CN110452868A - A kind of single step embryo medium - Google Patents
A kind of single step embryo medium Download PDFInfo
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Abstract
The invention discloses a kind of single step embryo mediums, single step embryo medium in the present invention, include the substance to play an important role to Embryo Culture and development: (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride and insulin, transferrins, selenium, Fetuin-A, platelet derived growth factor PDGF (Platelet derived growth factor, PDGF).The advantageous component being secreted into culture solution in Embryo Culture growth course had both been remained using single step embryo medium prepared by this group of substance; play the role of protection to promote cell development and inhibit cell disintegration using noxious products, the not high problem of Blastocyst formation rate caused by turn avoiding mechanical damage that embryo may be subject to during the transplantation process and stress reaction caused by culture environment changes.
Description
Technical field
The invention belongs to embryonic cell culture fields, and in particular to a kind of single step embryo medium.
Background technique
IVF, in vitro fertilization (in vitro fertilization): also known as test-tube baby refers to ovum and essence respectively
After son takes out, being placed in test tube makes its fertilization, then by embryonic precursors --- zygote transplation returns in maternal uterine and develops into fetus.
IVF culture is generally divided into two kinds of processes i.e. two kinds of culture solution series: whole (list/single step culture solution series) and dividing
Journey (double culture solution series).Using double culture solution series processes to embryo's early and late stagess provide different culture solutions at
Part, but it is also lost the important effective ingredient being secreted into culture solution by embryo, such as the embryotrophy factor.Whole culture solution system
Column process remains these important effective ingredients, but also remains harmful decomposition product in culture solution.
Summary of the invention
In view of the above-mentioned problems, the purpose of the present invention is to provide a kind of single step embryos for embryo in vitro culture development
Important substance needed for culture solution preparation.Single step embryo medium of the invention remains embryo and is secreted into having in culture solution
Ingredient is imitated, can play the role of that cell is protected to promote the growth and development of embryo's high efficiency and cell disintegration is inhibited to utilize noxious products.
To achieve the above object, the technical solution of the present invention is as follows:
It is used to prepare the important composition of single step embryo medium, the composition is by (±)-alpha-lipoic acid, acetyl L-
Carnitine or acetyl L-carnitine hydrochloride and insulin, transferrins, selenium, Fetuin-A, platelet derived growth factor PDGF
(Platelet derived growth factor, PDGF) composition.
Critical component is serum substitute, by insulin, transferrins, selenium, Fetuin-A, human blood platelets source growth factor
PDGF composition:
Insulin is by the beta Cell of islet in pancreas by endogenous or exogenous material such as glucose, lactose, ribose, essence
The stimulation of propylhomoserin, glucagon etc. and a kind of proteohormone secreted, can promote cell to glucose in culture solution
Metabolism utilizes, and promotes the fat in cell, protein to synthesize, promotes K+, Mg in the culture solution of periphery2+Enter across cell membrane thin
It is intracellular, promote the synthesis of DNA (DNA), ribonucleic acid (RNA) and atriphos (ATP), to embryonic cell body
Outer development has important facilitation.
Transferrins is main siderophillin in blood plasma, is responsible for iron and dropped by red blood cell that delivery is absorbed by digest tube
The iron for solving release, provides required iron for cell internalizing and cell metabolism.All cell growths all be unable to do without transferrins.No
But maintain normal cell metabolism depend in the form of bioactivity existing for iron, the confactor of iron or some enzymes, such as
RNA polymerase, DNA synzyme, while being also the important component of hemoglobin.
Selenium is the important composition of some antioxidases (glutathione peroxidase) and selenium-P albumen in animal and human body
Part plays a part of balance oxidation reduction atmosphere in vivo, improves the immunity of the human body.Selenium content in tissue is ten million
/ mono-, but it determines the presence of life, and the great function to human health is that other substances are irreplaceable, selenium deficiency
Human immunological competence's decline can be directly resulted in, by as the essential trace elements of the human body.
Fetuin-A is the glycoprotein of 59kD synthesized by liver a kind of, belongs to cystatin superfamily protein (half Guang ammonia
Pepsin inhibitor family) in a member and serum in one of most important albumen, there is multiple biological function, packet
Include bone formation, fetal brain and tissue development, external promotion cell Proliferation etc..
PDGF is a kind of important factor,mitogenic, has the ability of stimulation specific cells group division growth.
(±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, insulin, transferrins, selenium,
Fetuin-A, platelet derived growth factor PDGF molal weight ratio be 550.0-980.0:440.0-780.0:0.0138-
0.0197:0.0006-0.0008:0.0003-0.0005:0.0002-0.0004:0.00000 02-0.0000005.
The second object of the present invention is to provide a kind of single step embryo medium, by focusing on to Embryo Culture and develop
The weight ratio of the composition and basic culture solution to be acted on composition, composition and basic culture solution is 0.1%-1%.
Preferably, 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.
Preferably, the inorganic salt buffer, amino acid, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine,
Taurine, glucose, D-glucitol, N-acetyl-L-cysteine, N- (2- ethoxy) piperazine-N'-2- ethane sulfonic acid/4- hydroxyl
Ethyl piperazidine ethanesulfonic acid, 3- (N- morpholinyl) propane sulfonic acid sodium salt molar ratio be 98.303-120.12:1.2-3.8:0.009-
0.012:9.459-11.561:24.741-30.239:0.432-0.528:0.9-1.1:0.2 07-0.254:0.702-
0.858:8.125-10.239: 8.125-10.239。。
The inorganic salt buffer includes sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate
With the solution of sodium bicarbonate composition；Sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate and carbonic acid
The molar ratio of hydrogen sodium is 89.937-109.923:5-6.083:0.9-1.1:1.62-1.98:0.135-0.165:0.1 35-
0.165:0.576-0.704。
Preferably, the content of the human serum albumins is 0.45-0.55g/L, and the content of fungicide is 5.0-
20.0mg/L。
Preferably, the fungicide is gentamicin.
The third object of the present invention is to provide a kind of preparation method of single step embryo medium, comprising the following steps:
1) it is prepared under the conditions of hundred grades, according to the above ratio successively by sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, di(2-ethylhexyl)phosphate
Hydrogen sodium, potassium dihydrogen phosphate, sodium bicarbonate, N- (2- ethoxy) piperazine-N'-2- ethane sulfonic acid/4- hydroxyethyl piperazineethanesulfonic acid
HEPES, 3- (N- morpholinyl) propane sulfonic acid sodium salt MOPS-Na, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, ox sulphur
Acid, glucose, D-glucitol, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, amino acid are added to ultrapure
In water, solution I is obtained after mixing；The amino acid includes a. essential amino acid: Valine, L-Leu, l-Isoleucine, L-
One of phenylalanine, L-Trp, L-Methionine, L-lysine, L-threonine or several or its salt form；b.
Nonessential amino acid includes: glycine, l-Alanine, L-PROLINE, Serine, l-tyrosine, L-cysteine, L- asparagus fern
One of amide, L-Glutamine, L-Aspartic acid, Pidolidone, L-arginine, L-Histidine or several or its salt
Form.
2) CO2 protection gas is rushed in solution I；
3) human serum albumin, insulin, transferrins, selenium, Fetuin-A, blood platelet source is added after rushing CO2 protection gas
Growth factor PDGF obtains solution II after mixing；
4) 5-10 will be balanced in CO2 incubator after the membrane filtration degerming of 0.2um of step 3) configured solution II
Hour；
5) solution II for having balanced step 4) is aseptic subpackaged in CO2 protection gas, obtains single step embryo medium.
Preferably, the middle punching of step 2) is the CO2 that percentage by volume is 6-7%；It is volume hundred that CO2, which protects gas, in step 5)
Score is the CO2 of 6-7%.
Preferably, the condition balanced in incubator in step 4) are as follows: 37 DEG C, balance 5-10 is small in 6% CO2 incubator
When, PH is adjusted in 7.20-7.40.
Beneficial effects of the present invention:
1) single step embryo medium provided by the invention remains the effective component that embryo is secreted into culture solution, can rise
To protection cell and inhibit to decompose the effect for utilizing noxious products, avoid the mechanical damage that may be subject to during embryo transfer and
The not high problem of Blastocyst formation rate caused by stress reaction caused by culture environment is different.Single step Embryo Culture of the invention
The embryonic blastula formation rate of liquid culture can be up to 95%；
2) single step embryo medium provided by the invention is added to serum substitute: insulin, transferrins, selenium,
Fetuin-A, human blood platelets source growth factor PDGF, simulation human serum ingredient and in vivo pregnant environment, using restructuring material,
Utmostly protect and promote the safe, normal of embryo in vitro, fast-growth development.
Specific embodiment
Technical effect in order to further illustrate the present invention is specifically described the present invention below by embodiment.
Embodiment 1
Single step embryo medium
| Name of material | Formulation content |
| Sodium chloride | 99.937mmol/L |
| Potassium chloride | 5.531mmol/L |
| Magnesium sulfate | 1.0mmol/L |
| Glucose | 1.0mmol/L |
| Gentamicin | 9.5mg/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 |
| (±)-alpha-lipoic acid | 0.078mmol/L |
| Acetyl L-carnitine hydrochloride or acetyl L-carnitine | 0.078mmol/L |
| N-acetyl-L-cysteine | 0.785mmol/L |
| 4- hydroxyethyl piperazineethanesulfonic acid HEPES | 9.225mmol/L |
| 3- (N- morpholinyl) propane sulfonic acid sodium salt MOPS-Na | 9.165mmol/L |
| Essential amino acid | 1.6mmol/L |
| Nonessential amino acid | 0.9mmol/L |
| Seralbumin | 0.5g/L |
| Serum substitute solution | 0.95%, v/v |
Embodiment 2
Single step embryo medium
| Name of material | Formulation content |
| Sodium chloride | 89.937mmol/L |
| Potassium chloride | 5.013mmol/L |
| Magnesium sulfate | 0.898mmol/L |
| Glucose | 0.899mmol/L |
| Gentamicin | 5.0mg/L |
| Ethylenediamine tetra-acetic acid | 0.009mmol/L |
| Sodium Pyruvate | 9.459mmol/L |
| Alanyl glutamine | 24.741mmol/L |
| Taurine | 0.432mmol/L |
| D-glucitol | 0.207mmol/L |
| Calcium chloride | 1.621mmol/L |
| Sodium dihydrogen phosphate | 0.135mmol/L |
| Potassium dihydrogen phosphate | 0.135mmol/L |
| Sodium bicarbonate | 0.576mmol/L |
| (±)-alpha-lipoic acid | 0.055mmol/L |
| Acetyl L-carnitine hydrochloride or acetyl L-carnitine | 0.044mmol/L |
| N-acetyl-L-cysteine | 0.702mmol/L |
| 4- hydroxyethyl piperazineethanesulfonic acid HEPES | 8.125mmol/L |
| 3- (N- morpholinyl) propane sulfonic acid sodium salt MOPS-Na | 8.125mmol/L |
| Essential amino acid | 0.7mmol/L |
| Nonessential amino acid | 0.5mmol/L |
| Seralbumin | 0.45g/L |
| Serum substitute solution | 0.85%, v/v |
Embodiment 3
Single step embryo medium
Comparative example 1
| Name of material | Formulation content |
| Sodium chloride | 99.937mmol/L |
| Potassium chloride | 5.531mmol/L |
| Magnesium sulfate | 1.0mmol/L |
| Glucose | 1.0mmol/L |
| Gentamicin | 9.5mg/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 |
| (±)-alpha-lipoic acid | 0.048mmol/L |
| Acetyl L-carnitine hydrochloride or acetyl L-carnitine | 0.081mmol/L |
| N-acetyl-L-cysteine | 0.785mmol/L |
| 4- hydroxyethyl piperazineethanesulfonic acid HEPES | 9.225mmol/L |
| 3- (N- morpholinyl) propane sulfonic acid sodium salt MOPS-Na | 9.165mmol/L |
| Essential amino acid | 1.6mmol/L |
| Nonessential amino acid | 0.9mmol/L |
| Seralbumin | 0.5g/L |
| Serum substitute solution | 0.95%, v/v |
Comparative example 2
Comparative example 3
Comparative example 4
| Name of material | Formulation content |
| Sodium chloride | 99.937mmol/L |
| Potassium chloride | 5.531mmol/L |
| Magnesium sulfate | 1.0mmol/L |
| Glucose | 1.0mmol/L |
| Gentamicin | 9.5mg/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 |
| (±)-alpha-lipoic acid | 0 |
| Acetyl L-carnitine hydrochloride or acetyl L-carnitine | 0.078mmol/L |
| N-acetyl-L-cysteine | 0.785mmol/L |
| 4- hydroxyethyl piperazineethanesulfonic acid HEPES | 9.225mmol/L |
| 3- (N- morpholinyl) propane sulfonic acid sodium salt MOPS-Na | 9.165mmol/L |
| Essential amino acid | 1.6mmol/L |
| Nonessential amino acid | 0.9mmol/L |
| Seralbumin | 0.5g/L |
| Serum substitute solution | 0.95%, v/v |
One, the single step embryo medium system of the preparation method embodiment 1-3 and comparative example 1-4 of single step embryo medium
Preparation Method, comprising the following steps:
1) configuration vessel are passed through 250 DEG C, 30min is xeothermic to remove heat source.
2) it is prepared under the conditions of hundred grades, in above-mentioned respective ratio, successively material precise is added in ultrapure water, mixing
Solution I is obtained afterwards；
3) 6-7%CO is rushed in solution I2Protect gas；
4) human serum albumin and serum substitute of respective component are added after rushing 6-7%CO2 protection gas, is obtained after mixing molten
Liquid II；
5) 37 DEG C, 6% CO will placed after 0.2 μm of the membrane filtration degerming of the configured solution II of step 3)2Training
It supports and is balanced 5-10 hours in case, this step is used to adjust PH within the scope 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.
Two, single step embryo medium Indexs measure
1, carrying out detection to single step embryo medium of the invention takes the single step culture solution dispensed to detect, and examines
Survey project is pH value, osmotic pressure, cytotoxicity, intradermal stimulation, sensitization, sterile, pyrogen, bacterial endotoxin, the test of external mouse embryo
To judge whether spilting of an egg culture solution is qualified.
2, single step culture solution detection method and result
(1) pH value detection takes a certain amount of finished product to be detected with blood gas analyzer, records detected value three times and is averaged
It is worth the final ph as single step embryo medium, is qualified within the scope of 7.20-7.40.
(2) osmotic pressure
Taking 25 μ L liquid instillation 0.5mlEP pipe to be put into osmometer (freezing point osmotic pressure gauge) probe waits detected value to stablize,
Readout value, same method detect three times and are averaged the osmotic pressure as the single step embryo medium, close in range
Lattice.
(3) cytotoxicity is carried out by the regulation of GB/T 16886.5, and cytotoxicity score should be no more than 1 point.
(4) intradermal stimulation 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 is carried out using the membrane-filter procedure of the Sterility testing of Chinese Pharmacopoeia, and filtered filter membrane is in bacterium
Answering asepsis growth with culture in the culture medium of fungi is sterile qualification.
(7) pyrogen
According to two annex of Pharmacopoeia of People's Republic of China version in 2010, Ⅺ D prescriptive procedure, liquid in vitro fertilization answers apyrogeneity.
(8) detection of bacterial endotoxin is carried out using the reagents gel method in the endotoxic detection method of Chinese Pharmacopoeia, is sentenced
It is set to and is less than 1EU/mL qualification；
(9) external mouse embryo test
1) superfecundation
6~8 week old female mices are selected, only through intraperitoneal injection PMSG 10IU/；After 48h only through intraperitoneal injection hCG 10IU/,
Injection hCG same day female mice mates overnight with strain hero mouse.
2) liquid for preparing 30~50 μ L size of certain amount before preparation culture dish culture embryo in Tissue Culture Dish is micro-
Drop, surface covering culture oil, pre-equilibrates 4~18 hours in CO2 incubator.
3) mouse embryo acquires
The next morning inspection mating situation is mated, selection is shown in that bolt mouse is spare.
1- cell stage is collected: being put to death after 18 to 22 hours after injection hCG and is seen bolt female mice, collects in ampulla of uterine tube
1- cell stage.The cotton-shaped fertilized eggs group being collected into is placed into 37 DEG C of hyaluronidases preheated in advance, when embryo's week
The ovarian cumulus and granular cell enclosed be digested separation after shift, clean immediately after, pick out the fertilized embryo of normal morphology, be transferred to
In test sample spilting of an egg culture solution droplet, for the detection test of 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, cultivates in 37 DEG C, 6%CO2,5%O2, the incubator of saturated humidity；
Every group of mouse embryo number is no less than 50.
5) test result
1- cell stage records blastaea quantity after cultivating 96 hours respectively in vitro.
Observation index: blastaea morphologic observation.
Acceptable value: the Blastocyst formation rate of positive controls is substantially less than negative control group through statistical analysis；It is negative
Blastocyst formation rate >=80% of control group.
Three, the experiment of culture solution culture fertilized eggs Blastocyst formation utilizes the culture solution culture of embodiment 1-3 and comparative example 1-4
100 fertilized eggs record Blastocyst formation result:
1. the culture solution culture fertilized eggs that embodiment 1 is prepared are observed as a result, the following are three groups of repetition experimental datas:
| Fertilized eggs number | Blastocyst formation number |
| 100 | 93 |
| 100 | 95 |
| 100 | 91 |
2. the culture solution culture fertilized eggs that embodiment 2 is prepared are observed as a result, the following are three groups of repetition experimental datas:
| Fertilized eggs number | Blastocyst formation number |
| 100 | 90 |
| 100 | 89 |
| 100 | 89 |
3. the culture solution culture fertilized eggs that embodiment 3 is prepared are observed as a result, the following are three groups of repetition experimental datas:
| Fertilized eggs number | Blastocyst formation number |
| 100 | 88 |
| 100 | 89 |
| 100 | 89 |
4. the culture solution culture fertilized eggs that comparative example 1 is prepared are observed as a result, the following are three groups of repetition experimental datas:
| Fertilized eggs number | Blastocyst formation number |
| 100 | 75 |
| 100 | 72 |
| 100 | 73 |
5. the culture solution culture fertilized eggs that comparative example 2 is prepared are observed as a result, the following are three groups of repetition experimental datas:
| Fertilized eggs number | Blastocyst formation number |
| 100 | 60 |
| 100 | 63 |
| 100 | 61 |
6. 3 addition (±)-alpha-lipoic acids of comparative example, and additive amount is incremented by successively, see the table below data and result:
7. 4 addition acetyl L-carnitines of comparative example or acetyl L-carnitine hydrochloride, and additive amount is incremented by successively, see the table below number
According to and result:
According to the above experimental result as it can be seen that culture solution of the invention material (±)-alpha-lipoic acid, acetyl during the cultivation process
L-carnitine or acetyl L-carnitine hydrochloride and serum substitute: insulin, transferrins, selenium, Fetuin-A, human blood platelets source
It is complementary, the especially content direct relation Blastocyst formation of material (±)-α-lipoic acid between growth factor PDGF
Rate, and each material is linear related in a certain range；Without material (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-
The culture solution Blastocyst formation rate of carnitine hydrochloride is low, and < 63%, it is seen that material (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl
L-carnitine hydrochloride has apparent physiological action to Blastocyst formation.The blastaea shape of single step embryo medium culture of the invention
At rate height, Blastocyst formation rate is up to 95%.
Finally, it should be noted that the above examples are only used to illustrate the technical scheme of the present invention rather than limits, although ginseng
Technical solution of the present invention is described in detail according to preferred embodiment, it will be appreciated by those skilled in the art that can be to this
The technical solution of invention is modified or replaced equivalently, and without departing from the purpose and scope of the invention, should all be covered at this
In the protection scope of invention.
Claims (10)
1. a kind of single step embryo medium, characterized by comprising:
A, the composition to play an important role to Embryo Culture and development
B, basic culture solution
The composition, including (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, insulin, turn iron egg
White, selenium, Fetuin-A, platelet derived growth factor PDGF.
2. single step embryo medium according to claim 1, which is characterized in that the composition and basic culture solution
Volume ratio is 0.1%-1%.
3. single step embryo medium according to claim 1, which is characterized in that (±)-α-sulphur is pungent in the composition
Acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, insulin, transferrins, selenium, Fetuin-A, platelet derived growth factor
The molal weight ratio of PDGF is 550.0-980.0:440.0-780.0:0.0138-0.0197:0.0006-0.0008:0.00 03-
0.0005:0.0002-0.0004:0.0000002-0.0000005.
4. single step embryo medium according to claim 1, which is characterized in that the basic culture solution includes inorganic salts
Buffer, amino acid, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, taurine, glucose, D-glucitol, N-
Acetyl-L-cysteine, N- (2- ethoxy) piperazine-N'-2- ethane sulfonic acid/4- hydroxyethyl piperazineethanesulfonic acid, 3- (N- morpholine
Base) propane sulfonic acid sodium salt, human serum albumins 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, half Guang ammonia of N- acetyl-L-
Acid, N- (2- ethoxy) piperazine-N'-2- ethane sulfonic acid/4- hydroxyethyl piperazineethanesulfonic acid, 3- (N- morpholinyl) propane sulfonic acid sodium salt
Molar ratio is 98.303-120.12:1.2-3.8:0.009-0.012:9.459-11.561:24.741-30 .239:0.432-
0.528:0.9-1.1:0.207-0.254:0.702-0.858:8.125-10.239:8.125-10.239.
6. single step embryo medium according to claim 4, which is characterized in that the content of the human serum albumins is
0.45-0.55g/L, the content of fungicide are 5.0-20.0mg/L.
7. single step embryo medium according to claim 4, which is characterized in that the fungicide is gentamicin.
8. the preparation method of the single step embryo medium as described in any one of claim 1-7 right, which is characterized in that including
Following steps:
1) it is prepared under the conditions of hundred grades, according to the above ratio successively by sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, biphosphate
Sodium, potassium dihydrogen phosphate, sodium bicarbonate, N- (2- ethoxy) piperazine-N'-2- ethane sulfonic acid/4- hydroxyethyl piperazineethanesulfonic acid
HEPES, 3- (N- morpholinyl) propane sulfonic acid sodium salt MOPS-Na, ethylenediamine tetra-acetic acid, Sodium Pyruvate, alanyl glutamine, ox sulphur
Acid, glucose, D-glucitol, (±)-alpha-lipoic acid, acetyl L-carnitine or acetyl L-carnitine hydrochloride, amino acid are added to ultrapure
In water, solution I is obtained after mixing；
2) CO is rushed in solution I2Protect gas；
3) CO is being rushed2Protect gas after be added human serum albumin, insulin, transferrins, selenium, Fetuin-A, platelet derived growth because
Sub- PDGF obtains solution II after mixing；
4) by after 0.22 μm of the membrane filtration degerming of the configured solution II of step 3) 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) are as follows: 37
DEG C, 6% CO2It is balanced 5-10 hours in incubator, adjusts PH in 7.20-7.40.
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