CN104855371B - Realize that embryo is packed into system, freezing carrier and the freezing and storing method of freezing carrier - Google Patents
Realize that embryo is packed into system, freezing carrier and the freezing and storing method of freezing carrier Download PDFInfo
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- CN104855371B CN104855371B CN201410062556.3A CN201410062556A CN104855371B CN 104855371 B CN104855371 B CN 104855371B CN 201410062556 A CN201410062556 A CN 201410062556A CN 104855371 B CN104855371 B CN 104855371B
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Abstract
The present invention discloses a kind of system, freezing carrier and freezing and storing method realized embryo and be packed into freezing carrier, wherein ovum or embryo vitrification freezing carrier include: carrier body, the drawbar of the carrier body is integrally connected, drawn with the carrier body, wherein the carrier body is grid supporting body, and the grid supporting body has radian on longitudinal section；Since the grid supporting body of ovum or embryo vitrification freezing carrier has radian on longitudinal section; after grid supporting body and culture dish bottom form liquid bridge; the liquid on the big grid supporting body of potential energy height, pressure can be continued to be pressed into culture dish bottom by siphon principle, so as to reduce cryoprotector volume around package ovum/embryo when embryo is packed into vitrified frozen vector.
Description
Technical field
The present invention relates to a kind of field of biotechnology, are more precisely related to a kind of realizing that embryo is packed into freezing carrier and is
System, freezing carrier and freezing and storing method.
Background technique
Refrigeration Technique has become indispensable link in Issues of Human Assisted Reproductive Technologies, continuous with Refrigeration Technique
Development, slow freezing is gradually replaced glass freezing.Cooling rate (the > that vitrification passes through superelevation
2000 DEG C/min) so that the ovum/embryo being wrapped in high concentration cryoprotector is quickly formed glassy state, avoid intracellular ice
Crystalline substance generates damaging cells, to increase substantially ovum/embryo survival, anabiosis rate and potentiality of development.
In general, high speed temperature-fall period may be implemented by two aspects: first is that by the temperature of temperature-reducing medium (by opening
It puts carrier and puts into liquid nitrogen realization rapidly)；Second is that cryoprotector volume is the smaller the better around control package ovum/embryo.
But for second point, in real work, by the operation of experimenter by ovum/embryo together with cryoprotector one
It rises and is transferred on vitrifying carrier, wrapping up cryoprotector volume around ovum/embryo is entirely the skilled journey by operator
Degree determine and without objective standard.
Summary of the invention
Realize that embryo is packed into the system of freezing carrier, freezing carrier and cold the technical problem to be solved by the present invention is to provide a kind of
Freezing method for storing, to reduce cryoprotector volume around package ovum/embryo when embryo is packed into vitrified frozen vector.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme:
A kind of system realized embryo and be packed into freezing carrier comprising: culture dish and ovum or embryo vitrifying freeze carry
Body, wherein the ovum or embryo vitrification freezing carrier include:
Carrier body is integrally connected with the carrier body, draws the drawbar of the carrier body, wherein the carrier sheet
Body is grid supporting body, and the grid supporting body has radian on longitudinal section；
The radian most bottom surface of the grid supporting body of the ovum or embryo vitrification freezing carrier is bonded culture dish bottom, borrows
The drawbar of ovum or embryo vitrification freezing carrier is helped, the grid supporting body of the ovum or embryo vitrification freezing carrier is with drawbar
In culture dish bottom, movement makes the freezing liquid around the ovum carried in grid supporting body or embryo be transferred to training along drawing direction
Support ware bottom.
Further, the radian is semi arch.
Further, the grid aperture of grid supporting body is 80-100um.
In addition, invention additionally discloses a kind of ovum or embryo vitrification freezing carriers comprising:
Carrier body is integrally connected with the carrier body, draws the drawbar of the carrier body, wherein the carrier sheet
Body is grid supporting body, and the grid supporting body has radian on longitudinal section.
Further, the radian is semi arch.
Further, the grid aperture of grid supporting body is 80-100um.
In addition, invention additionally discloses a kind of ovum or embryo vitrifying freeze store methods, comprising: ovum or embryo's balance
Operating procedure, glass freezing liquid cleaning step, embryo are packed into carrier step, wherein the embryo is packed into carrier step and includes
Have:
Step 11: the grid supporting body of ovum or embryo vitrification freezing carrier is placed in+15% dimethyl of 15% ethylene glycol
It is infiltrated in the glass freezing liquid of sulfoxide；
Step 12: the grid supporting body of the ovum for invading profit completely or embryo vitrification freezing carrier is placed on culture dish
It is interior；
Step 13: the embryo after the cleaning in glass freezing operating procedure being sucked into glass pipette, transfers to ovum
Or on the grid supporting body of embryo vitrification freezing carrier, and remaining liqs all in glass pipette are all blown and beaten to grid and are held
Carrier surface；
Step 14: the drawbar of dragging ovum or embryo vitrification freezing carrier carries the ovum or embryo vitrifying freeze
The grid supporting body of body is mobile in culture dish bottom, is transferred to the freezing liquid on grid supporting body around embryo along drawing direction
Culture dish bottom；
Step 15: after the completion of dragging, the grid supporting body of ovum or embryo vitrification freezing carrier is placed in liquid nitrogen, it is complete
At refrigeration operation.
Further, the grid supporting body of ovum or embryo vitrification freezing carrier is placed in culture dish in step 12
It is that culture dish bottom is bonded with the radian of grid supporting body most bottom surface.
Further, ovum or embryo's balancing run step specifically:
Embryo is placed in the culture solution of+7.5% dimethyl sulfoxide of 7.5% ethylene glycol and is placed 10 minutes or so；Period embryo
First undergo the dehydration of shrinkage；The hydrone for being restored to life size from collapsed condition is undergone to be frozen the process of protective agent displacement again.
Further, glass freezing liquid cleaning step specifically: embryo is placed in+15% dimethyl sulfoxide of 15% ethylene glycol
Glass freezing liquid in, cleaning 5-10 is all over until be wrapped in the low concentration around embryo in ovum or embryo's balancing run step
Equilibrium liquid cleans up.
Compared with prior art, the invention has the following beneficial effects:
Ovum or embryo vitrification freezing carrier include: carrier body in the present invention, be integrally connected with the carrier body,
The drawbar of the carrier body is drawn, wherein the carrier body is grid supporting body, the grid supporting body is on longitudinal section
With radian；The radian most bottom surface of the grid supporting body of the ovum or embryo vitrification freezing carrier is bonded culture dish bottom,
By ovum or the drawbar of embryo vitrification freezing carrier, the grid supporting body of the ovum or embryo vitrification freezing carrier is with dragging
Bar is transferred to the freezing liquid around the ovum carried in grid supporting body or embryo along drawing direction in the movement of culture dish bottom
Culture dish bottom.Since the grid supporting body of ovum or embryo vitrification freezing carrier has radian on longitudinal section, because
This, in dragging process, the stiction of fluid is transferred to the freezing liquid thick liquid of bottommost on grid supporting body completely
The culture dish bottom of no liquid simultaneously generates pull of vacuum with dragging；And liquid bridge is formed in grid supporting body and culture dish bottom
Liang Hou can continue the liquid on the big grid supporting body of potential energy height, pressure to be pressed into culture dish bottom by siphon principle；And base
All liquid will be made to have the tendency that concentration in surface tension of liquid, generating all liq has equally distributed trend and make
Frozen liq on grid supporting body around embryo is transferred to culture dish bottom all into the segment set of bottommost, can be with
Cryoprotector volume around package ovum/embryo is reduced when embryo is packed into vitrified frozen vector.
Detailed description of the invention
Fig. 1 is that the present invention realizes that embryo is fitted into one kind of ovum or embryo vitrification freezing carrier in the system of freezing carrier
Specific embodiment structural schematic diagram；
Fig. 2 is the schematic diagram that carrier body 1 is grid supporting body in the present invention；
Fig. 3 is the schematic illustration that the present invention carries out freezing liquid transfer according to siphon principle；
Fig. 4 is that the present invention realizes that embryo is packed into the original state schematic diagram of the system of freezing carrier；
Fig. 5 is that the system that present invention realization embryo is packed into freezing carrier realizes that reducing freezing around package ovum/embryo protects
Protect the schematic illustration of agent.
Specific embodiment
The present invention is based on vitrification methods, specifically, the present invention realizes the system that embryo is packed into freezing carrier, packet
It includes: culture dish and ovum or embryo vitrification freezing carrier, unlike the prior art, the ovum or embryo's glass in the present invention
Changing freezing carrier includes:
Carrier body 1 is integrally connected with the carrier body 1, draws the drawbar 2 of the carrier body 1, wherein the load
Body ontology 1 is grid supporting body (refer to Fig. 2), and for different embryo's diameters, the grid pore size of grid supporting body can be with
Variation, for example, the grid aperture of grid supporting body is 80-100um, and with the growth of embryo, grid aperture is also in the present embodiment
It can be set more greatly, which is not described herein again.
In addition, grid supporting body described in the present embodiment has radian on longitudinal section, it is mainly benefit that the radian, which is arranged,
With the reason of siphon principle and surface tension of liquid, when carrying out embryo transfer, the freezing being wrapped in around embryo can be effectively reduced
Liquid when specific implementation, can be various forms of radians, for example, radian is semi arch in the present embodiment, it is of course also possible to root
Radian is adjusted according to actual state, which is not described herein again.
In addition, in the present embodiment, the radian most bottom of the grid supporting body of the ovum or embryo vitrification freezing carrier
Face is preferably bonded culture dish bottom, cold by ovum or the drawbar 2 of embryo vitrification freezing carrier, the ovum or embryo vitrifying
The grid supporting body of jelly carrier is with drawbar 2 around the mobile ovum for making to carry in grid supporting body in culture dish bottom or embryo
Freezing liquid is transferred to culture dish bottom along drawing direction.
The following detailed description of the principle of the present invention.
Siphon principle: being exactly the principle of linker, and the pressure being added in closed container on liquid is all equal everywhere.And rainbow
Water is filled in suction pipe, not gas, Lai Shuiduan water level is high, and water outlet palm or other objects close.At this time at intraductal pressure
Locate equal.After all are placed, water outlet is opened, although the atmospheric pressure on both sides is equal, the water level for carrying out water end (W.E.) is high, pressure
Greatly, water is pushed to continuously outflow water outlet.Highest point liquid is formed true under the effect of gravity toward movement at low level nozzle in managing
Sky, under vacuum, the liquid of high-order nozzle are sucked into highest point, form siphonage.
With reference to Fig. 1-Fig. 2, grid supporting body 1 has a radian on longitudinal section in the present invention, and the ovum or embryo's glass
The radian most bottom surface of the grid supporting body of glass freezing carrier is bonded culture dish bottom, in addition, with reference to Fig. 3-Fig. 5, when initial
It waits and is enclosed with freezing liquid 12 around embryo 11,16 bottom of culture dish is also added with freezing liquid 15, is pulled by drawbar, dragged
Cheng Zhong, the stiction of fluid make the thick liquid of the freezing liquid of bottommost on grid supporting body 14 by the resistance of stiction
And it is transferred to the culture dish bottom 16 of clean no liquid, the dragging process, which is similar in siphon principle, needs initially siphon pipe
Apply a pull of vacuum, further after grid supporting body 14 and culture dish bottom 16 form liquid bridge by mesh 13,
That is the mesh of grid supporting body and culture dish bottom is a liquid bridge, similar to the vacuum in the siphon pipe in siphon principle
Channel (specifically please refers to Fig. 3), and can be continued the freezing liquid on the big grid supporting body of potential energy height, pressure by siphon principle
It is pressed into culture dish bottom, therefore, the freezing liquid on the mesh 13 of grid supporting body 14 is constantly shifted down by liquid bridge；And
All liquid will be made to have the tendency that concentration based on surface tension of liquid, i.e., can generate all liq have it is equally distributed become
Gesture and make the frozen liq on grid supporting body 14 around embryo all into the segment set of bottommost, and then be transferred to culture dish bottom
The freezing liquid wrapped up around embryo is finally shifted the extra freezing liquid 17 for culture dish bottom by portion 16.
Illustrate a kind of ovum or embryo vitrifying freeze store method that the present invention realizes, tool below based on above-mentioned principle
Body includes: ovum or embryo's balancing run step, glass freezing liquid cleaning step, embryo's loading carrier step, wherein above-mentioned
Ovum or embryo's balancing run step, glass freezing liquid cleaning step are similarly to the prior art, wherein ovum or embryo are flat
Embryo is specifically mainly placed in the culture solution of+7.5% dimethyl sulfoxide of 7.5% ethylene glycol by weighing apparatus operating procedure places 10 minutes left sides
It is right；Period embryo first undergoes the dehydration of shrinkage；The hydrone for being restored to life size from collapsed condition is undergone to be frozen protective agent again
Displaced process.And embryo is specifically mainly placed in+15% dimethyl sulfoxide of 15% ethylene glycol by glass freezing liquid cleaning step
Glass freezing liquid in, cleaning 5-10 is all over until be wrapped in the low concentration around embryo in ovum or embryo's balancing run step
Equilibrium liquid cleans up, and which is not described herein again.
And carrier step is packed into for the embryo, it is main in the present invention comprising the following specific steps
Step 11: the grid supporting body of ovum or embryo vitrification freezing carrier is placed in+15% dimethyl of 15% ethylene glycol
It is infiltrated in the glass freezing liquid of sulfoxide；
Step 12: the grid supporting body of the ovum for invading profit completely or embryo vitrification freezing carrier is placed on culture dish
Interior, when specific implementation, it is with net that preferably the grid supporting body of ovum or embryo vitrification freezing carrier, which is placed in culture dish,
The radian of lattice supporting body most bottom surface is bonded culture dish bottom；
Step 13: the embryo after the cleaning in glass freezing operating procedure being sucked into glass pipette, transfers to ovum
Or on the grid supporting body of embryo vitrification freezing carrier, and remaining liqs all in glass pipette are all blown and beaten to grid and are held
Carrier surface；
Step 14: the drawbar of dragging ovum or embryo vitrification freezing carrier carries the ovum or embryo vitrifying freeze
The grid supporting body of body is mobile in culture dish bottom, is transferred to the freezing liquid on grid supporting body around embryo along drawing direction
Culture dish bottom；
Step 15: after the completion of dragging, the grid supporting body of ovum or embryo vitrification freezing carrier is placed in liquid nitrogen, it is complete
At refrigeration operation.
Although presently preferred embodiments of the present invention is disclosed above, the present invention is not limited to above-described embodiment, any skill
Technical staff in art field, do not depart from disclosed herein in the range of, when can make a little change and adjustment.
Claims (5)
1. a kind of system realized embryo and be packed into freezing carrier characterized by comprising culture dish and ovum or embryo vitrifying
Freezing carrier, wherein the ovum or embryo vitrification freezing carrier include:
Carrier body is integrally connected with the carrier body, draws the drawbar of the carrier body, wherein the carrier body is
Grid supporting body, the grid supporting body have radian on longitudinal section；
The radian most bottom surface of the grid supporting body of the ovum or embryo vitrification freezing carrier is bonded culture dish bottom, by ovum
The grid supporting body of the drawbar of son or embryo vitrification freezing carrier, the ovum or embryo vitrification freezing carrier is being trained with drawbar
Supporting the movement of ware bottom makes the freezing liquid around the ovum carried in grid supporting body or embryo be transferred to culture dish along drawing direction
Bottom；Wherein, the radian is semi arch；The grid aperture of grid supporting body is 80-100um.
2. a kind of ovum or embryo vitrifying freeze store method, comprising: ovum or embryo's balancing run step, glass freezing
Liquid cleaning step, embryo are packed into carrier step, which is characterized in that the embryo is packed into carrier step and includes:
Step 11: it is sub- that the grid supporting body of ovum or embryo vitrification freezing carrier being placed in+15% dimethyl of 15% ethylene glycol
It is infiltrated in the glass freezing liquid of sulfone；
Step 12: the grid supporting body of the ovum for invading profit completely or embryo vitrification freezing carrier is placed in culture dish；
Step 13: the embryo after the cleaning in glass freezing operating procedure being sucked into glass pipette, transfers to ovum or embryo
On the grid supporting body of tire vitrified frozen vector, and all grid supporting body is arrived in piping and druming by remaining liqs all in glass pipette
Surface；
Step 14: the drawbar of dragging ovum or embryo vitrification freezing carrier, by the ovum or embryo vitrification freezing carrier
Grid supporting body is mobile in culture dish bottom, and the freezing liquid on grid supporting body around embryo is made to be transferred to culture along drawing direction
Ware bottom；
Step 15: after the completion of dragging, the grid supporting body of ovum or embryo vitrification freezing carrier being placed in liquid nitrogen, is completed cold
Freeze operation.
3. according to the method described in claim 2, it is characterized in that, by ovum or embryo vitrification freezing carrier in step 12
It is to be bonded culture dish bottom with the radian of grid supporting body most bottom surface that grid supporting body, which is placed in culture dish,.
4. according to the method described in claim 2, it is characterized in that, ovum or embryo's balancing run step specifically:
Embryo is placed in the culture solution of+7.5% dimethyl sulfoxide of 7.5% ethylene glycol and is placed 10 minutes or so；Period embryo is first
Undergo the dehydration of shrinkage；The hydrone for being restored to life size from collapsed condition is undergone to be frozen the process of protective agent displacement again.
5. according to the method described in claim 2, it is characterized in that, glass freezing liquid cleaning step specifically: set embryo
In the glass freezing liquid of+15% dimethyl sulfoxide of 15% ethylene glycol, cleaning 5-10 is all over until ovum or embryo's balancing run
The low concentration equilibrium liquid being wrapped in around embryo in step cleans up.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410062556.3A CN104855371B (en) | 2014-02-24 | 2014-02-24 | Realize that embryo is packed into system, freezing carrier and the freezing and storing method of freezing carrier |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410062556.3A CN104855371B (en) | 2014-02-24 | 2014-02-24 | Realize that embryo is packed into system, freezing carrier and the freezing and storing method of freezing carrier |
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| CN104855371A CN104855371A (en) | 2015-08-26 |
| CN104855371B true CN104855371B (en) | 2019-05-21 |
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| CN115777683A (en) * | 2021-09-10 | 2023-03-14 | 深圳拜尔洛克生物技术有限公司 | Device for cryopreservation or thawing recovery of biological tissue |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003065014A1 (en) * | 2002-01-30 | 2003-08-07 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Sample support for the cryoconservation of biological samples |
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN204273039U (en) * | 2014-02-24 | 2015-04-22 | 张孝东 | Realize system, freezing carrier that embryo loads freezing carrier |
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- 2014-02-24 CN CN201410062556.3A patent/CN104855371B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003065014A1 (en) * | 2002-01-30 | 2003-08-07 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Sample support for the cryoconservation of biological samples |
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN204273039U (en) * | 2014-02-24 | 2015-04-22 | 张孝东 | Realize system, freezing carrier that embryo loads freezing carrier |
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