CN104140950A - Cleavage culture solution and preparation method thereof - Google Patents
Cleavage culture solution and preparation method thereof Download PDFInfo
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- CN104140950A CN104140950A CN201410390997.6A CN201410390997A CN104140950A CN 104140950 A CN104140950 A CN 104140950A CN 201410390997 A CN201410390997 A CN 201410390997A CN 104140950 A CN104140950 A CN 104140950A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000003776 cleavage reaction Methods 0.000 title abstract 6
- 230000007017 scission Effects 0.000 title abstract 6
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 14
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 238000012545 processing Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 78
- 235000015097 nutrients Nutrition 0.000 claims description 72
- 239000007788 liquid Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- 210000001161 mammalian embryo Anatomy 0.000 claims description 11
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 9
- 239000002158 endotoxin Substances 0.000 claims description 8
- 230000003204 osmotic effect Effects 0.000 claims description 7
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 7
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 7
- 239000012498 ultrapure water Substances 0.000 claims description 7
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 6
- 229930182566 Gentamicin Natural products 0.000 claims description 6
- 230000003013 cytotoxicity Effects 0.000 claims description 6
- 231100000135 cytotoxicity Toxicity 0.000 claims description 6
- 229960002518 gentamicin Drugs 0.000 claims description 6
- 206010070834 Sensitisation Diseases 0.000 claims description 5
- 230000008313 sensitization Effects 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 235000020776 essential amino acid Nutrition 0.000 claims description 4
- 239000003797 essential amino acid Substances 0.000 claims description 4
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims description 4
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
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- 239000002904 solvent Substances 0.000 claims description 3
- 230000000638 stimulation Effects 0.000 claims description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 2
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 2
- 235000004279 alanine Nutrition 0.000 claims description 2
- 108010044940 alanylglutamine Proteins 0.000 claims description 2
- 229960005261 aspartic acid Drugs 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- NGSFWBMYFKHRBD-UHFFFAOYSA-N sodium;2-hydroxypropanoic acid Chemical compound [Na+].CC(O)C(O)=O NGSFWBMYFKHRBD-UHFFFAOYSA-N 0.000 claims description 2
- 229960003080 taurine Drugs 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000003899 bactericide agent Substances 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 8
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- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
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- 210000002459 blastocyst Anatomy 0.000 description 2
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- 230000004720 fertilization Effects 0.000 description 2
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- 101100515508 Arabidopsis thaliana XI-D gene Proteins 0.000 description 1
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- 206010042573 Superovulation Diseases 0.000 description 1
- 241000239222 Tachypleus Species 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a cleavage culture solution and a preparation method thereof. The cleavage culture solution comprises a composition for processing and cultivating oosperms, a bactericide, a selectively added indicator and selectively added human serum albumin. Various indexes of the cleavage culture solution disclosed by the invention achieve the standard of an in vitro culture solution, the cleavage culture solution is safe to use, strong guarantee is provided for an assisted reproduction technology, and whether the culture solution goes bad or not is easily judged by adding the indicator, therefore, the cleavage culture solution is safe and convenient to use.
Description
Technical field
The present invention relates to human assistance reproductive technology, relate in particular to a kind of spilting of an egg nutrient solution and preparation method thereof.
Background technology
Spilting of an egg nutrient solution is cultivated and is widely used in tube-test baby techniques the division of zygote.The object of spilting of an egg nutrient solution is: one is to provide the nutrition of development of fertilized ova; The 2nd, for the growth of zygote provides necessary environment.The purposes of spilting of an egg nutrient solution: laboratory or clinical zygote first day to the cultivation of three days to people under carbon dioxide environment.
At present, the spilting of an egg nutrient solution dependence on import finished product in China's auxiliary procreation technology, the quality of the validity of the high and solution of cost can not well show, if still continue to use after nutrient solution is rotten, will cause irreversible result to experiment or clinical manipulation.
Summary of the invention
The standard that the object of the present invention is to provide a kind of indices to reach vitro culture liquid used safely, be easy to the spilting of an egg nutrient solution that judges that whether nutrient solution is rotten, for auxiliary procreation technology provides strong guarantee.
Spilting of an egg nutrient solution of the present invention, comprises zygote is processed to composition, the sterilant of cultivating.
Described sterilant is gentamicin, concentration 9.5-10.5mg/L.
In described composition, comprise sodium-chlor 90.25-99.75mmol/L, Repone K 2.375-2.625 mmol/L, magnesium sulfate 0.38-0.42mmol/L, glucose 0.19-0.21mmol/L, calcium chloride 0.19-0.21 mmol/L, ethylenediamine tetraacetic acid (EDTA) 0.012-0.014mmol/L, Sodium.alpha.-hydroxypropionate 1.4176-1.5669g/L, Sodium.alpha.-ketopropionate 0.19-0.21mmol/L, sodium bicarbonate 23.75-26.25mmol/L, Dipeptiven 0.95-1.05mmol/L, taurine 0.38-0.42mmol/L, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES 2.375-2.625mmol/L, non-essential amino acid 0.95-1.05%(v/v), gentamicin 9.5-10.5mg/L.
Described non-essential amino acid is one or several in ALANINE, altheine, L-Aspartic acid, Pidolidone, glycine, L-PROLINE, Serine.
The solvent of spilting of an egg nutrient solution adopts ultrapure water.
In spilting of an egg nutrient solution, contain human serum albumin 9.5-10.5%, v/v, can add or the front interpolation of packing in use.
In described spilting of an egg nutrient solution, can also selectivity add indicator phenol red, concentration 4.75-5.25mg/L.
The pH=7.20-7.40 of spilting of an egg nutrient solution of the present invention, osmotic pressure 260-290mOsm/Kg.
The preparation method of spilting of an egg nutrient solution of the present invention, comprises step below:
(1) preparation of basal liquid
A, elder generation proportionally weigh each component, and separated;
B, first load weighted component (except sterilant, selectivity add indicator, sodium bicarbonate, human serum albumin) is dissolved in ultrapure water, in dissolution process, follows liquid after first solid, NaHCO
3the principle finally adding;
C, to adding in step b gained solution load weighted sterilant, selectivity to add indicator and NaHCO
3;
D, the basal liquid that configures is placed to 37 ℃, 5.5% CO
2balance 6-12 hour in incubator, this step is used for regulating PH within the scope of 7.20-7.40.
(2) stoste of spilting of an egg nutrient solution preparation
A, the basal liquid of spilting of an egg nutrient solution is utilized under Bechtop to the membrane filtration degerming of 0.2 μ m, obtain filtrate A;
B, in filtrate A, add human serum albumin, shake or be uniformly mixed, obtain the stoste of spilting of an egg nutrient solution; Or omit this step, the stoste using filtrate A as spilting of an egg nutrient solution, adds human serum albumin as required voluntarily when doctor uses.
(3) processing of spilting of an egg nutrient solution stoste
Under gnotobasis, the stoste of spilting of an egg nutrient solution is carried out to coating-dividing sealing, obtain commercially available spilting of an egg nutrient solution, placed in refrigerator or freezer and preserve.
(4) spilting of an egg nutrient solution detects
Get the spilting of an egg nutrient solution minute installing and detect, test item is that pH value, osmotic pressure, cytotoxicity, intracutaneous stimulation, sensitization, aseptic, pyrogen, bacterial endotoxin, external mouse embryo test to judge that whether spilting of an egg nutrient solution is qualified.
The invention has the beneficial effects as follows, 1, the raw material of spilting of an egg nutrient solution of the present invention is all materials of verified its security, in preparation process, its solvent adopts ultrapure water, and (TOC) is ultralow for the total organic carbon of ultrapure water, has avoided too much affecting because of impurity the result of mensuration.2, use the usually growth of anti-bacteria of antibiosis, if gentamicin is the microbiotic of a kind of wide spectrum, Heat stability is good, in spilting of an egg nutrient solution, add the effectively growth of anti-bacteria of gentamicin.3, the domestic nutrient solution for tube-test baby techniques that also there is no moulding, this formula and preparation method provide strong assurance for domestic tube-test baby techniques, the not clear factor of having avoided external product to bring because of long-distance transport, blending process is simple to operate rationally simultaneously, detect tightly, can provide fresh nutrient solution for reproductive center or scientific research institutions.4, in spilting of an egg nutrient solution, selectivity adds indicator phenol red, phenol red when pH changes colour-change obvious, pH is at 7.20-7.40, in scope, phenol red solution is that pink is to orange red, it when pH value is less than 6.8, is yellow, it when pH value is greater than 8.4, is redness, variable color is obvious, the pH7.20-7.40 of spilting of an egg nutrient solution solution within the scope of this is that pink is to orange red, when spilting of an egg nutrient solution color is not inconsistent therewith, illustrate that this spilting of an egg nutrient solution is rotten, can not be for experiment or clinical, thus avoided using rotten spilting of an egg nutrient solution and the risk that produces.If do not contained phenol red indicator, solution is transparent, limpid, can guarantee before the deadline aseptic and normally use, and needs special concern product stock, transport condition and validity period.
Embodiment
The present invention is described further by the following examples.
One, spilting of an egg nutrient solution formula and preparation method
Table 1 spilting of an egg nutrient solution formula
Note: phenol red in formula is " 0 ", represents not contain in spilting of an egg nutrient solution phenol red; Human serum albumin is " 0 ", represents not add human serum albumin before packing, during use, adds.
The preparation method of spilting of an egg nutrient solution of the present invention, comprises step below:
(1) preparation of basal liquid
A, elder generation proportionally weigh each component, and separated;
B, first load weighted component (except sterilant, selectivity add indicator, sodium bicarbonate, human serum albumin) is dissolved in ultrapure water, in dissolution process, follows liquid after first solid, NaHCO
3the principle finally adding;
C, to adding in step b gained solution load weighted sterilant, selectivity to add indicator and NaHCO
3;
D, the basal liquid that configures is placed to 37 ℃, 5.5% CO
2balance 6-12 hour in incubator, this step is used for regulating PH within the scope of 7.20-7.40.
(2) stoste of spilting of an egg nutrient solution preparation
A, the basal liquid of spilting of an egg nutrient solution is utilized under Bechtop to the membrane filtration degerming of 0.2 μ m, obtain filtrate A;
B, in filtrate A, add human serum albumin, shake or be uniformly mixed, obtain the stoste of spilting of an egg nutrient solution; Or omit this step, the stoste using filtrate A as spilting of an egg nutrient solution, adds human serum albumin as required voluntarily when doctor uses.
(3) processing of spilting of an egg nutrient solution stoste
A, under gnotobasis, the stoste of spilting of an egg nutrient solution is carried out to coating-dividing sealing, obtain commercially available spilting of an egg nutrient solution, placed in refrigerator or freezer and preserve.
(4) spilting of an egg nutrient solution detects
Get the spilting of an egg nutrient solution minute installing and detect, test item is that pH value, osmotic pressure, cytotoxicity, intracutaneous stimulation, sensitization, aseptic, pyrogen, bacterial endotoxin, external mouse embryo test to judge that whether spilting of an egg nutrient solution is qualified.
two, spilting of an egg nutrient solution detection method and result
(1) pH value detects
Get a certain amount of finished product and carry out the value of detection record three times and average as the pH value of final liquid with pH meter, be qualified in scope.
(2) osmotic pressure
Get and on the filter paper that 10 μ L fluid drips enter osmometer (dew point osmometer) probe, probe is pushed to instrument and wait for that detected value is stable, read numerical value, same method detects to average for three times and is the osmotic pressure of this liquid, is qualified in scope.
(3) cytotoxicity
By the regulation of GB/T 16886.5, undertaken, cytotoxicity score should be no more than 1 minute.
(4) intracutaneous stimulates
By the regulation of GB/T 16886.10, undertaken, without intracutaneous irritant reaction.
(5) sensitization
By the regulation of GB/T 16886.10, undertaken, should be without sensitivity response.
(6) aseptic detection
Adopt the membrane-filter procedure of the aseptic detection of Chinese Pharmacopoeia to carry out, the filter membrane after filtration is cultivated and answered asepsis growth in the substratum of bacterium and fungi is aseptic qualified.
(7) pyrogen
According to two appendix XI D prescriptive procedures of Pharmacopoeia of People's Republic of China version in 2010, liquid in vitro fertilization is answered pyrogen-free.
(8) detection of bacterial endotoxin
Adopt the tachypleus amebocyte lysate gel method in the endotoxic detection method of Chinese Pharmacopoeia to carry out, be judged to that to be surely less than 1.0EU/mL qualified;
(9) external mouse embryo test
1. superovulation
Select female mouse in 6 ~ 8 week age, through abdominal injection PMSG 10 IU/ only; 48 h by abdominal injection hCG 10 IU/ only, the injection hCG same day female mouse with the male mouse of strain, mate and spend the night.
2. prepare culture dish
Cultivate the front liquid droplet of preparing some amount 30～50 μ L sizes in Tissue Culture Dish of embryo, surface coverage is cultivated with oil, at CO
2in incubator, pre-equilibration is 4 ~ 18 hours.
3 mouse embryos gather
Mate and check mating situation the next morning, select to see that bolt mouse is standby.
1-cell stage is collected: after injection hCG, after 18 to 22 hours, put to death and see the female mouse of bolt, at ampulla of uterine tube, collect 1-cell stage.The cotton-shaped zygote group of collecting is placed into 37 ℃ to shift to an earlier date in preheated Unidasa, when ovarian cumulus around of embryo and granulosa cell is digested shift immediately, clean after separated after, pick out the fertilization embryo of normal morphology, transfer in trial-product spilting of an egg nutrient solution droplet, for 1-cell mouse embryo, detect test.
4 vitro culture
Employing micro drop method is cultivated, and the mouse embryo of collecting is divided into a positive controls, a negative control group and a trial-product group at random, is placed in Balanced nutrient solution, in 37 ℃, and 5% CO
2, in the incubator of saturated humidity, cultivate.Every group of mouse embryo number is no less than 50.
5 test-results
The external cultivation respectively after 96 hours of 1-cell stage recorded blastaea quantity.
Observation index: blastaea morphologic observation.
Acceptable value:
1) the Blastocyst formation rate of positive controls by statistics Epidemiological Analysis significantly lower than negative control group;
2) Blastocyst formation rate >=80 % of negative control group.
(10) detected result
| Project | Formula 1 | Formula 2 | Formula 3 | Formula 4 |
| PH value | 7.33 | 7.35 | 7.35 | 7.33 |
| Osmotic pressure mOsm/Kg | 265 | 273 | 280 | 270 |
| Cytotoxicity | Qualified | Qualified | Qualified | Qualified |
| Intracutaneous stimulates | Without intracutaneous, stimulate | Without intracutaneous, stimulate | Without intracutaneous, stimulate | Without intracutaneous, stimulate |
| Sensitization | Without sensitivity response | Without sensitivity response | Without sensitivity response | Without sensitivity response |
| Pyrogen | Pyrogen-free | Pyrogen-free | Pyrogen-free | Pyrogen-free |
| Aseptic detection | Aseptic | Aseptic | Aseptic | Aseptic |
| Detection of bacterial endotoxin | Qualified | Qualified | Qualified | Qualified |
| External mouse embryo test | Blastaea qualification rate 82% | Blastaea qualification rate 86% | Blastaea qualification rate 81% | Blastaea qualification rate 85% |
Claims (8)
1. spilting of an egg nutrient solution, is characterized in that: comprise zygote is processed to composition, the sterilant of cultivating.
2. spilting of an egg nutrient solution according to claim 1, is characterized in that, described sterilant is gentamicin, concentration 9.5-10.5mg/L.
3. spilting of an egg nutrient solution according to claim 1, it is characterized in that, in described composition, comprise sodium-chlor 90.25-99.75mmol/L, Repone K 2.375-2.625 mmol/L, magnesium sulfate 0.38-0.42mmol/L, glucose 0.19-0.21mmol/L, calcium chloride 0.19-0.21 mmol/L, ethylenediamine tetraacetic acid (EDTA) 0.012-0.014mmol/L, Sodium.alpha.-hydroxypropionate 1.4176-1.5669g/L, Sodium.alpha.-ketopropionate 0.19-0.21mmol/L, sodium bicarbonate 23.75-26.25mmol/L, Dipeptiven 0.95-1.05mmol/L, taurine 0.38-0.42mmol/L, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES 2.375-2.625mmol/L, non-essential amino acid 0.95-1.05%, v/v, gentamicin 9.5-10.5mg/L.
4. spilting of an egg nutrient solution according to claim 3, is characterized in that, described non-essential amino acid is one or several in ALANINE, altheine, L-Aspartic acid, Pidolidone, glycine, L-PROLINE, Serine.
5. spilting of an egg nutrient solution according to claim 1, is characterized in that, the solvent of spilting of an egg nutrient solution adopts ultrapure water.
6. according to the spilting of an egg nutrient solution described in claim 1-5 any one, it is characterized in that, in described spilting of an egg nutrient solution, contain human serum albumin 9.5%-10.5%, v/v, can add or the front interpolation of packing in use.
7. according to the spilting of an egg nutrient solution described in claim 1-5 any one, it is characterized in that, in described spilting of an egg nutrient solution, selectivity adds indicator phenol red, phenol red concentration 4.75-5.25mg/L.
8. the method for preparing claim 1-7 item spilting of an egg nutrient solution, is characterized in that comprising step below:
(1) preparation of basal liquid
A, elder generation proportionally weigh each component, and separated;
B, first the load weighted component the indicator adding except sterilant, selectivity, sodium bicarbonate, human serum albumin is dissolved in ultrapure water;
C, to adding in step b gained solution load weighted sterilant, selectivity to add indicator and NaHCO
3;
D, the basal liquid that configures is placed to 37 ℃, 5.5% CO
2balance 6-12 hour in incubator;
(2) stoste of spilting of an egg nutrient solution preparation
A, the basal liquid of spilting of an egg nutrient solution is utilized under Bechtop to the membrane filtration degerming of 0.2 μ m, obtain filtrate A;
B, in filtrate A, add human serum albumin, shake or be uniformly mixed, obtain the stoste of spilting of an egg nutrient solution; Or omit this step, the stoste using filtrate A as spilting of an egg nutrient solution, adds human serum albumin as required voluntarily when doctor uses;
(3) processing of spilting of an egg nutrient solution stoste
Under gnotobasis, the stoste of spilting of an egg nutrient solution is carried out to coating-dividing sealing, obtain commercially available spilting of an egg nutrient solution, placed in refrigerator or freezer and preserve;
(4) spilting of an egg nutrient solution detects
Get the spilting of an egg nutrient solution minute installing and detect, test item is that pH value, osmotic pressure, cytotoxicity, intracutaneous stimulation, sensitization, aseptic, pyrogen, bacterial endotoxin, external mouse embryo test to judge that whether spilting of an egg nutrient solution is qualified.
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| CN201410390997.6A CN104140950A (en) | 2014-08-08 | 2014-08-08 | Cleavage culture solution and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105994037A (en) * | 2016-05-24 | 2016-10-12 | 福建师范大学 | Goldfish egg indoor hatching culture method |
| CN108728403A (en) * | 2018-06-05 | 2018-11-02 | 瑞柏生物（中国）股份有限公司 | A kind of spilting of an egg culture solution and preparation method thereof |
| CN108753690A (en) * | 2018-06-05 | 2018-11-06 | 瑞柏生物（中国）股份有限公司 | One step culture solution of one kind and preparation method thereof |
| CN108753691A (en) * | 2018-06-05 | 2018-11-06 | 瑞柏生物（中国）股份有限公司 | A kind of blastocyst culture liquid and preparation method thereof |
| 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 |
| CN113755429A (en) * | 2021-09-29 | 2021-12-07 | 中国科学院苏州生物医学工程技术研究所 | Composition for preparing cleavage culture solution, cleavage culture solution and preparation method |
-
2014
- 2014-08-08 CN CN201410390997.6A patent/CN104140950A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 秦妍: ""不同培养液体系对小鼠卵母细胞孤雌激活的影响"", 《中国优秀硕士学位论文全文数据库-医药卫生科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105994037A (en) * | 2016-05-24 | 2016-10-12 | 福建师范大学 | Goldfish egg indoor hatching culture method |
| CN108728403A (en) * | 2018-06-05 | 2018-11-02 | 瑞柏生物（中国）股份有限公司 | A kind of spilting of an egg culture solution and preparation method thereof |
| CN108753690A (en) * | 2018-06-05 | 2018-11-06 | 瑞柏生物（中国）股份有限公司 | One step culture solution of one kind and preparation method thereof |
| CN108753691A (en) * | 2018-06-05 | 2018-11-06 | 瑞柏生物（中国）股份有限公司 | A kind of blastocyst culture liquid and preparation method thereof |
| 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 |
| CN113755429A (en) * | 2021-09-29 | 2021-12-07 | 中国科学院苏州生物医学工程技术研究所 | Composition for preparing cleavage culture solution, cleavage culture solution and preparation method |
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