CN102771472A - Freezing liquid for preserving embryo, preparation method and application thereof - Google Patents
Freezing liquid for preserving embryo, preparation method and application thereof Download PDFInfo
- Publication number
- CN102771472A CN102771472A CN2012102797813A CN201210279781A CN102771472A CN 102771472 A CN102771472 A CN 102771472A CN 2012102797813 A CN2012102797813 A CN 2012102797813A CN 201210279781 A CN201210279781 A CN 201210279781A CN 102771472 A CN102771472 A CN 102771472A
- Authority
- CN
- China
- Prior art keywords
- freezing liquid
- bsa
- ethylene glycol
- embryo
- serum albumin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses a freezing liquid for preserving embryo, especially a freezing liquid for preserving sampled embryo. The freezing liquid comprises ethylene glycol and sucrose, and is characterized by comprising 0.4-5g of arabinogalactan, 75-85mL of ethylene glycol, 30-40g of sucrose, and 910-930mL of PBS buffer solution containing 0.0021 g/mL bovine serum albumin (BSA). A preparation method of the freezing liquid comprises the steps of dissolving ethylene glycol, sucrose and arabinogalactan in the BSA-containing PBS buffer solution. The freezing liquid fully utilizes synergic effects of the four components that ethylene glycol, sucrose, arabinogalactan and bovine serum albumin are coordinated and supported one another, and has higher development rate of thawed sampled embryo in comparison with conventional ethylene glycol freezing liquid.
Description
Technical field
The present invention relates to a kind of freezing liquid that is used to preserve the embryo, particularly a kind of freezing liquid that is used to preserve the sampling embryo.The invention still further relates to the preparation method and the application of said freezing liquid.
Background technology
The freezing of embryo is embryo's store method, and the embryo is placed freezing liquid protection and takes special cooling process, makes the embryo under ultralow temperature (liquid nitrogen-196 ℃, liquid helium-269 ℃), stop metabolism.When needing to use the embryo, make it recover survival ability through specific thaw routine again, thereby reach long preservation embryo's purpose.
For sex identification and other genetic test and analysis, usually need utilize micromanipulative technique that sub-fraction under the embryonic tissue cutting is used for analyzing, the freezing preservation of remainder is subsequent use.
But the embryo can cause damage to the embryo after being cut sampling, reduces, destroys cell connection etc. like oolemma breakage, embryonic cell quantity, and the embryo is reduced freezing tolerance.Embryo after freezing no matter at the form natural rate of interest, freeze the back rate of recovery, still all not ideal enough at aspects such as embryo development rates.
Freezing liquid mainly comprises two kinds: sequencing freezing liquid and glass freezing liquid at a slow speed.It is freezing that conventional freezing is exactly sequencing at a slow speed, and the composition of sequencing freezing liquid and proportioning are not changeless at a slow speed, and at present the most frequently used is ethylene glycol+sucrose or adds a little other compositions again, and generally research is more on ox embryo freezing.Because embryo's quality, kind, developmental stage and condition of culture is different, makes refrigerating effect there are differences, the composition of freezing liquid also can change thereupon, but the effect that is used to preserve the sampling embryo preferably freezing liquid rarely have report.
Nineteen eighty-three Lehn-Jensen finds that oolemma plays a kind of physical barriers effect in the freeze-thaw process, can resist virus and microorganism.At present, less to the report of sampling embryo cryopreservation, and relevant report, freezing efficiency is all very general.And the conventional ethylene glycol freezing liquid of being made up of ethylene glycol and sucrose because the cryoprotector selection is improper, mostly exists developmental rate and the lower problem of transplanting succeed rate after the low and embryo that takes a sample of freezing sampling embryo efficient is frozen.Therefore, be badly in need of a kind of freezing liquid of preserving the sampling embryo.
Arabinogalactan (Arabinogalactan, AG), molecular weight is 530000; For HMW impermeability freezeproof protectant, more at medicine and Applications in Food Industry, often be used as food additives; And have immune isoreactivity, so pair cell toxicity is less, but its report as cryoprotector is not almost had; And in conventional freezing protection liquid, add HMW impermeability freezeproof protectant, have no precedent report before this.
Summary of the invention
Be demand and the deficiency that solves above-mentioned field, the present invention provides a kind of freezing liquid that is used to preserve the embryo, its preparation method and application.Freezing liquid of the present invention has solved conventional ethylene glycol freezing liquid and has been used for the low problem of freezing sampling embryo development rate through in conventional embryo cryopreservation liquid, adding the HMW impermeability freezeproof protectant arabinogalactan of certain concentration.
A kind of freezing liquid that is used to preserve the embryo contains ethylene glycol and sucrose, it is characterized by the buffer solution PBS that also contains Arabic galactose, contains bovine serum albumin(BSA), and its proportioning is:
Arabic galactose 0.4～5g: ethylene glycol 75～85ml: sucrose 30～40g:: the buffer solution PBS 910～930ml that contains bovine serum albumin(BSA).
Among the said buffer solution PBS that contains bovine serum albumin(BSA), the content of said bovine serum albumin(BSA) in buffer solution is 0.0021g/ml;
During preparation, ethylene glycol, sucrose and Arabic galactose are dissolved among the buffer solution PBS that contains bovine serum albumin(BSA).
When said freezing liquid the ratio of each component is: Arabic galactose 0.46～4.6g: ethylene glycol 80ml: sucrose 30～32g: when containing the buffer solution PBS920ml of bovine serum albumin(BSA); Freezing liquid embryo development rate of the present invention is higher than the embryo development rate of control group; According to this area general knowledge, freezing liquid general effect of the present invention is superior to control group.
When said freezing liquid the ratio of each component is: Arabic galactose 2.76～3.68g: ethylene glycol 80ml: sucrose 30～32g: when containing the buffer solution PBS920ml of bovine serum albumin(BSA); According to correction data; Freezing liquid general effect of the present invention is preferable; Freezing liquid difference on effect in this is interval is not remarkable, and refrigerating effect is more stable, does not have variation fluctuated.The concentration of arabinogalactan is improved or reduces, then all can not reach so good effect.
When said freezing liquid the ratio of each component is: Arabic galactose 2.76g: ethylene glycol 80ml: sucrose 30～32g: when containing the buffer solution PBS 920ml of bovine serum albumin(BSA), it is best that refrigerating effect reaches.
The consumption of said each component of freezing liquid is: Arabic galactose 0.0276g, and ethylene glycol 0.8ml, sucrose 0.315008g contains the buffer solution PBS9.2ml of bovine serum albumin(BSA), is optimum ratio, and under this ratio, freezing liquid effect of the present invention is best.
In the above-mentioned freezing liquid, said embryo is the sampling embryo through cutting sampling.
The preparation method of above-mentioned freezing liquid comprises the steps:
(1) arabinogalactan is added in the container, add buffer solution PBS again, 4 ℃ leave standstill dissolving;
(2) add sucrose again, 4 ℃ leave standstill dissolving;
(3) add bovine serum albumin(BSA) BSA again, do not shake, 4 ℃ leave standstill dissolving;
(4) add buffer solution PBS again, obtain mixed liquor;
(5) get step (4) gained mixed liquor and mix mutually with ethylene glycol, sealing, 4 ℃ of refrigerators leave standstill;
The proportioning of above-mentioned each composition is: Arabic galactose 0.4～5g: ethylene glycol 75～85ml: sucrose 30～40g: contain the buffer solution PBS910～930ml of bovine serum albumin(BSA), the content of said bovine serum albumin(BSA) in buffer solution is 0.0021g/ml.
The application of above-mentioned freezing liquid in preserving the sampling embryo.
Technique effect:
The present invention is the macromolecule freezeproof protectant arabinogalactan that in conventional freezing liquid, adds impermeability, BSA (bovine serum albumin(BSA)) etc.; These materials can not permeate through cell membranes, and its viscosity increases in the rapid cooling process, and the damage surface of a wound is had the colloidality protective effect; And extracellular fluid concentration is high; Help intracellular fluid to deviate from, prevent the formation of the big ice crystal of cell interior, so the embryo is played a protective role.
Arabinogalactan is a kind of important vegetable active polysaccharide, in food and daily-use chemical industry, makes additive in early days, and it has tangible immunocompetence discovered in recent years, becomes a kind of novel immunopotentiating agent.Arabinogalactan pair cell toxicity is little; And it has the speciality of colloidality; When the embryo being carried out when freezing, reduce pair cell and do not damage thereby cell interior just can not form big ice crystal, even maybe be when reaching suitable concentration; Can form special diaphragm in outside, stop extracellular ice crystal pair cell to cause damage.Between frost free period, arabinogalactan is a buffering osmotic pressure material, and the speed of expansion of cell reduces the generation of permeability damage when thawing through reduction.Ethylene glycol is low-molecular-weight permeability cryoprotector, and in several kinds of low-molecular-weight cryoprotectors commonly used, ethylene glycol toxicity is minimum; Permeability is best; Interpolation sucrose can reduce the toxicity of solution, and serum contains many compositions, comprises energy matrix, amino acid, vitamin, hormone, growth factor and cell factor; As embryo's nutrition and energy substrate, be that other material can not substituted optimal selection.But because calf serum is prone to cause the animal pathogenic fungi pollution, the present invention uses bovine serum albumin(BSA) (BSA) to substitute calf serum, exclude the problem of pathogen contamination, and wherein composition does not have great changes.Freezing liquid of the present invention is the basis with conventional freezing liquid (ethylene glycol+sucrose); Add the arabinogalactan as HMW impermeability freezeproof protectant of certain concentration; The present invention makes full use of ethylene glycol; Sucrose, cooperatively interacting, support each other, acting synergistically between arabinogalactan and the bovine serum albumin(BSA).Freezing liquid of the present invention is used to the freezing of embryo of taking a sample, and the developmental rate behind the sampling embryo cryopreservation is high.
This area general knowledge, a kind of embryo development rate of freezing liquid is higher than the embryo development rate of control group, and so this freezing liquid effect just is superior to control group.
The impaired embryo's conventional freezing of oolemma liquid adds arabinogalactan, to the influence of refrigerating effect
Associative list 1 and table 2; Can find out when the ratio of the weight of arabinogalactan in the solution and the volume of total freezing liquid is respectively 0.00276g/ml and 0.00368g/ml; It is better that refrigerating effect reaches; And difference is not remarkable between the refrigerating effect of these two kinds of freezing liquids, and refrigerating effect is more stable, does not have variation fluctuated.The concentration of arabinogalactan is improved or reduces, then all can not reach so good effect.Between frost free period; Arabinogalactan has the effect that forms ice crystal when thawing that stops as the HMW cryoprotector; And with the higher permeable pressure head of sucrose synergy buffering fluid of inside and outside cell, prevent that cell transition expands when the cell interior freezant does not remove fully.In addition, so, can significantly improve the cultivation developmental rate behind the embryo thawing with adding an amount of arabinogalactan in the conventional ethylene glycol freezing liquid of embryo.
Freezing and the bright embryo of the impaired embryo's secondary of oolemma is freezing, and refrigerating effect relatively
Long term studies shows that the embryo can receive a series of injury in freezing and course of defrosting, can cause the variation of cell membrane fat phase like low temperature, thereby influences the metabolism of cell; Mainly be that the permeability cryoprotector can produce chemical toxic action to the embryo in the cryoprotector; Temperature is high more, action time is longer, concentration is big more; Then toxicity will be big more; This toxicity can cause cell to become fragile to burst apart, polymerization and depolymerization, chromosome and the dna damage etc. of oolemma sclerosis, microtubule microfilament, and the ice crystal damage of embryo cryopreservation when thawing etc.
Confirm that through the contrast experiment freezing liquid of the present invention has following advantage:
1. cultivate developmental rate behind the sampling embryo thawing and be significantly higher than conventional freezing liquid;
2. when the ratio of the volume of the weight of arabinogalactan and total freezing liquid is between 0.00276～0.00368g/ml; Refrigerating effect is better; And refrigerating effect is more stable; Do not have variation fluctuated, concentration raising or reduction with arabinogalactan then all can not reach so good effect;
3. freezing liquid of the present invention is especially better to sampling embryo cryopreservation effect.
Description of drawings
Fig. 1 embryo cryopreservation tubulature sketch map
Embodiment
It is in order further to understand the present invention better that following embodiment is provided; Be not limited to said preferred forms; Content of the present invention and protection domain are not constituted restriction; Anyone makes up with the characteristic of other prior aries and the identical or akin product of any and the present invention that draws under enlightenment of the present invention or with the present invention, all drops within protection scope of the present invention.
The conventional ethylene glycol freezing liquid that control group uses in the present invention's test is made up of ethylene glycol and sucrose, and the ethylene glycol in the embodiment of the invention is pure liquid ethylene glycol.
Employed frigorimeter is the embryo cryopreservation appearance in the present invention's test, model: CL5500, producer: Australia.
Agents useful for same of the present invention source:
Arabinogalactan is purchased the Sigma company in the U.S., specifications and models: 10830-25G.
PBS buffer solution (pH7.2～7.4): NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
410mmol/L, KH
2PO
42mmol/L;
The BSA bovine serum albumin(BSA) is available from U.S. Hyclone company, among the embodiment ethylene glycol available from U.S. Sigma company (E-9129), specifications and models: 102466-500mL, wherein ethylene glycol density is 1.110g/mL at25 ° C;
The conventional ethylene glycol freezing liquid (EG FREEZE) of control group is import EG freezing liquid, purchases the BIONICHE company in the U.S., specifications and models: 50mL, EVM034.
The preparation of embodiment 1.0.03EAS freezing liquid:
The 0.03g arabinogalactan is placed the 10ml volumetric flask, add PBS to nearly graduation mark, 4 ℃ leave standstill dissolving; After treating to dissolve fully, add 0.3424g sucrose, 4 ℃ leave standstill dissolving; After treating to dissolve fully, add 0.021gBSA (not shaking anti-the foaming), 4 ℃ leave standstill dissolving; After treating to dissolve fully, add PBS and be settled to graduation mark; Behind the liquid mixing, solution 9.2ml mixes with the pure liquid ethylene glycol of 0.8ml mutually in the measuring bottle of trying to please in the volumetric flask, sealing, and refrigerator leaves standstill for 4 ℃, and is for use.
The preparation of embodiment 2.0.005EAS freezing liquid:
Preparation steps is with embodiment 1, adds the arabinogalactan of 0.005g, and the amount of sucrose, pure ethylene glycol and bovine serum albumin(BSA) is the same.
The preparation of embodiment 3.0.01EAS freezing liquid:
Preparation steps is with embodiment 1, adds the arabinogalactan of 0.01g, and the amount of sucrose, pure ethylene glycol and bovine serum albumin(BSA) is the same.
The preparation of embodiment 4.0.02EAS freezing liquid:
Preparation steps is with embodiment 1, adds the arabinogalactan of 0.02g, and the amount of sucrose, pure ethylene glycol and bovine serum albumin(BSA) is the same.
The preparation of embodiment 5.0.04EAS freezing liquid:
Preparation steps is with embodiment 1, adds the arabinogalactan of 0.04g, and the amount of sucrose, pure ethylene glycol and bovine serum albumin(BSA) is the same.
The preparation of embodiment 6.0.05EAS freezing liquid:
Preparation steps is with embodiment 1, adds the arabinogalactan of 0.05g, and the amount of sucrose, pure ethylene glycol and bovine serum albumin(BSA) is the same.
The method for using of embodiment 7. freezing liquids of the present invention:
The present invention adopts sequencing at a slow speed freezing, and method for using is following:
1. the frigorimeter assembling is inserted power supply, pour liquid nitrogen into, 0 program of selection frigorimeter is carried out freezing, and this program is: plant ice for-6 ℃, cooling rate is 0.5 ℃/min, and-35 ℃ are dropped into liquid nitrogen.
2. six sections method tubulatures are adopted in tubulature, the present invention, the two sections freezing liquids of packing into earlier, and the 3rd section embryo that packs into then, the back reinstalls freezing liquid for three sections, fills in labeled tubule plug at last.(like Fig. 1)
3. the embryo puts into freezing liquid balance 5min; After the tubulature tubule put into the frigorimeter that is cooled to-6 ℃ and plant ice (plant freezing point and see Fig. 1) behind the balance 5min once more, press RUN key (instrument begins to begin to lower the temperature with the speed of 0.5 ℃/min behind the 10min) after planting ice; Ice is planted in inspection behind the 5min; When the frigorimeter temperature drops to-35 ℃, tubule dropped into and put into liquid nitrogen container among the liquid nitrogen and the CANE that packs into and preserve.
Embodiment 8.EAS series freezing liquid (adding the arabinogalactan freezing liquid) and conventional ethylene glycol freezing liquid (EG FREEZE) carry out the impaired embryo's secondary of oolemma freeze-thaw to be observed
With embodiment 1-6 gained freezing liquid, to the impaired embryo of oolemma carry out secondary freezing, thaw, CO
2The incubator culture experiment is a control group with conventional ethylene glycol freezing liquid (EG FREEZE) simultaneously, relatively the refrigerating effect of freezing liquid of the present invention and conventional freezing liquid (EG FREEZE).
The impaired embryo's secondary of table 1 oolemma freeze-thaw cultivation results
Annotate: the identical person's difference of same column shoulder mark lowercase is remarkable (P＞0.05) not, shoulder mark lowercase difference person's significant difference (P＜0.05).
As shown in table 1, aspect embryo's rate of recovery, have only 0.01EAS and 0.03EAS to be higher than control group (P＞0.05), in all the other four kinds of freezing liquids, embryo's rate of recovery of 0.05EAS is minimum; Cultivate the 24h developmental rate and cultivate the 48h developmental rate, have only 0.01EAS to be lower than control group (P＞0.05), all the other five kinds of freezing liquids all are higher than conventional ethylene glycol freezing liquid (EG FREEZE), but difference not significantly (P＞0.05).Therefore, generally speaking, the effect of freezing liquid of the present invention all is superior to conventional freezing liquid.
Result in conjunction with the impaired embryo's secondary of the oolemma freeze-thaw viewing test of EAS series freezing liquid picks out two kinds of freezing liquids of 0.03EAS and 0.04EAS and carries out follow-up bright embryo refrigeration test.
The impaired bright embryo freeze-thaw of embodiment 9. oolemmas is cultivated
Be contrast still with conventional ethylene glycol freezing liquid (EG FREEZE), with 0.03EAS freezing liquid and 0.04EAS freezing liquid, to the impaired bright embryo of oolemma carry out freezing, thaw, CO
2Incubator culture experiment, the relatively refrigerating effect of freezing liquid of the present invention and conventional ethylene glycol freezing liquid (EG FREEZE).
Table 2
Annotate: the identical person's difference of same column shoulder mark lowercase is remarkable (P＞0.05) not, shoulder mark lowercase difference person's significant difference (P＜0.05), and shoulder mark capitalization difference person difference is remarkable (P＜0.01) extremely.
As shown in table 2, aspect embryo's rate of recovery, two kinds of freezing liquids picking out are compared with control group, and difference is not significantly (P＞0.05); Cultivate the 24h developmental rate with cultivate two kinds of freezing liquids of 48h developmental rate all the utmost point be significantly higher than conventional ethylene glycol freezing liquid (EGFREEZE) (P＜0.01), compare between two kinds of freezing liquids, difference is significantly (P＞0.05) not, but 0.03EAS best results wherein.
Cultivation 24h developmental rate through more several kinds of freezing liquids can be found out with cultivation 48h developmental rate; The freezing liquid refrigerating effect that adds arabinogalactan is better; This maybe be relevant with the colloidality speciality of arabinogalactan; Reduce the formation of damaging ice crystal or formed diaphragm, more effectively prevented the injury of extracellular ice crystal pair cell.
Therefore, add an amount of arabinogalactan in the sampling embryo conventional freezing liquid, can obtain reasonable refrigerating effect; Add the arabinogalactan freezing liquid, only better to sampling embryo cryopreservation effect, for complete embryo, EG FREEZE refrigerating effect is better.
Claims (8)
1. a freezing liquid that is used to preserve the embryo contains ethylene glycol and sucrose, it is characterized by the buffer solution PBS that also contains Arabic galactose, contains bovine serum albumin(BSA), and its proportioning is:
Arabic galactose 0.4～5g: ethylene glycol 75～85ml: sucrose 30～40g: the buffer solution PBS 910～930ml that contains bovine serum albumin(BSA);
Among the said buffer solution PBS that contains bovine serum albumin(BSA), the content of said bovine serum albumin(BSA) in buffer solution is 0.0021g/ml;
During preparation, ethylene glycol, sucrose and Arabic galactose are dissolved among the buffer solution PBS that contains bovine serum albumin(BSA).
2. according to the said freezing liquid of claim 1, it the ratio of each component is: Arabic galactose 0.46～4.6g: ethylene glycol 80ml: sucrose 30～32g: the buffer solution PBS 920ml that contains bovine serum albumin(BSA).
3. according to the said freezing liquid of claim 2, it the ratio of each component is: Arabic galactose 2.76～3.68g: ethylene glycol 80ml: sucrose 30～32g: the buffer solution PBS 920ml that contains bovine serum albumin(BSA).
4. according to the said freezing liquid of claim 3, it the ratio of each component is: Arabic galactose 2.76g: ethylene glycol 80ml: sucrose 30～32g: the buffer solution PBS 920ml that contains bovine serum albumin(BSA).
5. according to the said freezing liquid of claim 4, the consumption of its each component is: Arabic galactose 0.0276g, and ethylene glycol 0.8ml, sucrose 0.315008g contains the buffer solution PBS 9.2ml of bovine serum albumin(BSA).
6. the arbitrary described freezing liquid of claim 1～5, said embryo is the sampling embryo through cutting sampling.
7. the preparation method of the arbitrary said freezing liquid of claim 1～5 comprises the steps:
(1) arabinogalactan is added in the container, add buffer solution PBS again, 4 ℃ leave standstill dissolving;
(2) add sucrose again, 4 ℃ leave standstill dissolving;
(3) add bovine serum albumin(BSA) BSA again, do not shake, 4 ℃ leave standstill dissolving;
(4) add buffer solution PBS again, obtain mixed liquor;
(5) get step (4) gained mixed liquor and mix mutually with ethylene glycol, sealing, 4 ℃ of refrigerators leave standstill;
The proportioning of above-mentioned each composition is: Arabic galactose 0.4～5g: ethylene glycol 75～85ml: sucrose 30～40g: contain the buffer solution PBS 910～930ml of bovine serum albumin(BSA), the content of said bovine serum albumin(BSA) in buffer solution is 0.0021g/ml.
8. the application of the arbitrary said freezing liquid of claim 1～5 in preserving the sampling embryo.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210279781.3A CN102771472B (en) | 2012-04-25 | 2012-08-07 | Freezing liquid for preserving embryo, preparation method and application thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210125210.4 | 2012-04-25 | ||
| CN201210125210 | 2012-04-25 | ||
| CN201210279781.3A CN102771472B (en) | 2012-04-25 | 2012-08-07 | Freezing liquid for preserving embryo, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102771472A true CN102771472A (en) | 2012-11-14 |
| CN102771472B CN102771472B (en) | 2014-04-23 |
Family
ID=47116789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210279781.3A Expired - Fee Related CN102771472B (en) | 2012-04-25 | 2012-08-07 | Freezing liquid for preserving embryo, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102771472B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103783034A (en) * | 2014-01-20 | 2014-05-14 | 广西大学 | Swine spermatogonia stem cell cryopreserved liquid and use method thereof |
| CN104206376A (en) * | 2014-09-17 | 2014-12-17 | 南宁培元基因科技有限公司 | Lowline embryo freezing liquid, freezing method and unfreezing method |
| CN106857498A (en) * | 2016-12-27 | 2017-06-20 | 科兴（大连）疫苗技术有限公司 | A kind of cell cryopreservation protection liquid and its application without dimethyl sulfoxide (DMSO) |
| WO2018064976A1 (en) * | 2016-10-04 | 2018-04-12 | Transwell Biotech Co., Ltd. | Compositions and methods for cell cryopreservation |
| CN110604128A (en) * | 2019-10-21 | 2019-12-24 | 内蒙古金草原牧业有限公司 | Beef cattle propagation embryo cryopreservation liquid and freezing method |
| CN111616138A (en) * | 2019-12-31 | 2020-09-04 | 青岛德瑞骏发生物科技股份有限公司 | Horse embryo refrigerating fluid formula |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997014785A2 (en) * | 1995-10-19 | 1997-04-24 | Advanced Reproduction Technologies, Inc. | Methods and compositions to improve germ cell and embryo survival and function |
| CN1302590A (en) * | 1999-10-22 | 2001-07-11 | 朱士恩 | Simple and efficient freeze-thaw method for vitrification of embryo |
| CN1322467A (en) * | 2000-05-08 | 2001-11-21 | 朱士恩 | One-step process for embryo vitrifying freeze storage |
| CN1727473A (en) * | 2004-07-31 | 2006-02-01 | 青岛六和牧业发展有限公司 | Technique for freezing bare embryo |
| US7411113B2 (en) * | 2004-02-25 | 2008-08-12 | Pioneer Hi-Bred International, Inc. | Modulating myo-inositol catabolism in plants |
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN101897330A (en) * | 2010-08-31 | 2010-12-01 | 湖南光琇高新生命科技有限公司 | Low-toxicity vitrified frozen solution of human embryonic stem cells (hESCs) and method of using same |
-
2012
- 2012-08-07 CN CN201210279781.3A patent/CN102771472B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997014785A2 (en) * | 1995-10-19 | 1997-04-24 | Advanced Reproduction Technologies, Inc. | Methods and compositions to improve germ cell and embryo survival and function |
| CN1302590A (en) * | 1999-10-22 | 2001-07-11 | 朱士恩 | Simple and efficient freeze-thaw method for vitrification of embryo |
| CN1322467A (en) * | 2000-05-08 | 2001-11-21 | 朱士恩 | One-step process for embryo vitrifying freeze storage |
| US7411113B2 (en) * | 2004-02-25 | 2008-08-12 | Pioneer Hi-Bred International, Inc. | Modulating myo-inositol catabolism in plants |
| CN1727473A (en) * | 2004-07-31 | 2006-02-01 | 青岛六和牧业发展有限公司 | Technique for freezing bare embryo |
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN101897330A (en) * | 2010-08-31 | 2010-12-01 | 湖南光琇高新生命科技有限公司 | Low-toxicity vitrified frozen solution of human embryonic stem cells (hESCs) and method of using same |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103783034A (en) * | 2014-01-20 | 2014-05-14 | 广西大学 | Swine spermatogonia stem cell cryopreserved liquid and use method thereof |
| CN103783034B (en) * | 2014-01-20 | 2015-10-21 | 广西大学 | One boar stem spermatogonium cryopreserving liquid and using method thereof |
| CN104206376A (en) * | 2014-09-17 | 2014-12-17 | 南宁培元基因科技有限公司 | Lowline embryo freezing liquid, freezing method and unfreezing method |
| WO2018064976A1 (en) * | 2016-10-04 | 2018-04-12 | Transwell Biotech Co., Ltd. | Compositions and methods for cell cryopreservation |
| CN109843052A (en) * | 2016-10-04 | 2019-06-04 | 全崴生技股份有限公司 | The composition and method saved for cell freezing |
| US10815456B2 (en) | 2016-10-04 | 2020-10-27 | Transwell Biotech Co., Ltd. | Composition, kit and method for cryopreserving cells |
| CN106857498A (en) * | 2016-12-27 | 2017-06-20 | 科兴（大连）疫苗技术有限公司 | A kind of cell cryopreservation protection liquid and its application without dimethyl sulfoxide (DMSO) |
| CN106857498B (en) * | 2016-12-27 | 2021-05-14 | 科兴（大连）疫苗技术有限公司 | Cell cryopreservation protective solution without dimethyl sulfoxide and application thereof |
| CN110604128A (en) * | 2019-10-21 | 2019-12-24 | 内蒙古金草原牧业有限公司 | Beef cattle propagation embryo cryopreservation liquid and freezing method |
| CN111616138A (en) * | 2019-12-31 | 2020-09-04 | 青岛德瑞骏发生物科技股份有限公司 | Horse embryo refrigerating fluid formula |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102771472B (en) | 2014-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102771472B (en) | Freezing liquid for preserving embryo, preparation method and application thereof | |
| CN102771473A (en) | Freezing liquid for preserving embryo, preparation method and application thereof | |
| CN108207930B (en) | Cocktail type cryoprotectant and application thereof | |
| Mazur | Principles of cryobiology | |
| CN102648708B (en) | Freezing liquid for embryo or cells and application thereof | |
| CN107094753A (en) | A kind of candidate stem cell frozen stock solution and candidate stem cell cryopreservation methods | |
| CN107996558A (en) | Cells frozen storing liquid and its application | |
| ES2762966T3 (en) | Cryopreservation procedure of cells with therapeutic objective | |
| CN104222068B (en) | Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium | |
| CN104839144A (en) | Vitrification freezing fluid of oocytes | |
| EP0594651A1 (en) | Dried blood cells and method of preparation. | |
| WO2006029467A1 (en) | Rapid freeze drying process | |
| CN105432780A (en) | Compound biological preservative suitable for keeping mulberries fresh and preparation method and application thereof | |
| US20210084891A1 (en) | Vitreous state stabilizing agent for animal cell cryopreservation solution | |
| CN105505778B (en) | The ultralow temperature method for preserving of the original parent species of Pleurotus eryngii | |
| CN116636525A (en) | Freezing solution and freezing method for long-term preservation of stem cells and products thereof | |
| CN114190368B (en) | Serum-free immune cell cryopreservation liquid, preparation method and immune cell cryopreservation method | |
| CN106386787A (en) | Breast cancer stem cell cryopreservation protection agent and prepared cryopreservation protection kit | |
| CN110115265A (en) | A kind of embryo vitrifying freeze liquid | |
| CN102258007A (en) | Refrigerant and method for storing Pacific codfish sperms | |
| KR20210052451A (en) | Compositions for transport and/or cryopreservation of cells and/or tissues | |
| CN101342192B (en) | Quick freezing conservation method for bastard halibut spermatozoon | |
| CN110074106A (en) | Rosaceous plant anti-freezing composition, antifreeze injection and preparation method thereof and antifreeze infusion medicament | |
| WO2012028967A2 (en) | Method for cryopreservation of human spermatozoa free from seminal plasma using a fast and simple aseptic vitrification-devitrification process; portable kit for carrying out the method; and use of the same for treatment of disorders related to reproductive failures | |
| CN108552161A (en) | A kind of the Ultra-cryofreezing preservation liquid and its Cryopreservation methods and applications of loach sperm |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Free format text: FORMER OWNER: LI SHUJING Effective date: 20140717 Owner name: SHIJIAZHUANG TIANQUAN GOOD DAIRY COW CO., LTD. Free format text: FORMER OWNER: YU WENLI Effective date: 20140717 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100107 CHAOYANG, BEIJING TO: 050200 SHIJIAZHUANG, HEBEI PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20140717 Address after: The town of Luquan city of Hebei province Shijiazhuang City Han village 050200 Patentee after: SHIJIAZHUANG TIANQUAN LIANGZHONG COW CO.,LTD. Address before: 100107, Beijing, Chaoyang District Beiyuan Road red Camp Road, Tian Lang Park proud City, finance center B block, room 2406 Patentee before: Yu Wenli Patentee before: Li Shujing |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140423 Termination date: 20210807 |