CN108244100A - A kind of glass freezing liquid and its freezing method - Google Patents
A kind of glass freezing liquid and its freezing method Download PDFInfo
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- CN108244100A CN108244100A CN201810287794.2A CN201810287794A CN108244100A CN 108244100 A CN108244100 A CN 108244100A CN 201810287794 A CN201810287794 A CN 201810287794A CN 108244100 A CN108244100 A CN 108244100A
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- freezing liquid
- glass
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- taxol
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention belongs to supplementary reproduction fields, and in particular to a kind of glass freezing liquid.The glass freezing liquid of the present invention is used as cryoprotector by adding taxol, and combines taxol addition manner, and the glass freezing liquid processing of acquisition can improve blastocyst rate in vitro fertilization after egg mother cell thaws.It can play the role of reducing the freezing injury of mature oocyte using taxol as the additive in freezing liquid and improve after fertilization Embryo viability.And taxol addition concentration is studied, obtain the optimum concentration of addition manner of the present invention.The glass freezing liquid of the present invention can increase substantially anabiosis rate and blastocyst rate after mature oocyte freeze-thaw, and play a protective role to cell ultrastructure.
Description
Technical field
The invention belongs to supplementary reproduction fields, and in particular to a kind of glass freezing liquid for adding taxol.
Background technology
The gamete of the mankind and the freezen protective of embryo have been a necessary parts in artificial field of reproduction.The past this
In 20 years, the development of freezen protective technology can also reflect the development degree of Artificial reproduction technology.
Since egg mother cell volume is big, water content is more, intracellular ice crystal is easily formed in refrigerating process, cell is caused
Mechanical injuries, therefore the key that egg mother cell preserves is to reduce the formation of intracellular ice crystal in refrigerating process.Refrigerating process
The osmotic injury that the protection liquid of mechanical damage and high concentration caused by middle intracellular ice crystal is formed generates cell causes ovum female thin
The transparent desmorrhexis of born of the same parents, hardening, cortical granule discharge, and are difficult to after micro-pipe, depolymerization of microfilaments from assembling, chromosome aberration etc., this is directly
It influences survival rate, rate in vitro fertilization, body early embryo survival rate and the pregnancy rate of defrosting egg mother cell or even influences offspring's
Health.
Current Refrigeration Technique can be used in the embryo of freezen protective sperm, ovum and different times, especially vitrifying
Refrigeration Technique (Vitrification) has not only obtained medical field and has widely approved, and the slow art freezing of substitution tradition gradually
Method, and as the optimal selection of Refrigeration Technique, even by this Technology application in highly difficult ovum, ovary tissue and human embryos
In the freezing of stem cell.Glass freezing is cured using high concentration protection agent solution when enduring cold, and is increased by the extreme of viscosity
The characteristics of, become structureless glassy state from liquid, this glassy state can keep its solution state molecule and ion
Separation distribution.Since vitrification is quick, low cost, the features such as intracellular ice crystal is avoided to be formed, so as ovum
The method of mother cell freezing.
Although after having been obtained after the chilled processing of egg mother cell and immature oocyte in many stages of ripeness normally
Generation, but its success rate is still very low.Most of egg mother cells will all cause in form and function not during glass freezing
With the damage of degree.And it is considered as freezing efficiency drop that freezing, which causes cytoskeleton injury (microtubule depolymerization leads to spindle disorder),
Low one of the major reasons.
Therefore a kind of glass freezing liquid for adding taxol is developed, it will be to stablizing cytoskeleton and protection cell ultra micro
Structure plays a role, and reduces damage of the glass freezing process to cell.
Invention content
In view of this, the purposes one of the objects of the present invention is to provide a kind of taxol in glass freezing liquid,
It plays a role for the survival rate after improving the freezing of egg mother cell and after fertilization embryo development rate.
To achieve the above object, the technical scheme is that：
The ice crystal that existing vitrification is formed in refrigerating process damages egg mother cell skeleton.Mesh
Before, many scholars think that spindle is a key factor for influencing mature oocyte refrigerating effect.Mature oocyte is spun
Hammer body is formed by two kinds of tubulin polymerizations of α and β, is the temporary dynamical structure of micro-pipe, normal mature is divided, is fertilized,
Polar body is discharged and pronuclear migration is all essential.The polymerization of micro-pipe maintains dynamic with depolymerization under specific physiological condition and puts down
Weighing apparatus, this balance are highly susceptible to the influence of various physic-chemical changes (such as cool down or be exposed to cryoprotector).Therefore micro-pipe
Formed it is environmentally sensitive, and be easy to occur microtubule depolymerization, cause spindle formation be obstructed.
For taxol as a kind of Cytoskeleton Stabilizer, it has dependence and invertibity, this work to the combination of micro-pipe
With the critical concentration for reducing the tubulin needed for polymerization, the direction movement that dynamic equilibrium is made to be assembled towards micro-pipe promotes micro-
Pipe re-assemblying in vitro.Taxol treatment can increase substantially blastocyst rate after mature oocyte freeze-thaw,
And it plays a protective role to cell ultrastructure.
The present invention provides purposes of the taxol in glass freezing liquid, to stablizing cytoskeleton and improving egg mother cell
Blastaea incidence play a role.
The second object of the present invention is to provide a kind of glass freezing liquid for adding taxol, female thin for improving ovum
Survival rate and after fertilization embryo development rate after the freezing of born of the same parents play a role.
To achieve the above object, the technical scheme is that：
Taxol is added in glass freezing liquid.
Taxol is previously mentioned that as Cytoskeleton Stabilizer, the effect that may be played in egg mother cell freezing,
Therefore taxol is added in glass freezing liquid.
As a preferred option, in above-mentioned glass freezing liquid taxol a concentration of 0.1~1.5 μm of ol/L.
The present invention is by the addition concentration of taxol in experimental verification glass freezing liquid, when paclitaxel concentration is 0.5 μ
Best results during mol/L.
Except paclitaxel concentration problem, this invention also solves the taxol addition manners during glass freezing.Experiment
The result shows that it only can not show a candle in pretreatment and freezing liquid one, freezing liquid two in the effect of preprocessing process addition taxol
Add the effect of taxol.Therefore it is equal in basic freezing liquid, freezing liquid one, freezing liquid two during the glass freezing of the present invention
Add taxol.
As a preferred option, above-mentioned glass freezing liquid includes basic freezing liquid, freezing liquid one and freezing liquid two；Wherein
Basic freezing liquid includes M199, fetal calf serum, buffer and taxol；
Freezing liquid one adds permeability cryoprotector on the basis of basic freezing liquid；
Freezing liquid two adds permeability cryoprotector and sucrose on the basis of basic freezing liquid.
Further, it is preferable that above-mentioned basis freezing liquid ingredient includes：M199 culture solutions are as basic liquid, NaHCO35~
10~14mmol/L of 10mmol/L, HEPES, 10~15mmol/L of HEPES-Na, 0.065~0.077g/L of penicillin, strepto-
0.1~1.5 μm of 0.05~0.09g/L of element, fetal calf serum (volume content) 10%~20%, taxol ol/L.
Preferably, above-mentioned permeability cryoprotector is ethylene glycol and dimethyl sulfoxide (DMSO).
Preferably, the permeability cryoprotector of the above-mentioned addition of freezing liquid one is：The second of volume content 5.5%~7.5%
The dimethyl sulfoxide (DMSO) of glycol and volume content 6.0%~7.5%.
Preferably, the permeability cryoprotector of the above-mentioned addition of freezing liquid two is：The second two of volume content 15%~20%
The dimethyl sulfoxide (DMSO) of alcohol and volume content 15%~20%；Addition sucrose concentration is 0.5~1.0mol/L.
The third object of the present invention is to provide adds in a kind of above-mentioned basic freezing liquid, freezing liquid one, freezing liquid two
The preparation method of the glass freezing liquid of taxol.
To achieve the above object, the technical scheme is that：
The preparation side of the glass freezing liquid of taxol is added in above-mentioned basis freezing liquid, freezing liquid one, freezing liquid two
Method includes the following steps：
1) basic freezing liquid is prepared；
2) permeability cryoprotector is added on the basis of the basic freezing liquid of step 1), obtains freezing liquid one；
3) permeability cryoprotector and sucrose are added on the basis of the basic freezing liquid of step 1), obtains freezing liquid two.
The fourth object of the present invention is to provide adds in a kind of above-mentioned basic freezing liquid, freezing liquid one, freezing liquid two
The application method of the glass freezing liquid of taxol, in the method glass freezing liquid of the invention freezing egg mother cell acquirement
Peak efficiency.
To achieve the above object, the technical scheme is that：
The glass freezing liquid that taxol is added in above-mentioned basis freezing liquid, freezing liquid one, freezing liquid two is female thin to ovum
The method that born of the same parents carry out glass freezing, includes the following steps：
1) by basic freezing liquid, freezing liquid one, freezing liquid two in equilibrium at room temperature 25min；
2) egg mother cell is moved on into 5~8min of processing in basic freezing liquid；
3) egg mother cell is moved into after handling, 20~40s is balanced in freezing liquid one；
4) it moves into freezing liquid two, preserves egg mother cell sucking OPS pipes, the rapid liquid nitrogen frozen that puts into 25s.
The beneficial effects of the present invention are：The glass freezing liquid of addition taxol provided by the invention, to stablizing cell
Skeleton and protection cell ultrastructure play a role.For egg mother cell, the survival rate of egg mother cell, embryo in vitro fertilization after freezing
Tire developmental rate, cleavage rates and mulberry blastocyst rate are improved, for anabiosis rate, rate of fertilization, embryo after the freezing of egg mother cell
The raising of developmental potency plays an important role.
Description of the drawings
Fig. 1 is the part 1- cells of embodiment 2, wherein：A is control group, and B is experimental group 1, and C is experimental group 2.
Fig. 2 is the part 2- cells of embodiment 2, wherein：A is control group, and B is experimental group 1, and C is experimental group 2.
Fig. 3 is the part blastomere of embodiment 2, wherein：A is control group, and B is experimental group 1, and C is experimental group 2.
Fig. 4 is the part 2- cells of embodiment 3, wherein：A is control group, and B is 0.5 μm of ol/L taxol group, and C is 1.0 μ
Mol/L taxol groups, D are 1.5 μm of ol/L taxol groups.
Fig. 5 is the part blastomere of embodiment 3, wherein：：A is control group, and B is 0.5 μm of ol/L taxol group, and C is
1.0 μm of ol/L taxol groups, D are 1.5 μm of ol/L taxol groups.
Specific embodiment
The preferred embodiment of the present invention will be described in detail (with reference to attached drawing) below.Tool is not specified in preferred embodiment
The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition, illustrated embodiment are to preferably be said to present disclosure
It is bright, but be not that present disclosure is only limitted to illustrated embodiment.So those skilled in the art are according to foregoing invention
Content carries out nonessential modifications and adaptations to embodiment, still falls within protection scope of the present invention.
The preparation of 1 glass freezing liquid of embodiment
1. prepare cryoprotective extender (pretreatment fluid):
1) M199 culture solutions are as solvent, NaHCO35~10mmol/L, 10~14mmol/L of HEPES, HEPES-Na 10
~15mmol/L, 0.065~0.077g/L of penicillin, 0.05~0.09g/L of streptomysin, fetal calf serum (volume content) 10%~
20%th, 0.1~1.5 μm of ol/L of taxol；
2) mixing, 0.22 μm of disposable filter filtering packing, for use.
2. freezing liquid one：6.5% (v/v) ethylene glycol and 7% (v/v) DMSO are added in cryoprotective extender.
3. freezing liquid two：18% (v/v) ethylene glycol+18% (v/v) DMSO and 0.75mol/ is added in cryoprotective extender
L sucrose.
The addition manner of taxol in 2 glass freezing liquid of embodiment
1. grouping
With the glass freezing liquid for not adding taxol as a control group, it is only used as and tests added with taxol in pretreatment fluid
Added with the conduct experimental group 2 of taxol in group 1 and pretreatment fluid, freezing liquid one, freezing liquid two.
2. experimental method
Three groups of glass freezing liquid freeze for mature oocyte, are carried out using OPS (openpulled straw) method
Glass freezing：
1) by basic freezing liquid, freezing liquid one, freezing liquid two in equilibrium at room temperature 25min；
2) egg mother cell is moved on into 5~8min of processing in basic freezing liquid；
3) egg mother cell is moved into after handling, 20~40s is balanced in freezing liquid one；
4) it moves into freezing liquid two, preserves egg mother cell sucking OPS pipes, the rapid liquid nitrogen frozen that puts into 25s.
3. experimental result
Survival rate, developmental rate of in-vitro fertilization embryo, cleavage rates and mulberry after record mouse mature oocyte glass freezing
Blastocyst rate the results are shown in Table 1, and picture is shown in attached drawing 1~3.Wherein：1- cells are the fertilized eggs after thawing；2- cells are cell
Mitosis is carried out, splits into two cells；Blastaea is the 5th day, has a blastocoele for expansion among blastaea, inside has one
A circle trophocyte has been enclosed in close inner cell mass, blastaea outside.
The freezing liquid handling result of 1 taxol difference addition manner of table
As shown in Table 1, after thawing wherein in pretreatment fluid, freezing liquid one, freezing liquid two added with the processing group of taxol
Survival rate, developmental rate of in-vitro fertilization embryo, cleavage rates and mulberry blastocyst rate, which are significantly higher than not in only pretreatment fluid, adds Japanese yew
Alcohol processing group (P<0.05).Therefore, Japanese yew is added in the pretreatment fluid in glass freezing liquid, freezing liquid one, freezing liquid two
Alcohol has egg mother cell better protecting effect.
The addition concentration of taxol in 3 glass freezing liquid of embodiment
1. grouping
With the glass freezing liquid for not adding taxol as a control group, the vitrifying of various concentration taxol is cold there are three types of adding
Freeze liquid as experimental group.
2. experimental method
Four groups of glass freezing liquid freeze for mouse mature oocyte：Using OPS (open pulled straw) method
Glass freezing is carried out, egg mother cell is moved on to 25min is handled in cryoprotective extender first, then moved into freezing liquid one and balance
30s, then move into glass freezing liquid two, egg mother cell and a small amount of freezing liquid sucking OPS input liquid nitrogen frozens are protected in 25s
It deposits.
3. experimental result
Survival rate, developmental rate of in-vitro fertilization embryo, cleavage rates and mulberry after record mouse mature oocyte glass freezing
Blastocyst rate the results are shown in Table 2, and picture is shown in attached drawing 4,5.
Table 2 adds various concentration taxol freezing liquid to the influence after mature oocyte glass freezing
Processing group post-thaw survival rate, developmental rate of in-vitro fertilization embryo, cleavage rates and mulberry blastaea hair added with taxol
The rate of educating, which is significantly higher than, does not add taxol treatment group (P<0.05) best results, and during 0.5 μm of ol/L.
To sum up, when the glass freezing liquid of the present invention adds Japanese yew in pretreatment fluid, freezing liquid one, freezing liquid two
Alcohol, and paclitaxel concentration be 0.5 μm of ol/L when, it is best for the protective effect of egg mother cell.The glass freezing liquid of the present invention
Middle addition taxol, the post-thaw survival rate of egg mother cell, developmental rate of in-vitro fertilization embryo, cleavage rates and mulberry blastocyst rate are equal
It significantly improves.
The glass freezing liquid of the addition taxol of the present invention in supplementary reproduction field, for improve success rate have it is important
Meaning.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail in good embodiment, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (10)
1. purposes of the taxol in glass freezing liquid is used to prepare.
2. a kind of glass freezing liquid for adding taxol.
3. glass freezing liquid according to claim 2, which is characterized in that taxol is dense in the glass freezing liquid
It spends for 0.1~1.5 μm of ol/L.
4. glass freezing liquid according to claim 2, characteristic are, the glass freezing liquid includes basis and freezes
Liquid, freezing liquid one and freezing liquid two；
Wherein described basic freezing liquid includes M199 culture solutions, fetal calf serum, buffer and taxol；
The freezing liquid one adds permeability cryoprotector on the basis of basic freezing liquid；
The freezing liquid two adds permeability cryoprotector and sucrose on the basis of basic freezing liquid.
5. glass freezing liquid according to claim 4, which is characterized in that the basis freezing liquid ingredient includes：M199
Culture solution is as basic liquid, NaHCO35~10mmol/L, 10~14mmol/L of HEPES, 10~15mmol/L of HEPES-Na,
0.065~0.077g/L of penicillin, 0.05~0.09g/L of streptomysin, fetal calf serum (volume content) 10%~20%, taxol
0.1~1.5 μm of ol/L.
6. glass freezing liquid according to claim 4, which is characterized in that the infiltration of the freezing liquid one and freezing liquid two
Property cryoprotector be ethylene glycol and dimethyl sulfoxide (DMSO).
7. glass freezing liquid according to claim 4, which is characterized in that the permeability freezing that the freezing liquid one adds
Protective agent is：The ethylene glycol of volume content 5.5%~7.5% and the dimethyl sulfoxide (DMSO) of volume content 6.0%~7.5%.
8. glass freezing liquid according to claim 4, which is characterized in that the permeability freezing that the freezing liquid two adds
Protective agent is：The ethylene glycol of volume content 15%~20% and the dimethyl sulfoxide (DMSO) of volume content 15%~20%；
Addition sucrose concentration is 0.5~1.0mol/L.
9. the preparation method of glass freezing liquid described in claim 4, which is characterized in that include the following steps：
1) basic freezing liquid is prepared；
2) permeability cryoprotector is added on the basis of the basic freezing liquid of step 1), obtains freezing liquid one；
3) permeability cryoprotector and sucrose are added on the basis of the basic freezing liquid of step 1), obtains freezing liquid two.
10. the method that the glass freezing liquid described in claim 4 carries out egg mother cell glass freezing, which is characterized in that
Include the following steps：
1) by basic freezing liquid, freezing liquid one, freezing liquid two in equilibrium at room temperature 25min；
2) egg mother cell is moved on into 5~8min of processing in basic freezing liquid；
3) egg mother cell is moved into after handling, 20~40s is balanced in freezing liquid one；
4) it moves into freezing liquid two, preserves egg mother cell sucking OPS pipes, the rapid liquid nitrogen frozen that puts into 25s.
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| CN201810287794.2A CN108244100A (en) | 2018-04-03 | 2018-04-03 | A kind of glass freezing liquid and its freezing method |
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| CN201810287794.2A CN108244100A (en) | 2018-04-03 | 2018-04-03 | A kind of glass freezing liquid and its freezing method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111149795A (en) * | 2020-01-14 | 2020-05-15 | 成都艾伟孚生物科技有限公司 | Cryoprotectant and application thereof, sperm freezing liquid and preparation method thereof |
| CN111183972A (en) * | 2020-01-14 | 2020-05-22 | 成都艾伟孚生物科技有限公司 | Composition and application thereof, sperm refrigerating fluid and preparation method thereof |
| CN111602653A (en) * | 2020-07-02 | 2020-09-01 | 深圳韦拓生物科技有限公司 | Vitrified refrigerating fluid suit and preparation method thereof |
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| CN105052894A (en) * | 2015-08-26 | 2015-11-18 | 中国农业科学院特产研究所 | GV-stage oocyte cryopreservation fluid and cryopreservation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111149795A (en) * | 2020-01-14 | 2020-05-15 | 成都艾伟孚生物科技有限公司 | Cryoprotectant and application thereof, sperm freezing liquid and preparation method thereof |
| CN111183972A (en) * | 2020-01-14 | 2020-05-22 | 成都艾伟孚生物科技有限公司 | Composition and application thereof, sperm refrigerating fluid and preparation method thereof |
| CN111602653A (en) * | 2020-07-02 | 2020-09-01 | 深圳韦拓生物科技有限公司 | Vitrified refrigerating fluid suit and preparation method thereof |
| CN111602653B (en) * | 2020-07-02 | 2021-03-02 | 深圳韦拓生物科技有限公司 | Vitrified refrigerating fluid suit and preparation method thereof |
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