CN101066052A - Livestock embryo vitrifying freeze process on metal surface - Google Patents
Livestock embryo vitrifying freeze process on metal surface Download PDFInfo
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Abstract
The present invention is livestock embryo vitrifying freeze process on metal surface, and belongs to the field of biotechnology. The livestock embryo vitrifying freeze process includes replacing freeze protectant for liquid inside and outside the embryo cell and fast freezing of the embryo liquid drop. During the fast freezing, the embryo liquid drop is dropped onto the exposed upper surface of one metal block with lower part soaked inside liquid nitrogen. The present invention can cool embryo in high speed while avoiding contamination of impurity in liquid nitrogen to the embryo. Besides, adding cytochalasin B as cytoskeleton protectant into the frozen liquid can raise the freezeproof capacity of the embryo and raise vitrification freezing effect. The present invention is used for freezing livestock embryo.
Description
Technical field
The present invention relates to the embryo biotechnology field, particularly relate to the livestock embryo vitrifying freeze process.
Background technology
Along with the development of superovulation technology, in vitro fertilization, embryo transfer technology and animal cloning technology, livestock embryo has become the commercialization product.It is very important for the popularization of embryo's commercial applications and clone technology that embryo cryopreservation is preserved technology, and the livestock embryo Refrigeration Technique has become the research focus in the embryo engineering technology.In recent years embryo's freezing preservation technology particularly vitrifying freeze process obtained rapidly development, the glass freezing technology is the result that snap frozen combines with the high concentration cryoprotector.The high concentration cryoprotector is forming transparent vitreous solid in the cooling procedure fast; viscosity is very high in the cooling procedure; form but there is ice crystal, avoided the damage of ice crystal pair cell lipid film and cytoskeletal structure in the refrigerating process, keep normal molecule of fluid of inside and outside cell body and ion distribution.The glass freezing operating procedure is simple, comprising: the inside and outside solution of cryoprotector displacement cell of high concentration, fast cooling makes the solution stickiness extremely improve and solidify, and places liquid nitrogen and preserves.The factor that influences the glass freezing effect mainly comprises: (1) cryoprotector.With a kind of histocyte, the concentration of cryoprotector and kind are most important influence factors.The extracellular cryoprotector generally adopts macromolecular substances, as polysaccharide (sucrose, trehalose) polyethylene glycol, fetal bovine serum albumin (BAS) and Percoll etc.; Cryoprotector is the small-molecule substance with good penetration in the cell, as dimethyl sulfoxide (DMSO), ethylene glycol and glycerine.(2) temperature decrease speed.The faster the better for cooling rate, and the method that can directly drop into liquid nitrogen is a micro drop method, and its cooling rate is about 2500 ℃/min.(3) embryo's carrier.To the requirement of embryo's carrier is that freezing preservative agent volume around the embryo should be as far as possible little, few as far as possible at interval with liquid nitrogen, easy and simple to handle and be difficult for losing the embryo.
Present glass freezing mainly adopt microtubule method and micro drop method (MICRO-DROP VITRIFICATION, MDV).Because the existence of microtubule makes embryo's chilling rate reduce, influenced the efficient of freezing preservation in the microtubule method.And the operation of micro drop method routine is directly to immerse embryo's drop in the liquid nitrogen, owing to may contain some pathogenic microorganism in the liquid nitrogen, so embryo's drop directly contacts the pollution that can cause the embryo with liquid nitrogen, embryo's drop splashes in the liquid nitrogen simultaneously, because the relation of the temperature difference, liquid nitrogen around embryo's drop gasifies easily and forms air mass, thereby influenced cooling velocity.In the embryo cryopreservation process, low temperature can cause certain infringement to embryo's cytoskeleton, and this has weakened embryo's freezing tolerance to a certain extent.
Summary of the invention
Defective at above-mentioned field; the invention provides a kind of embryo metal surface vitrifying freeze process (Metal SurfaceVitrification; MSV); can improve embryo's cooling velocity greatly; in freezing liquid, add cytoskeleton protectant---cytochalasin B simultaneously; strengthen embryo's freezing tolerance, improved the glass freezing effect.It is freezing that the present invention is used for livestock embryo, can make recovery rate behind the blastaea freeze-thaw in vitro fertilization more than 96%, incubation rate 80-85%.
Livestock embryo metal surface vitrifying freeze process; the snap frozen step that comprises inside and outside liquid of cryoprotector displacement embryonic cell and frozen embryo drop; it is characterized in that: snap frozen is the upper surface that the frozen embryo drop is dropped in a metal derby; described metal derby soaks or is suspended in the liquid nitrogen, and its upper surface is exposed to the liquid nitrogen surface.
The preferred copper billet of the material of described metal derby.
The inside and outside liquid of described cryoprotector displacement embryonic cell carries out on 39 ℃ constant temperature platform, adopt following steps: (1) put into the equilibrium liquid balance 10-20 minute with the embryo, (2) embryo is moved in the VS1 liquid 3-5 minute, (3) embryo is moved into 20-30 second in the VS2 liquid, suck with the VS2 of 2ul with through the embryo that cryoprotector was handled then and form the frozen embryo drop in the glass pipette; Described equilibrium liquid is the cytochalasin B that has added 5 μ g/ml in the DPBS basal liquid; Described VS1 liquid is that to have added volumn concentration in equilibrium liquid be 7.5%DMSO, 7.5% ethylene glycol; Described VS2 liquid is that to have added volumn concentration in equilibrium liquid be that 16.5%DMSO, 16.5% ethylene glycol and final concentration are 0.5M sucrose.
Also added volumn concentration in the described equilibrium liquid and be 20% serum and 1% antibiotic.
Described livestock embryo is ox blastaea in vitro fertilization or body-cell neucleus transplanting blastaea.
The defreezing method of described frozen embryo carries out on 39 ℃ constant temperature platform, adopt following steps: (1) puts into frozen embryo DPBS basal liquid 1-3 minute that has added 0.25M sucrose, (2) embryo is moved in the DPBS basal liquid added 0.15M sucrose 5-10 minute, (3) move into the embryo in the DPBS basal liquid and got final product in 5-10 minute.
The present invention adopts the metal surface vitrifying freeze process, and (Metal Surface Vitrification MSV), will directly drop in the metal surface (as shown in Figure 1) of immersing liquid nitrogen through the embryo's drop after the frozen solution displacement.Because metal is immersed in the liquid nitrogen, it maintains the low temperature characteristics of liquid nitrogen solution, the fast characteristic of metal material heat transfer is arranged again simultaneously, when the frozen embryo drop directly drops in the metal surface, it does not pass liquid nitrogen solution, air mass in the time of can not forming liquid nitrogen gasification has so just improved chilling rate greatly.Impurity such as the difficult processing such as bacterium, suspended particulate substance in the liquid nitrogen simultaneously is clean, easily makes embryo's freezing environment contaminated, thereby has reduced the survival after embryo cryopreservation thaws, and has also influenced embryo's commercial applications simultaneously.And the metal derby surface is that fine processing is clean.Metal derby is regardless of being in the immersion liquid nitrogen or being suspended in the liquid nitrogen as long as its upper surface is exposed at liquid nitrogen outward.The preferred copper billet of metal derby, its heat transfer rate is the fastest.
The inside and outside liquid step of cryoprotector displacement embryonic cell was divided into for three steps among the present invention; at first in equilibrium liquid after the balance; add again in low concentration freezeproof protectant dimethyl sulfoxide (DMSO) (DMSO) and the ethylene glycol and replace, displacement fast in high concentration freezeproof protectant dimethyl sulfoxide (DMSO) (DMSO) and ethylene glycol and extracellular fluid freezeproof protectant sucrose then.The present invention also is added with cytoskeleton stabilizing agent cytochalasin B in equilibrium liquid, further improve embryo's freezing tolerance, also can add serum and antibiotic simultaneously.
The frozen embryo drop cools off formation vitrifying globule after dropping in the metal surface fast, and liquid nitrogen is preserved in the cryovial of these globules can being packed into then.
Freeze-substitution of the present invention and thaw routine are all finished on 39 ℃ constant temperature platform, and the temperature condition that this homogeneous is constant makes easy to operation.Experiment showed, the ox blastaea in vitro fertilization after the inventive method is freezing, the recovery rate after thawing is more than 96%, incubation rate 80-85%; Recovery rate behind the bovine somatic cells clone blastaea freeze-thaw reaches 94.2%, incubation rate 80.7%, improved the survival rate after embryo cryopreservation thaws greatly, for the freezing preservation of livestock embryo provides a new method and thinking, thereby further promoted the commercial applications of livestock embryo, and the application aborning of cloning domestic animal technology, improved the economic benefit of livestock embryo.
Description of drawings
Fig. 1 livestock embryo of the present invention is at metal surface vitrifying freeze process schematic diagram
1-frozen embryo drop, 2-metal derby, 3-liquid nitrogen, 4-liquid nitrogen container.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
With freezing ten pieces of ox blastaeas is that example illustrates refrigerating process of the present invention:
1. the preparation of freezing liquid:
1.1 the manufacturer and the article No. of used each reagent chemicals are as follows
DPBS(Gibco，15240-013)
DMSO(Sigma，D-5879)
Ethylene glycol (Sigma, E-9129)
Cytochalasin B (Sigma, C-6762)
Antibiotic (Gibco, 15240-062)
Sucrose (Sigma, S-1888)
Serum FBS (Hyclone, SH30070.03)
1.2DPBS basal liquid: with 39.5ml DPBS, filter behind 10ml FBS and the 0.5ml antibiotic mixing, refrigerate 4 ℃ standby
Equilibrium liquid: filter behind the cytochalasin B mixing with 10ml DPBS basal liquid and 50 μ g, refrigerate 4 ℃ standby
VS1:, frozen standby in-20 ℃ with filtering behind 8.5ml DPBS basal liquid, 0.75mlDMSO, 0.75ml ethylene glycol and the 50 μ g cytochalasin B mixings
VS2:, frozen standby in-20 ℃ with filtering behind the sucrose of 6.7ml DPBS basal liquid, 1.65mlDMSO, 1.65ml ethylene glycol, 1.7115g and the 50 μ g cytochalasin B mixings
0.25M the DPBS basal liquid of sucrose:, refrigerate in 4 ℃ standby with filtering behind the sucrose of 0.8558g and the 10ml DPBS basal liquid mixing
0.15M the DPBS basal liquid of sucrose:, refrigerate in 4 ℃ standby with filtering behind the sucrose of 0.5135g and the 10ml DPBS basal liquid mixing
2. refrigerating process
2.1. the constant temperature platform is opened, and constant temperature platform temperature is set at 39 ℃, with the DPBS basal liquid, equilibrium liquid, VS1, VS2, the DPBS basal liquid of 0.25M sucrose and the DPBS basal liquid of 0.15M sucrose place culture dish preheating on the constant temperature platform;
2.2. select ten pieces of the normal ox blastaeas of form at microscopically;
2.3 ten pieces of ox blastaeas were put into the equilibrium liquid balance 10 minutes at 39 ℃ constant temperature platform with the embryo;
2.4 the embryo was moved in the VS1 liquid 3 minutes;
2.5 the embryo is moved into 20-30 second in the VS2 liquid, drip on the copper billet that is immersed in the liquid nitrogen surface with the VS2 of 2 μ l with through ten pieces of ox blastaeas that cryoprotector was handled then, form frozen particle;
Be stored in the liquid nitrogen 2.6 will contain the frozen particle of ten pieces of ox blastaeas, until thawing.
3. course of defrosting
3.1, the frozen particle that contains ten pieces of ox blastaeas is put into the DPBS basal liquid 1 minute that has added 0.25M sucrose at 39 ℃ constant temperature platform;
3.2 ten pieces of ox blastaeas were moved in the DPBS basal liquid that has added the 0.15M sucrose 5 minutes;
3.3 ten pieces of ox blastaeas were moved in the DPBS basal liquids 5 minutes;
3.4 observe ox blastaea recovery situation, and carry out embryo transplantation.
From year January in January, 2006 to 2007, we have carried out the glass freezing experiment of ox blastaea in vitro fertilization and somatic cell clone blastaea respectively in the Inner Mongol and Beijing, and experimental result is as follows:
The metal surface glass freezing effect of table one ox blastaea in vitro fertilization and body-cell neucleus transplanting blastaea
| The blastaea type | Freezing blastaea quantity | Survival blastaea quantity (%) | Hatched blastocyst quantity (%) | The transplant recipient number | Conceived number (%) |
| Embryo in vitro fertilization | 116 | 112(96.6) | 98(84.4) | 36 | 11(30.6) |
| Clone embryos | 104 | 98(94.2) | 84(80.7) | 24 | 7(29.2) |
Fig. 1 is that livestock embryo of the present invention is at metal surface vitrifying freeze process schematic diagram, frozen embryo drop 1 drops in metal derby 2 upper surfaces, this metal derby 2 is immersed in the liquid nitrogen 3, and liquid nitrogen storage is in foam plastics container 4, the rapid freezing formation vitrifying globule of this frozen embryo drop.
Claims (6)
1. livestock embryo is at the metal surface vitrifying freeze process; the snap frozen step that comprises inside and outside liquid of cryoprotector displacement embryonic cell and frozen embryo drop; it is characterized in that: snap frozen is the upper surface that the frozen embryo drop is dropped in a metal derby; described metal derby soaks or is suspended in the liquid nitrogen, and its upper surface is exposed to the liquid nitrogen surface.
2. livestock embryo according to claim 1 is at the metal surface vitrifying freeze process, and the material of described metal derby is a copper billet.
3. livestock embryo according to claim 1 is at the metal surface vitrifying freeze process, the inside and outside liquid of described cryoprotector displacement embryonic cell carries out on 39 ℃ constant temperature platform, adopt following steps: (1) put into the equilibrium liquid balance 10-20 minute with the embryo, (2) embryo is moved in the VS1 liquid 3-5 minute, (3) embryo is moved into 20-30 second in the VS2 liquid, suck with the VS2 of 2ul with through the embryo that cryoprotector was handled then and form the frozen embryo drop in the glass pipette; Described equilibrium liquid is the cytochalasin B that has added 5 μ g/ml in the DPBS basal liquid; Described VS1 liquid is that to have added volumn concentration in equilibrium liquid be 7.5%DMSO, 7.5% ethylene glycol; Described VS2 liquid is that to have added volumn concentration in equilibrium liquid be that 16.5%DMSO, 16.5% ethylene glycol and final concentration are 0.5M sucrose.
4. livestock embryo according to claim 3 is at the metal surface vitrifying freeze process, also added volumn concentration in the described equilibrium liquid and be 20% serum and 1% antibiotic.
5. livestock embryo according to claim 4 is at the metal surface vitrifying freeze process, and described livestock embryo is ox blastaea in vitro fertilization or body-cell neucleus transplanting blastaea.
6. the defreezing method of frozen embryo, on 39 ℃ constant temperature platform, carry out, adopt following steps: the frozen embryo that (1) obtains the arbitrary method of claim 1-5 is put into DPBS basal liquid 1-3 minute that has added 0.25M sucrose, (2) embryo is moved in the DPBS basal liquid added 0.15M sucrose 5-10 minute, (3) move into the embryo in the DPBS basal liquid and got final product in 5-10 minute.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428910A (en) * | 2010-11-03 | 2012-05-02 | 江苏省北科生物科技有限公司 | Protecting liquid for freezing and storing human umbilical cord Wharton jelly tissue block |
| CN102648708A (en) * | 2011-02-25 | 2012-08-29 | 深圳华大方舟生物技术有限公司 | Freezing liquid for embryo or cells and application thereof |
| CN103947584A (en) * | 2014-04-04 | 2014-07-30 | 湖南苗王生物科技有限公司 | Thawing method for vitrified frozen sturgeon sperms |
| CN104012521A (en) * | 2014-06-10 | 2014-09-03 | 西安交通大学 | Non-contact type liquid drop method refrigerating device and refrigerating method |
| 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 |
| CN108402031A (en) * | 2018-03-13 | 2018-08-17 | 南京至力高企信息技术有限公司 | A kind of CIK cell cryopreservation device |
| CN108651440A (en) * | 2018-04-18 | 2018-10-16 | 中南大学 | A kind of supper-fast freezen protective system of human seminal fluid |
| ES2737681A1 (en) * | 2018-07-11 | 2020-01-15 | Univ Sevilla | DEVICE AND PROCEDURE FOR PREPARING CELLS FOR ULTRA-FAST VITRIFICATION BY DEHYDRATION (Machine-translation by Google Translate, not legally binding) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DE69913186T3 (en) * | 1998-10-14 | 2012-08-09 | Katrina T. Forest | METHOD FOR GLAZING A BIOLOGICAL SAMPLE |
| CN100348096C (en) * | 2004-09-15 | 2007-11-14 | 中国科学院海洋研究所 | Vitrifiation freezing preservation method for genine porgy embryo particles |
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- 2007-06-18 CN CNB2007101190041A patent/CN100459861C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428910A (en) * | 2010-11-03 | 2012-05-02 | 江苏省北科生物科技有限公司 | Protecting liquid for freezing and storing human umbilical cord Wharton jelly tissue block |
| CN102648708A (en) * | 2011-02-25 | 2012-08-29 | 深圳华大方舟生物技术有限公司 | Freezing liquid for embryo or cells and application thereof |
| CN102648708B (en) * | 2011-02-25 | 2014-08-13 | 深圳华大方舟生物技术有限公司 | Freezing liquid for embryo or cells and application thereof |
| CN103947584A (en) * | 2014-04-04 | 2014-07-30 | 湖南苗王生物科技有限公司 | Thawing method for vitrified frozen sturgeon sperms |
| CN104012521A (en) * | 2014-06-10 | 2014-09-03 | 西安交通大学 | Non-contact type liquid drop method refrigerating device and refrigerating method |
| 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 |
| CN108402031A (en) * | 2018-03-13 | 2018-08-17 | 南京至力高企信息技术有限公司 | A kind of CIK cell cryopreservation device |
| CN108651440A (en) * | 2018-04-18 | 2018-10-16 | 中南大学 | A kind of supper-fast freezen protective system of human seminal fluid |
| ES2737681A1 (en) * | 2018-07-11 | 2020-01-15 | Univ Sevilla | DEVICE AND PROCEDURE FOR PREPARING CELLS FOR ULTRA-FAST VITRIFICATION BY DEHYDRATION (Machine-translation by Google Translate, not legally binding) |
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