine is to exist
In a kind of internal natural material, especially content is very rich in muscle, brain and sperm, is participated in human body a series of heavy
The metabolic process wanted, it takes away acetyl group from cell and assists energy transfer.Acetyl L-carnitine is in central nervous system and man
It plays an important role in sexual reproduction.The main function of acetyl L-carnitine：1. transhipment aliphatic acid enters mitochondria and carries out B- herein
Oxidation；2. protect cell：Prevent it from damaging cell in vitro excessive acyl coenzyme A exclusion；3. prevent lactic acid accumulation.Fierceness fortune
A large amount of lactic acid are generated after dynamic, l-cn can reduce lactic acid production quantity, improve exercise tolerance；Meanwhile it also has promotion carbon aquation
Close the effects that matter energies such as object utilize, improve sperm number and vigor.
Acetyl L-carnitine can prevent the toxicity accumulation of acetyl coenzyme A in aliphatic acid and mitochondria in cytoplasm, while be line
The generation of energy provides enough acetyl coenzyme As in plastochondria.Acetyl L-carnitine and its hydrochloride have a variety of physiological activity, including
Trophism, neuroprotection, the analgesic effect of peripheral nervous system promote energetic supersession and by long-chain fat acid transporter
Oxidative phosphorylation is participated in mitochondrial inner membrane and generates ATP energy supplies, and the antioxidant reductase performance adjusted under pathological conditions is anti-oxidant
Effect and the effect in male reproductive system etc..
Critical component C：N-acetyl-L-cysteine
Free sulfhydryl group that n-acetyl-L-cysteine can directly have an effect with electrophilic oxide group (nucleophilic-
SH direct antioxidation) is played, molecular structure is rendered it susceptible to through cell membrane, is a kind of synthesizing glutathion (GSH)
Essential amino acid.Glutathione is intracellular most important protective agent, to keeping the integrality of cell function and cellular morphology
It is required, it can prevent cell from the damage of the oxygen radical and various cytotoxic substances of inside and outside.N- acetyl group-L- half
Cystine plays an important role in terms of appropriate GSH levels are kept, so as to help to protect cell not because of the horizontal mistakes of internal GSH
Cytotoxin damages caused by low.
Further, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, half Guangs of N- acetyl group-L-
The molar ratio of propylhomoserin is 0.065-0.098：0.054-0.078：0.752-0.858.
As a preferred embodiment, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, N- acetyl group-
The molar ratio of L-cysteine is 0.065-0.0875：0.064-0.078：0.752-0.805.
As a preferred embodiment, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, N- acetyl group-
The molar ratio of L-cysteine is 0.078：0.078：0.782.
(±)-alpha-lipoic acid content is too low to cause embryo development procedure intoxicating phenomenon occur, and embry ogenesis rate is low, poor morphology
Phenomena such as；If excessively high cause embryo can not proper splitting and death.Acetyl L-carnitine or acetyl L-carnitine hydrochloride content mistake
It is low to lead to Late Embryogenesis cell cracking, it is excessively high to cause embryo viability low.N-acetyl-L-cysteine content is too low similary
It can lead to cytotoxic phenomenon, particularly late stage of culture, culture solution is sticky, and form is not completed.If excessively high cause culture solution to ooze
Press thoroughly it is bigger than normal, phenomena such as cell shrinkage.
The second object of the present invention is to provide a kind of single step embryo medium, by (±)-alpha-lipoic acid, acetyl L- meat
Alkali or acetyl L-carnitine hydrochloride, n-acetyl-L-cysteine and basic culture solution composition, (±)-alpha-lipoic acid, second
Acyl-L-carnitine or acetyl L-carnitine hydrochloride, n-acetyl-L-cysteine and basic culture solution molar ratio be 0.055-
0.098：0.044-0.078：0.702-0.858：135.251-167.614.
Single step embryo medium refers to the whole culture solution for development of fertilized ova to blastaea, during development of fertilized ova
Nothing needs to change culture solution.The machine that may be subject to during embryo transfer is that of avoiding compared to tradition point formula culture solution its advantage
The problem of Blastocyst formation rate is not high caused by stress reaction caused by tool damage is different with culture environment.
As a preferred embodiment, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, N- acetyl group-
The molar ratio of L-cysteine and basic culture solution is 0.065-0.098：0.054-0.078：0.752-0.858：140.336-
167.614。
As a preferred embodiment, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, N- acetyl group-
The molar ratio of L-cysteine and basic culture solution is 0.065-0.0875：0.064-0.078：0.752-0.805：140.336-
160.413。
As a preferred embodiment, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, N- acetyl group-
The molar ratio of L-cysteine and basic culture solution is 0.078：0.078：0.782：140.336-160.413.
Further, the basic culture solution includes inorganic salt buffer, amino acid, ethylenediamine tetra-acetic acid, Sodium Pyruvate, third
Aminoacyl glutamine, taurine, glucose, D-glucitol, human serum albumins and fungicide.
As a preferred embodiment, the fungicide is gentamicin.
Further, the inorganic salt buffer, amino acid, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine,
Taurine, glucose, D-glucitol molar ratio be 98.303-120.12:1.2-3.8:0.009-0.012:9.459-
11.561:24.741-30.239:0.432-0.528:0.9-1.1:0.207-0.254。
The composition of the basic culture solution and the culture solution Blastocyst formation rate of proportioning configuration are high, and form is complete.
As a preferred embodiment, the inorganic salt buffer, amino acid, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl paddy ammonia
Amide, taurine, glucose, D-glucitol molar ratio be 105.128-120.12:1.5-3.5:0.01-0.012:
10.254-11.561:26.156-30.239:0.432-0.528:0.9-1.1:0.207-0.254。
Further, the content of the human serum albumins described in 100ml single step embryo mediums is 0.45-0.55g/L；
The content of fungicide is 45-55mg/L in 100ml single step embryo mediums.
As a preferred embodiment, the content of the human serum albumins described in 100ml single step embryo mediums is 0.48-
0.53g/L；The content of fungicide is 48-53mg/L in 100ml single step embryo mediums.
As a preferred embodiment, the content of the human serum albumins described in 100ml single step embryo mediums is 0.5g/L；
The content of fungicide is 50mg/L in 100ml single step embryo mediums.
Further, the inorganic salt buffer includes sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, phosphorus
The solution of acid dihydride potassium and sodium bicarbonate composition；Sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, biphosphate
The molar ratio of potassium and sodium bicarbonate is 89.937-109.923:5-6.083:0.9-1.1:1.62-1.98:0.135-0.165:
0.135-0.165:0.576-0.704。
As a preferred embodiment, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate and carbonic acid
The molar ratio of hydrogen sodium is 101.278-108.455:5.547-6.083:0.1-1.1:1.72-1.88:0.145-0.165:
0.145-0.165:0.612-0.704。
The second object of the present invention is to provide a kind of preparation method of above-mentioned single step embryo medium, and feature exists
In including the following steps：
1) it is prepared under the conditions of hundred grades, it is successively that sodium chloride, potassium chloride, magnesium sulfate, glucose, celebrating is big mould according to the above ratio
Element, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, taurine, D-glucitol, calcium chloride, sodium dihydrogen phosphate, phosphorus
Acid dihydride potassium, sodium bicarbonate, (±)-alpha-lipoic acid, acetyl L-carnitine hydrochloride or acetyl L-carnitine and half Guangs of N- acetyl group-L-
Propylhomoserin is added in aqueous medium, and solution I is obtained after mixing；
2) CO is rushed in solution I2Protect gas；
3) CO is being rushed2The amino acid and human serum albumins of above-mentioned component are added in during protection gas, is obtained after mixing molten
Liquid II；
4) by after 0.2 μm of membrane filtration degerming of the solution II that step 3) has been configured in CO25-10 is balanced in incubator
Hour；
5) solution II for having balanced step 4) is in CO2It protects gas aseptic subpackaged, obtains single step embryo medium.
Further, the middle punching of step 2) is the CO that percentage by volume is 6-7%2；CO in step 5)2Protection gas is volume hundred
Score is the CO of 6-7%2。
In 6-7%CO2It protects under gas effect, inorganic salts buffer composition should not decompose, and help quickly to adjust pH value, extend
The term of validity.
Further, the condition balanced in incubator in step 4) is：37 DEG C, 6% CO2It is small that 5-10 is balanced in incubator
When, PH is adjusted in 7.20-7.40.
As a preferred embodiment, configuration vessel are passed through 250 DEG C before step 1), 30min is xeothermic to remove heat source.
The third object of the present invention is to provide a kind of above-mentioned single step embryo medium in vitro in fertilization preparations
Using.
The present invention also aims to provide a kind of side using above-mentioned single step embryo medium extracorporeal culturing embryo
Method includes the following steps：
1) using micro drop method culture, the liquid droplet surface covering culture oil in culture vessel, in CO2In incubator
Balance 4~18 hours；
2) embryo is placed in Balanced culture solution, in 37 DEG C, 5%CO2, cultivate in the incubator of saturated humidity.
The beneficial effects of the present invention are：
1) there are the internal oxygen radical of removing, chelating provided by the present invention for preparing the composition of single step embryo medium
Metal ion regenerates other internal antioxidants to play antioxidation and protection mitochondrial function, adjust energetic supersession,
Prevent cytotoxic accumulation and the effect of protection cell.
2) single step embryo medium provided by the invention remains the active ingredient that embryo is secreted into culture solution, can rise
To protection cell and inhibit to decompose the effect using noxious products, avoid the mechanical damage that may be subject to during embryo transfer and
The problem of Blastocyst formation rate is not high caused by stress reaction caused by culture environment difference.The single step Embryo Culture of the present invention
The Blastocyst formation rate of liquid culture is high, Blastocyst formation rate >=94%.
3) this hair prepare single step embryo medium method it is easy to operate, 6-7%CO is rushed in preparation process2Protection
Gas, in 6-7%CO2It protects under gas effect, inorganic salts buffer composition should not decompose, and help quickly to adjust pH value, extend effective
Phase.
Specific embodiment
It detailed description of a preferred embodiment of the present invention will be given below.The reality of actual conditions is not specified in preferred embodiment
Proved recipe method, usually according to normal condition, illustrated embodiment are to preferably be illustrated to present disclosure, but are not
Present disclosure is only limitted to illustrated embodiment.So those skilled in the art according to foregoing invention content to embodiment party
Case carries out nonessential modifications and adaptations, still falls within protection scope of the present invention.
1 single step embryo medium of embodiment
2 single step embryo medium of embodiment
3 single step embryo medium of embodiment
1 single step embryo medium of comparative example
Comparative example 2
Critical component A, B, C are not added with, other are same as Example 1
| Material | Hundred grades of conditions |
| Sodium chloride | 99.93mmol/L |
| Potassium chloride | 5.53mmol/L |
| Magnesium sulfate | 1mmol/L |
| Glucose | 1mmol/L |
| Gentamicin | 50mg/L |
| Ethylenediamine tetra-acetic acid | 0.01mmol/L |
| Sodium Pyruvate | 10.51mmol/L |
| Alanyl glutamine | 27.49mmol/L |
| Taurine | 0.48mmol/L |
| D-glucitol | 0.23mmol/L |
| Calcium chloride | 1.8mmol/L |
| Sodium dihydrogen phosphate | 0.15mmol/L |
| Potassium dihydrogen phosphate | 0.15mmol/L |
| Sodium bicarbonate | 0.64mmol/L |
| Essential amino acid | 0.7mmol/L-2.3mmol/L |
| Nonessential amino acid | 0.5mmol/L-1.5mmol/L |
| Human serum albumins | 0.5g/L |
Comparative example 3
Only add critical component A, other are same as Example 1
Comparative example 4
Only add critical component B, other are same as Example 1
Comparative example 5
Only add critical component C, other are same as Example 1
The preparation method of 4 single step embryo medium of embodiment
The single step embryo medium of Examples 1 to 3 and comparative example 1~5 clicks method preparation, includes the following steps：
1) configuration vessel are passed through 250 DEG C, 30min is xeothermic to remove heat source.
2) it prepares under the conditions of hundred grades, is added in ultra-pure water in above-mentioned respective ratio successively material precise, mix
Solution I is obtained afterwards；
3) 6-7%CO is rushed in solution I2Protect gas；
4) 6-7%CO is being rushed2The amino acid and human serum albumins of above-mentioned component are added in during protection gas, after mixing
Obtain solution II；
5) 37 DEG C, 6% CO will placed after 0.2 μm of membrane filtration degerming of the solution II that step 3) has been configured2Training
It supports and is balanced 5-10 hours in case, this step is used for adjusting PH in the range of 7.20-7.40；
6) solution II for having balanced step 4) is in 6-7%CO2It protects gas aseptic subpackaged, obtains single step embryo medium.
5 single step embryo medium Indexs measure of embodiment
First, the single step embryo medium of the present invention is detected
The single step dispensed culture solution is taken to be detected, detection project is pH value, osmotic pressure, cytotoxicity, intradermal thorn
Sharp, sensitization, sterile, pyrogen, bacterial endotoxin, external mouse embryo are tested to judge whether spilting of an egg culture solution is qualified.
2nd, single step culture solution detection method and result
(1) pH value detects
A certain amount of finished product is taken to be detected the value recorded three times and the pH being averaged as final liquid with blood gas analyzer
Value is qualified in the range of.
(2) osmotic pressure
25 μ L liquid is taken to instill 0.5mlEP pipes and are put into osmometer (freezing point osmotic pressure gauge) probe waiting detection value stabilization,
Readout value, same method detect three times osmotic pressure being averaged as the liquid, are qualified in the range of.
(3) cytotoxicity
It is carried out by the regulation of GB/T 16886.5, cytotoxicity score should be no more than 1 point.
(4) intradermal stimulation
It is carried out by the regulation of GB/T 16886.10, without intradermal stimulate the reaction.
(5) sensitization
It is carried out by the regulation of GB/T 16886.10, it should be without sensitivity response.
(6) Sterility testing
It is carried out using the membrane-filter procedure of the Sterility testing of Chinese Pharmacopoeia, the filter membrane after filtering is in the culture of bacterium and fungi
It is sterile qualification that asepsis growth is answered in culture in base.
(7) pyrogen
According to two annex of Pharmacopoeia of People's Republic of China version in 2010, Ⅺ D prescriptive procedures, liquid in vitro fertilization answers apyrogeneity.
(8) detection of bacterial endotoxin
It is carried out using the reagents gel method in the endotoxic detection method of Chinese Pharmacopoeia, is judged to be less than 1EU/mL conjunctions surely
Lattice；
(9) external mouse embryo experiment
1. superfecundation
6~8 week old female mices are selected, through PMSG 10IU/ are injected intraperitoneally only；Through hCG 10IU/ are injected intraperitoneally only after 48h, note
HCG same day female mice is penetrated with being mated overnight with strain hero mouse.
2. prepare culture dish
The liquid droplet of 30~50 μ L sizes of certain amount, surface covering training are prepared before culture embryo in Tissue Culture Dish
Foster oil, in CO2It is pre-equilibrated 4~18 hours in incubator.
3. mouse embryo acquires
The next morning inspection mating situation is mated, selection is shown in that bolt mouse is spare.
1- cell stages are collected：It is put to death after 18 to 22 hours after injection hCG and sees bolt female mice, 1- is collected in ampulla of uterine tube
Cell stage.The cotton-shaped fertilized eggs group being collected into is placed into 37 DEG C of hyaluronidases preheated in advance, around embryo
Ovarian cumulus and granular cell be digested after separation after shifting, cleaning immediately, pick out the fertilized embryo of normal morphology, be transferred to confession
In test product spilting of an egg culture solution droplet, detect and test for 1- cell mouse embryo.
4. in vitro culture
Using micro drop method culture, by the mouse embryo being collected into be randomly divided into a positive controls, a negative control group and
One test sample group, is placed in Balanced culture solution, in 37 DEG C, 5%CO2, cultivate in the incubator of saturated humidity；Every group of mouse
Embryo number is no less than 50.
5. result of the test
1- cell stages record blastaea quantity after cultivating 96 hours respectively in vitro.
Observation index：Blastaea morphologic observation.
Acceptable value：
1) the Blastocyst formation rate of positive controls is substantially less than negative control group through statistical analysis；
2) Blastocyst formation rate >=80% of negative control group.
6 culture solution culture fertilized eggs Blastocyst formation of embodiment is tested
Utilize Examples 1 to 3 and 100 fertilized eggs record Blastocyst formation results of culture solution culture of comparative example 1~5：
1. the culture solution culture fertilized eggs that embodiment 1 is prepared, observation is as a result, be three groups of repetition experimental datas below：
| Fertilized eggs number | Blastocyst formation number |
| 100 | 97 |
| 100 | 98 |
| 100 | 96 |
2. the culture solution culture fertilized eggs that embodiment 2 is prepared, observation is as a result, be three groups of repetition experimental datas below：
| Fertilized eggs number | Blastocyst formation number |
| 100 | 95 |
| 100 | 94 |
| 100 | 96 |
3. the culture solution culture fertilized eggs that embodiment 3 is prepared, observation is as a result, be three groups of repetition experimental datas below：
| Fertilized eggs number | Blastocyst formation number |
| 100 | 94 |
| 100 | 95 |
| 100 | 94 |
4. the culture solution culture fertilized eggs that comparative example 1 is prepared, observation is as a result, be three groups of repetition experimental datas below：
| Fertilized eggs number | Blastocyst formation number |
| 100 | 78 |
| 100 | 76 |
| 100 | 78 |
5. comparative example 2 does not add the culture solution culture fertilized eggs of critical component A, B, C preparation, observation is as a result, be three below
Group repeats experimental data：
| Fertilized eggs number | Blastocyst formation number |
| 100 | 60 |
| 100 | 63 |
| 100 | 61 |
6. 3 addition critical component A of comparative example, and additive amount is incremented by successively, see the table below data and result：
7. 4 addition critical component B of comparative example, and additive amount is incremented by successively, see the table below data and result：
8. 5 addition critical component C of comparative example, and additive amount is incremented by successively, see the table below data and result：
According to more than experimental result as it can be seen that the culture solution of present invention critical component A in incubation, is phase between B, C
It is auxiliary to coordinate, the content direct relation Blastocyst formation rate of especially critical component A, and each material linear phase in a certain range
Close, without critical component A, the culture solution Blastocyst formation rate of B, C are low, ＜ 63%, it is seen that critical component A, B, C to Blastocyst formation all
There is apparent physiological action.Containing critical component A, the culture solution Blastocyst formation rate of B, C are high, Blastocyst formation rate >=94%.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail in good embodiment, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (10)
1. be used to prepare the composition of single step embryo medium, which is characterized in that the composition by (±)-alpha-lipoic acid,
Acetyl L-carnitine or acetyl L-carnitine hydrochloride, n-acetyl-L-cysteine composition；(±)-alpha-lipoic acid, acetyl L-
Carnitine or acetyl L-carnitine hydrochloride, the molar ratio of n-acetyl-L-cysteine are 0.055-0.098：0.044-0.078：
0.702-0.858。
2. composition according to claim 1, which is characterized in that (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl
L-carnitine hydrochloride, n-acetyl-L-cysteine molar ratio be 0.065-0.098：
0.054-0.078：0.752-0.858.
3. single step embryo medium, which is characterized in that by (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloric acid
Salt, n-acetyl-L-cysteine and basic culture solution composition, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L- meat
The molar ratio of alkali salt hydrochlorate, n-acetyl-L-cysteine and basic culture solution is 0.055-0.098：0.044-0.078：
0.702-0.858：135.251-167.614.
4. single step embryo medium according to claim 3, which is characterized in that the basic culture solution includes inorganic salts
Buffer solution, amino acid, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, taurine, glucose, D-glucitol, people
Seralbumin and fungicide.
5. single step embryo medium according to claim 4, which is characterized in that the inorganic salt buffer, amino acid,
Ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, taurine, glucose, D-glucitol molar ratio be 98.303-
120.12:1.2-3.8:0.009-0.012:9.459-11.561:24.741-30.239:0.432-0.528:0.9-1.1:
0.207-0.254。
6. single step embryo medium according to claim 4, which is characterized in that in 100ml single step embryo mediums
Described in human serum albumins content be 0.45-0.55g/L；The content of fungicide in 100ml single step embryo mediums
For 45-55mg/L.
7. single step embryo medium according to claim 4, which is characterized in that the inorganic salt buffer includes chlorination
The solution that sodium, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium bicarbonate form；Sodium chloride, chlorination
Potassium, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium bicarbonate molar ratio be 89.937-109.923:5-
6.083:0.9-1.1:1.62-1.98:0.135-0.165:0.135-0.165:0.576-0.704。
8. the preparation method of claim 3~7 any one of them single step embryo medium, which is characterized in that including following
Step：
1) prepared under the conditions of hundred grades, according to the above ratio successively by sodium chloride, potassium chloride, magnesium sulfate, glucose, gentamicin,
Ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, taurine, D-glucitol, calcium chloride, sodium dihydrogen phosphate, di(2-ethylhexyl)phosphate
Hydrogen potassium, sodium bicarbonate, (±)-alpha-lipoic acid, acetyl L-carnitine hydrochloride or acetyl L-carnitine and n-acetyl-L-cysteine
It is added in aqueous medium, solution I is obtained after mixing；
2) CO is rushed in solution I2Protect gas；
3) CO is being rushed2The amino acid and human serum albumins of above-mentioned component are added in during protection gas, solution II is obtained after mixing；
4) by after 0.2 μm of membrane filtration degerming of the solution II that step 3) has been configured in CO2It is balanced 5-10 hours in incubator；
5) solution II for having balanced step 4) is in CO2It protects gas aseptic subpackaged, obtains single step embryo medium.
9. preparation method according to claim 8, which is characterized in that it is 6-7% that punching, which is percentage by volume, in step 2)
CO2；CO in step 5)2Protection gas is the CO that percentage by volume is 6-7%2。
10. preparation method according to claim 8, which is characterized in that the condition balanced in incubator in step 4) is：37
DEG C, 6% CO2It is balanced 5-10 hours in incubator, adjusts PH in 7.20-7.40.
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| CN110452868A (en) * | 2019-05-14 | 2019-11-15 | 成都艾伟孚生物科技有限公司 | A kind of single step embryo medium |
| 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 |
| CN111172099A (en) * | 2020-03-06 | 2020-05-19 | 广州裕康生物科技有限公司 | Gamete buffer solution and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679895A (en) * | 2018-12-18 | 2019-04-26 | 西安交通大学 | The embryo medium and Embryo Culture method of the excretion body of source containing fallopian tubal |
| CN110452868A (en) * | 2019-05-14 | 2019-11-15 | 成都艾伟孚生物科技有限公司 | A kind of single step embryo medium |
| 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 |
| CN111172099A (en) * | 2020-03-06 | 2020-05-19 | 广州裕康生物科技有限公司 | Gamete buffer solution and preparation method thereof |
| CN111172099B (en) * | 2020-03-06 | 2023-12-29 | 广州辅康生物科技有限公司 | Gamete buffer solution and preparation method thereof |
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| CN108251354B (en) | 2021-04-06 |
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