CN110214776A - It is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method - Google Patents
It is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method Download PDFInfo
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- CN110214776A CN110214776A CN201910585668.XA CN201910585668A CN110214776A CN 110214776 A CN110214776 A CN 110214776A CN 201910585668 A CN201910585668 A CN 201910585668A CN 110214776 A CN110214776 A CN 110214776A
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- straw
- tip carrier
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- pig blastocyst
- freezing
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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
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
Abstract
The invention discloses it is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, the preparation of the embryo vitrifying freeze Tip carrier made by S1 based on straw, S2 freeze the freezing of the pig blastocyst of Tip carrier, S3 freeze Tip carrier pig blastocyst defrosting, to realize efficiently freezing and thaw for pig blastocyst.The present invention it is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, it is technically simple, it is convenient to operate, the great proficiency of operator is not needed, it is at low cost, high-efficient, the incidence for significantly reducing the embryo damage and loss in embryo vitrifying freeze and course of defrosting, can be advantageously applied to the Popularization And Development of human reproduction's medicine and animal husbandry.
Description
Technical field
It is specially a kind of that Tip carrier is freezed based on straw the present invention relates to bioengineering and embryo genetic technical field
Pig blastocyst freeze and defreezing method.
Background technique
With the development of medicine and biotechnology, no matter from human ancillary reproductive and precious mammal embryo of silkworms and embryo
Preservation or save the bio-diversity or the preservation of gene editing model animal etc. of region breeding stock, mammal embryo
Vitrification be all made that very big contribution.And using gamete and Embryo Cryopreservation Technology transport can it is more economical and
The risk of transmission can be reduced.
Rall W.F. in 1985 applies vitrification successfully to save 8- cell mouse embryo for the first time and is thawing
After so that embryo is continued development to blastaea, this is the embryo that the mankind successfully save mammal using vitrification for the first time
Tire.Vitrification is gradually applied to the cryo-conservation of the egg mother cells such as ox, sheep, pig and people and body early embryo later.
Vitrification is to make embryo quickly through " danger area " (ice crystal is formed) using the method for hypervelocity cooling, is made in cell
Water keeps a kind of glazed disordered state of class, and the formation for reducing ice crystal reduces freezing injury, to improve the survival rate of cell.
Micro drop method by reducing protection liquid product is developed in principle at this, and the various carriers of micro-droplet status can be kept outstanding
Be it is important, what is be currently mainly used mainly has a Cryotop, OPS, SOPS, nylon membrane, and surface of solids etc. is used as freezing carrier.Mesh
The mainly Cryotop of prehuman's egg mother cell and body early embryo frozen, what a large amount of freezings of commercialization ox embryo used
It is OPS and SOPS, in contrast Cryotop has higher success rate.But both carrier costs are excessively high, limit this skill
Popularization and application of the art in animal husbandry.
Current commercialized freezing carrier is at high cost, and purchase approach is few, and delivery date is long, Reusability is unable to, by the limit of carrier
System also has no idea to store a large amount of embryo.Meanwhile in the freezing and thaw of pig blastocyst, conventional method, technology is complicated, operation
It is cumbersome, need grasp freezing and deforst technique that worker is skilled, and common laborer is difficult quickly to grasp freezing skill, cell it is cold
Freeze, defrosting survival rate it is low, which limits this technology popularization and application.
Summary of the invention
It is an object of the invention to: provide it is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method,
To solve disadvantages described above.
To achieve the goals above, the invention provides the following technical scheme:
It is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, comprising the following steps:
S1: the preparation of the embryo vitrifying freeze Tip carrier based on straw production
A1, controllable constant-temperature thermal station, metal module, ultraviolet lamp, straw, ophthalmologic operation are cut, stapler, staple, lattice ruler
Wiped clean, and with 75% alcohol controllable constant-temperature thermal station, metal module, ophthalmologic operation are cut, stapler, staple, lattice ruler
Naturally dry after disinfection；
A2, controllable constant-temperature thermal station temperature is adjusted to 50~60 DEG C, after temperature is stablized, operator takes anti-scald hand
Set；
A3, using lattice ruler, measuring 0.15cm apart from the long position 1cm and 2cm in straw open end, and in straw opening
Width carries out simple marking respectively；
A4, it is marked according to previous step, cuts off straw using ophthalmologic operation, cut in straw open end and stay wide 0.15cm, length
The plastic strip of 1cm；In controllable constant-temperature thermal station, it will be flattened except the other parts straw of plastic strip using metal module, and upper
One step 2cm mark position is bound using stapler and staple；
A5, the straw for making previous step, in the UV lamp sterilize 12~18min, and randomly select two sterilizing after
Straw examine droplet solid performance complete the preparation of embryo vitrifying freeze Tip carrier after the assay was approved；
S2: the pig blastocyst of freezing Tip carrier freezes
B1, it is cultivated 7 days by PN phase pig zona-free oocytes micromanipulation degreasing, and in PZM3；
B2, the degreasing pig blastocyst of culture is cleaned 2 times in HM drop, at room temperature, balances 5~10min in ES liquid
It is handled；
B3, by previous step treated pig blastocyst, with 5 pieces in one group of immigration VS drop, immediately using moving embryo needle for VS
5 pieces of embryos in drop uniformly put the modeling of the wide 0.15cm, long 1cm of embryo vitrifying freeze Tip carrier made from S1 step
It in material strip, and will be mounted in the freezing Tip carrier investment liquid nitrogen of pig blastocyst, the time of whole process is no more than 60s, that is, completes
The pig blastocyst of entire freezing Tip carrier freezes；
S3: the defrosting of the pig blastocyst of freezing Tip carrier
C1, the freezing Tip carrier for being mounted with pig blastocyst is removed from liquid nitrogen, is rapidly inserted into the TS thawing solution of heating
In, after remaining stationary 15~25s, pig blastocyst and freezing Tip carrier thaw separation；
C2, by the pig blastocyst of previous step thaw separation, moved into DS dilution in 1min and balance 5min, then moved into
3min is balanced in WS1 liquid, is finally moved into WS2 liquid and is balanced 3min；
C3, the pig blastocyst by previous step through WS2 liquid Balance Treatment move into culture dish culture 8h in PZM3, then observe pig
The futtock situation of the blastocoele of blastaea completes the whole process of the defrosting of the pig blastocyst of freezing Tip carrier.
Preferably, in A1 step, the controllable constant-temperature thermal station, concrete model is CBL Model100.
Preferably, in A1 step, the straw, concrete model IMV005565.
Preferably, in B2 step, the room temperature, specific dimension is 20~28 DEG C.
Preferably, in B3 step, the time of the whole process is 45~60s.
Preferably, in C1 step, the temperature of the TS thawing solution is 37~41 DEG C.
The beneficial effects of the present invention are:
The present invention it is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, it is recessed using straw itself
The design feature of type produces the embryo vitrifying freeze Tip carrier based on straw production, with the Cryotop of sky high cost or
Person's SOPS carrier is compared to more meaningful, firstly, nontoxic, and the production of Tip carrier is used and is easiest to from the point of view of economy
The material of acquisition, it is easy to make, reduce the cost of embryo freezing, the popularization of embryo freezing commercially can be accelerated in this way and answered
With；Secondly, the use of Tip carrier, relative to Cryotop, because reducing casing, reducing volume can have in liquid nitrogen container
More embryos are stored in the space of limit, not only reduce cost, and increase the convenience of operation；Again, SOPS carrier
Due to the density issue of its own, it is easy to swim in liquid nitrogen surface during operation, increases operation difficulty and embryo
The risk of loss, and itself flat structure of Tip carrier increases support density very good solution this problem by staple.This
Invent it is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, it is technically simple, operate it is convenient, do not need
The great proficiency of operator, at low cost, high-efficient, the embryo significantly reduced in embryo vitrifying freeze and course of defrosting damages
Wound and the incidence lost, can be advantageously applied to the Popularization And Development of human reproduction's medicine and animal husbandry.
Detailed description of the invention
Fig. 1: the pig blastocyst of of the invention (Tip carrier, experimental group) and Cryotop carrier (control group) respectively before freezing,
After defrosting after 0h, defrosting 6h micrograph；
Fig. 2: the futtock rate figure of the pig blastocyst of (Tip carrier, experimental group) of the invention and Cryotop carrier (control group).
Specific embodiment
In order to facilitate the understanding of those skilled in the art, the present invention is further illustrated combined with specific embodiments below.
Embodiment 1:
It is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, comprising the following steps:
S1: the preparation of the embryo vitrifying freeze Tip carrier based on straw production
A1, by controllable constant-temperature thermal station (model CBL Model 100), metal module, ultraviolet lamp, straw (model
IMV005565), ophthalmologic operation cut, stapler, staple, lattice ruler wiped clean, and with 75% alcohol to controllable constant warm
Naturally dry after platform, metal module, ophthalmologic operation are cut, stapler, staple, lattice ruler sterilize；
A2, controllable constant-temperature thermal station temperature is adjusted to 50 DEG C, after temperature is stablized, operator takes anti-scald gloves；
A3, using lattice ruler, measuring 0.15cm apart from the long position 1cm and 2cm in straw open end, and in straw opening
Width carries out simple marking respectively；
A4, it is marked according to previous step, cuts off straw using ophthalmologic operation, cut in straw open end and stay wide 0.15cm, length
The plastic strip of 1cm；In controllable constant-temperature thermal station, it will be flattened except the other parts straw of plastic strip using metal module, and upper
One step 2cm mark position is bound using stapler and staple；
A5, the straw for making previous step, sterilize 12min in the UV lamp, and randomly selects the wheat after several sterilizings
Pipe examines droplet solid performance, wherein with the droplet shape on the plastic strip of straw at mellow and full, towering person is best, and droplet shape
Elongated, flat person is poor, thus the quality of the straw of this batch of the overall evaluation, straw after the assay was approved, that is, completes embryo's glass
The preparation of glassization freezing Tip carrier；
S2: the pig blastocyst of freezing Tip carrier freezes
B1, PN cell stage pig zona-free oocytes are operated into degreasing under the microscope, and in PZM3 (Porcine Zygote
Medium-3 pig zygophase embryo medium) middle culture 7 days；
It is B2, the degreasing pig blastocyst of previous step culture is clear in HM (Handling medium operate liquid) drop of 100 μ L
It washes 2 times, reusing shifting embryo needle, (Equilibration solution is flat by the ES of the 100 μ L of degreasing pig blastocyst immigration after cleaning
Weigh liquid) in liquid, 28 DEG C at room temperature balance 5min handled；
B3, by previous step treated pig blastocyst, the VS (Vitrification for being one group of 20 μ L of immigration with 5 pieces
Solution glass freezing liquid) in drop, it is using 200 μm of diameter of shifting embryo needle that 5 pieces of embryos in VS drop are uniform immediately
It puts the wide 0.15cm of embryo vitrifying freeze Tip carrier made from S1 step, on the plastic strip of long 1cm, and pig will be mounted with
In the freezing Tip carrier investment liquid nitrogen of blastaea, the time of whole process is no more than 60s, and concrete restriction is completed in 45~60s,
Realize freezing for the pig blastocyst of entire freezing Tip carrier；
S3: the defrosting of the pig blastocyst of freezing Tip carrier
C1, the freezing Tip carrier for being mounted with pig blastocyst is removed from liquid nitrogen, is rapidly inserted into equipped with 500 μ L heating
In TS (Thawing solution thawing solution) liquid that temperature is 37 DEG C, it is remain stationary 15s, realizes pig blastocyst and freezing Tip
Carrier thaw separation；
C2, by the pig blastocyst of previous step thaw separation, moved into 1min 500 μ L DS (Diluent solution dilution
Liquid) in balance 5min, then move into and balance 3min in WS1 liquid (Washing solution1 cleaning solution 1), finally move into WS2 liquid
3min is balanced in (Washing solution2 cleaning solution 2)；
C3, the pig blastocyst by previous step through WS2 liquid Balance Treatment move into culture dish culture 8h in PZM3, then observe pig
The futtock situation of the blastocoele of blastaea completes the whole process of the defrosting of the pig blastocyst of freezing Tip carrier.
Embodiment 2:
Operating procedure is same as Example 1 with method, and different is:
A2, controllable constant-temperature thermal station temperature is adjusted to 60 DEG C；
A5, sterilize 18min in the UV lamp；
In B2, ES liquid, 20 DEG C balance 10min at room temperature and are handled；
C1, the freezing Tip carrier for being mounted with pig blastocyst is removed from liquid nitrogen, is rapidly inserted into equipped with 500 μ L heating
In the TS liquid that temperature is 41 DEG C, it is remain stationary 25s, realizes pig blastocyst and freezing Tip carrier thaw separation.
Embodiment 3:
Operating procedure is same as Example 1 with method, and different is:
A2, controllable constant-temperature thermal station temperature is adjusted to 55 DEG C；
A5, sterilize 15min in the UV lamp；
In B2, ES liquid, 24 DEG C balance 8min at room temperature and are handled；
C1, the freezing Tip carrier for being mounted with pig blastocyst is removed from liquid nitrogen, is rapidly inserted into equipped with 500 μ L heating
In the TS liquid that temperature is 39 DEG C, it is remain stationary 20s, realizes pig blastocyst and freezing Tip carrier thaw separation.
Using above-described embodiment, a kind of pig blastocyst based on straw freezing Tip carrier of the present invention freeze and defreezing method,
Experiment is carried out altogether and repeats 5 batches (experimental group), and embodiment 1, embodiment 2, the quantity of embodiment 3 of every batch of use are identical；
The same period is control group using adding the Cryotop carrier glassization of rattan to freeze pig blastocyst, remaining operation is all the same, finally obtains
Experimental data average value such as the following table 1:
The development condition of the pig blastocyst of table 1, (experimental group) of the invention and Cryotop carrier (control group)
As shown in Figure 1, the pig blastocyst for (Tip carrier, experimental group) of the invention and Cryotop carrier (control group) exists respectively
Before freezing, after thawing after 0h, defrosting 6h micrograph；As shown in Fig. 2, for (Tip carrier, experimental group) of the invention and Cryotop
The futtock rate figure of the pig blastocyst of carrier (control group).Based on table 1, Fig. 1, Fig. 2 it is found that experiment statistics result Mean ± S.E.:
71% ± 12%.It is examined using t and carries out significance difference analysis, P=0.44 (P < 0.05 is significant difference).Illustrate fortune of the present invention
There is similar freezing efficiency with Tip carrier and commercialized Cryotop carrier.It is of the invention a kind of based on straw freezing Tip load
The pig blastocyst of body freeze and defreezing method, it is technically simple, operate it is convenient, do not need the great proficiency of operator, cost
It is low, high-efficient, the incidence of the embryo damage and loss in embryo vitrifying freeze and course of defrosting is significantly reduced, it can be fine
Ground is applied to the Popularization And Development of human reproduction's medicine and animal husbandry.
Above-mentioned is that invention is exemplarily described, it is clear that present invention specific implementation is not subject to the restrictions described above,
As long as using this insubstantial improvement that the inventive concept and technical scheme of the present invention carry out, or the not improved structure by invention
Think and technical solution directly applies to other occasions, it is within the scope of the present invention.
Claims (6)
1. it is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method, which is characterized in that including following step
It is rapid:
S1: the preparation of the embryo vitrifying freeze Tip carrier based on straw production
A1, controllable constant-temperature thermal station, metal module, ultraviolet lamp, straw, ophthalmologic operation are cut, the wiping of stapler, staple, lattice ruler
Completely, and with 75% alcohol to controllable constant-temperature thermal station, metal module, ophthalmologic operation it cuts, the disinfection of stapler, staple, lattice ruler
Naturally dry afterwards；
A2, controllable constant-temperature thermal station temperature is adjusted to 50~60 DEG C, after temperature is stablized, operator takes anti-scald gloves；
A3, using lattice ruler, measuring 0.15cm wide apart from the long position 1cm and 2cm in straw open end, and in straw opening
Degree, carries out simple marking respectively；
A4, it is marked according to previous step, cuts off straw using ophthalmologic operation, cut in straw open end and stay wide 0.15cm, long 1cm
Plastic strip；In controllable constant-temperature thermal station, it will be flattened except the other parts straw of plastic strip using metal module, and in previous step
2cm mark position is bound using stapler and staple；
A5, the straw for making previous step, sterilize 12~18min in the UV lamp, and randomly selects the wheat after two sterilizings
Pipe examines droplet solid performance to complete the preparation of embryo vitrifying freeze Tip carrier after the assay was approved；
S2: the pig blastocyst of freezing Tip carrier freezes
B1, it is cultivated 7 days by PN phase pig zona-free oocytes micromanipulation degreasing, and in PZM3；
B2, the degreasing pig blastocyst of culture is cleaned 2 times in HM drop, balances 5~10min at room temperature, in ES liquid and carries out
Processing；
B3, by previous step treated pig blastocyst, with 5 pieces in one group of immigration VS drop, immediately using moving embryo needle for VS drop
In 5 pieces of embryos uniformly put the plastic strip of the wide 0.15cm of embryo vitrifying freeze Tip carrier, long 1cm made from S1 step
On, and will be mounted in the freezing Tip carrier investment liquid nitrogen of pig blastocyst, the time of whole process is no more than 60s, that is, completes entire
Freeze freezing for the pig blastocyst of Tip carrier；
S3: the defrosting of the pig blastocyst of freezing Tip carrier
C1, the freezing Tip carrier for being mounted with pig blastocyst is removed from liquid nitrogen, is rapidly inserted into the TS thawing solution of heating, protected
After holding static 15~25s, pig blastocyst and freezing Tip carrier thaw separation；
C2, by the pig blastocyst of previous step thaw separation, moved into DS dilution in 1min and balance 5min, then move into WS1 liquid
Middle balance 3min is finally moved into WS2 liquid and is balanced 3min；
C3, the pig blastocyst by previous step through WS2 liquid Balance Treatment move into culture dish culture 8h in PZM3, then observe pig blastocyst
Blastocoele futtock situation, that is, complete freezing Tip carrier pig blastocyst defrosting whole process.
2. a kind of pig blastocyst based on straw freezing Tip carrier according to claim 1 freeze and defreezing method, it is special
Sign is, in A1 step, the controllable constant-temperature thermal station, concrete model is CBL Model 100.
3. a kind of pig blastocyst based on straw freezing Tip carrier according to claim 1 freeze and defreezing method, it is special
Sign is, in A1 step, the straw, and concrete model IMV005565.
4. a kind of pig blastocyst based on straw freezing Tip carrier according to claim 1 freeze and defreezing method, it is special
Sign is, in B2 step, the room temperature, specific dimension is 20~28 DEG C.
5. a kind of pig blastocyst based on straw freezing Tip carrier according to claim 1 freeze and defreezing method, it is special
Sign is, in B3 step, the time of the whole process is 45~60s.
6. a kind of pig blastocyst based on straw freezing Tip carrier according to claim 1 freeze and defreezing method, it is special
Sign is, in C1 step, the temperature of the TS thawing solution is 37~41 DEG C.
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| CN201910585668.XA CN110214776A (en) | 2019-07-01 | 2019-07-01 | It is a kind of based on straw freezing Tip carrier pig blastocyst freeze and defreezing method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098285A3 (en) * | 2003-05-08 | 2005-01-06 | Cellartis Ab | Cryopreservation of human blastocyst-derived stem cells by use of a closed straw vitrification method |
| CN102669088A (en) * | 2012-05-30 | 2012-09-19 | 河南省农业科学院 | Mammal embryo and oocyte vitrification freezing carrier |
| CN202603479U (en) * | 2012-06-14 | 2012-12-19 | 河南省农业科学院 | Vitrified freezing device for embryos and oocytes of mammal |
-
2019
- 2019-07-01 CN CN201910585668.XA patent/CN110214776A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098285A3 (en) * | 2003-05-08 | 2005-01-06 | Cellartis Ab | Cryopreservation of human blastocyst-derived stem cells by use of a closed straw vitrification method |
| CN102669088A (en) * | 2012-05-30 | 2012-09-19 | 河南省农业科学院 | Mammal embryo and oocyte vitrification freezing carrier |
| CN202603479U (en) * | 2012-06-14 | 2012-12-19 | 河南省农业科学院 | Vitrified freezing device for embryos and oocytes of mammal |
Non-Patent Citations (2)
| Title |
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| 张慧琴: "《生殖医学理论与实践》", 31 May 2014, 上海世界图书出版公司 * |
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