CN104855371A - System for feeding embryo into freezing carrier, freezing carrier and freezing preservation method - Google Patents
System for feeding embryo into freezing carrier, freezing carrier and freezing preservation method Download PDFInfo
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- CN104855371A CN104855371A CN201410062556.3A CN201410062556A CN104855371A CN 104855371 A CN104855371 A CN 104855371A CN 201410062556 A CN201410062556 A CN 201410062556A CN 104855371 A CN104855371 A CN 104855371A
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Abstract
The invention discloses a system for feeding embryos into a freezing carrier, the freezing carrier and a freezing preservation method. The ovum or embryo vitrification freezing carrier comprises a carrier body and a dragging rod, wherein the dragging rod is integrally connected with the carrier body and is used for dragging the carrier body; the carrier body is a grid bearing body; a longitudinal cross section of the grid bearing body has a radian. As the longitudinal cross section of the grid bearing body of the ovum or embryo vitrification freezing carrier has the radian, after a liquid bridge is formed at the bottoms of the grid bearing body and a petri dish, a liquid with high potential energy and large pressure intensity on the grid bearing body can be continuously pressed into the bottom of the petri dish by virtue of a siphon principle, and the volume of a freezing protecting agent wrapping the periphery of ova/embryos can be reduced when the embryos are fed into the vitrification freezing carrier.
Description
Technical field
The present invention relates to a kind of biological technical field, more precisely relate to and a kind ofly realize embryo and load the system of freezing carrier, freezing carrier and freezing and storing method.
Background technology
Refrigeration Technique has become indispensable link in Issues of Human Assisted Reproductive Technologies, along with the development of Refrigeration Technique, slow freezing gradually replace by glass freezing.Vitrification makes the ovum/embryo be wrapped in high concentration cryoprotector form glassy state rapidly by the cooling rate (> 2000 DEG C/min) of superelevation; avoid intracellular ice crystal to produce damaging cells, thus increase substantially ovum/embryo survival, anabiosis rate and potentiality of development.
General, high speed temperature-fall period can be realized by two aspects: one is the temperature (dropping into rapidly liquid nitrogen by open carrier to realize) by temperature-reducing medium; Two is that around controlling parcel ovum/embryo, cryoprotector volume is the smaller the better.
But for second point; in real work; ovum/embryo is transferred on vitrifying carrier together with cryoprotector by the operation of personnel by experiment, and around parcel ovum/embryo, cryoprotector volume is without objective standard completely by the qualification decision of operating personnel.
Summary of the invention
The technical problem that the present invention solves is to provide and a kind ofly realizes embryo and load the system of freezing carrier, freezing carrier and freezing and storing method, to reduce cryoprotector volume around parcel ovum/embryo when embryo loads vitrified frozen vector.
In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:
Realize embryo and load the system of freezing carrier, it comprises: culture dish and ovum or embryo vitrification freezing carrier, and wherein this ovum or embryo vitrification freezing carrier comprise:
Carrier body, the drawbar of the described carrier body that is integrally connected with described carrier body, draws, wherein said carrier body is grid supporting body, and described grid supporting body has radian on longitudinal section;
Bottom the most bottom surface laminating culture dish of the radian of the grid supporting body of described ovum or embryo vitrification freezing carrier, by the drawbar of ovum or embryo vitrification freezing carrier, the grid supporting body of this ovum or embryo vitrification freezing carrier with drawbar bottom culture dish mobile make to carry in grid supporting body ovum or embryo around freezing liquid transfer to bottom culture dish along drawing direction.
Further, described radian is semi arch.
Further, the grid aperture of grid supporting body is 80-100um.
In addition, the present invention also discloses a kind of ovum or embryo vitrification freezing carrier, and it comprises:
Carrier body, the drawbar of the described carrier body that is integrally connected with described carrier body, draws, wherein said carrier body is grid supporting body, and described grid supporting body has radian on longitudinal section.
Further, described radian is semi arch.
Further, the grid aperture of grid supporting body is 80-100um.
In addition, the present invention also discloses a kind of ovum or embryo vitrifying freeze store method, comprising: ovum or embryo's balancing run step, glass freezing liquid cleaning step, embryo's loading carrier step, and wherein, described embryo loads carrier step and includes:
Step 11: the glass freezing liquid that the grid supporting body of ovum or embryo vitrification freezing carrier is placed in 15% ethylene glycol+15% dimethyl sulfoxide (DMSO) is infiltrated;
Step 12: the grid supporting body of the ovum or embryo vitrification freezing carrier of invading profit is completely placed in culture dish;
Step 13: the embryo after the cleaning in glass freezing operating procedure is sucked glass pipette, transfers on the grid supporting body of ovum or embryo vitrification freezing carrier, and remaining liqs all in glass pipette are are all blown and beaten grid supporting body surface;
Step 14: the drawbar dragging ovum or embryo vitrification freezing carrier, moves the grid supporting body of this ovum or embryo vitrification freezing carrier, makes the freezing liquid on grid supporting body around embryo transfer to bottom culture dish along drawing direction bottom culture dish;
Step 15: after having dragged, is placed in liquid nitrogen by the grid supporting body of ovum or embryo vitrification freezing carrier, completes refrigeration operation.
Further, being placed in culture dish by the grid supporting body of ovum or embryo vitrification freezing carrier in step 12 is with bottom the most bottom surface laminating culture dish of the radian of grid supporting body.
Further, ovum or embryo's balancing run step are specially:
Culture fluid embryo being placed in 7.5% ethylene glycol+7.5% dimethyl sulfoxide (DMSO) is placed about 10 minutes; Period embryo first experiences the dehydration of shrinkage; Experience the process that the hydrone returning to life size from collapsed condition is frozen protectant displacement again.
Further, glass freezing liquid cleaning step is specially: glass freezing liquid embryo being placed in 15% ethylene glycol+15% dimethyl sulfoxide (DMSO), cleaning 5-10 all over until in ovum or embryo's balancing run step the low concentration equilibrium liquid be wrapped in around embryo clean up.
Compared with prior art, the present invention has following beneficial effect:
In the present invention, ovum or embryo vitrification freezing carrier comprise: carrier body, the drawbar of the described carrier body that is integrally connected with described carrier body, draws, and wherein said carrier body is grid supporting body, and described grid supporting body has radian on longitudinal section; Bottom the most bottom surface laminating culture dish of the radian of the grid supporting body of described ovum or embryo vitrification freezing carrier, by the drawbar of ovum or embryo vitrification freezing carrier, the grid supporting body of this ovum or embryo vitrification freezing carrier with drawbar bottom culture dish mobile make to carry in grid supporting body ovum or embryo around freezing liquid transfer to bottom culture dish along drawing direction.Because the described grid supporting body of ovum or embryo vitrification freezing carrier has radian on longitudinal section, therefore, in dragging process, bottom the culture dish that the stiction of fluid makes the freezing liquid thick liquid of bottommost on grid supporting body transfer to clean no liquid and along with dragging, produce pull of vacuum; And form liquid bridge bottom grid supporting body and culture dish after, can continue bottom press-in culture dish by the liquid on potential energy is high, grid supporting body that pressure is large by siphon principle; And have concentrated trend based on surface tension of liquid by making all liquid; produce all liq have equally distributed trend and make frozen liq on grid supporting body around embryo all toward in the segment set of bottommost; and then transfer to bottom culture dish, cryoprotector volume around parcel ovum/embryo can be reduced when embryo loads vitrified frozen vector.
Accompanying drawing explanation
Fig. 1 is a kind of specific embodiment structural representation that the present invention realizes ovum or embryo vitrification freezing carrier in the system of embryo's loading freezing carrier;
Fig. 2 is that in the present invention, carrier body 1 is the schematic diagram of grid supporting body;
Fig. 3 is that the present invention carries out the principle schematic of freezing liquid transfer according to siphon principle;
Fig. 4 is the original state schematic diagram that the present invention realizes the system of embryo's loading freezing carrier;
Fig. 5 is that the present invention realizes the system that embryo loads freezing carrier and realizes reducing the principle schematic of cryoprotector around parcel ovum/embryo.
Embodiment
The present invention is based on vitrification method, concrete, the present invention realizes the system that embryo loads freezing carrier, and it comprises: culture dish and ovum or embryo vitrification freezing carrier, unlike the prior art, in the present invention, this ovum or embryo vitrification freezing carrier comprise:
Carrier body 1, be connected with described carrier body 1 one, draw the drawbar 2 of described carrier body 1, wherein said carrier body 1 is grid supporting body (with reference to figure 2), and for different embryo's diameters, the grid pore size of grid supporting body can change, such as, in the present embodiment, the grid aperture of grid supporting body is 80-100um, along with the growth of embryo, grid aperture can also arrange larger, repeats no more here.
In addition, the supporting body of grid described in the present embodiment has radian on longitudinal section, arranging described radian mainly utilizes the reason of siphon principle and surface tension of liquid when carrying out embryo's transfer, can effectively reduce the freezing liquid be wrapped in around embryo, during specific implementation, it can be various forms of radian, such as, in the present embodiment, radian is semi arch, certainly, also can adjust radian according to actual state, repeat no more here.
In addition, in the present embodiment, the most bottom surface of radian of the grid supporting body of described ovum or embryo vitrification freezing carrier is preferably fitted bottom culture dish, by the drawbar 2 of ovum or embryo vitrification freezing carrier, the grid supporting body of this ovum or embryo vitrification freezing carrier with drawbar 2 bottom culture dish mobile make to carry in grid supporting body ovum or embryo around freezing liquid transfer to bottom culture dish along drawing direction.
The following detailed description of principle of the present invention.
Siphon principle: the principle being exactly linker, is added in the pressure in closed container on liquid, all equal everywhere.And in siphon pipe, fill water, do not have gas, carry out water end (W.E.) water level high, delivery port palm or other objects close.Now intraductal pressure is equal everywhere.After all place, open delivery port, although the atmospheric pressure on both sides is equal, the water level carrying out water end (W.E.) is high, and pressure is large, promotes water and continuously outflows delivery port.In pipe, peak liquid moves toward low level mouth of pipe place under gravity, and form vacuum, under vacuum, the liquid of the high-order mouth of pipe is sucked into peak, forms siphonage.
With reference to figure 1-Fig. 2, in the present invention, grid supporting body 1 has radian on longitudinal section, and bottom the radian of the grid supporting body of described ovum or embryo vitrification freezing carrier most bottom surface laminating culture dish, in addition, with reference to figure 3-Fig. 5, freezing liquid 12 is enclosed with around embryo 11 time initial, also freezing liquid 15 is added with bottom culture dish 16, pulled by drawbar, in dragging process, the stiction of fluid make the thick liquid of the freezing liquid of bottommost on grid supporting body 14 be subject to stiction resistance and bottom the culture dish transferring to clean no liquid 16, described dragging process is similar in siphon principle needs an initial applying pull of vacuum to siphon pipe, further bottom grid supporting body 14 and culture dish 16 form liquid bridge by mesh 13 after, namely be a liquid bridge bottom the mesh of grid supporting body and culture dish, be similar to the vacuum passage (specifically please refer to Fig. 3) in the siphon pipe in siphon principle, and can be high by potential energy by siphon principle, freezing liquid on the grid supporting body that pressure is large continues bottom press-in culture dish, therefore, freezing liquid on the mesh 13 of grid supporting body 14 is constantly down shifted by liquid bridge, and have concentrated trend based on surface tension of liquid by making all liquid, namely can produce all liq have equally distributed trend and make frozen liq on grid supporting body 14 around embryo all toward in the segment set of bottommost, and then to transfer to bottom culture dish 16, the freezing liquid transfer of namely wrapping up around embryo is the most at last the unnecessary freezing liquid 17 bottom culture dish.
Based on above-mentioned principle, a kind of ovum that the present invention realizes or embryo vitrifying freeze store method are described below, specifically comprise: 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 all similarly to the prior art, wherein, embryo is specifically mainly placed in the culture fluid placement about 10 minutes of 7.5% ethylene glycol+7.5% dimethyl sulfoxide (DMSO) by ovum or embryo's balancing run step; Period embryo first experiences the dehydration of shrinkage; Experience the process that the hydrone returning to life size from collapsed condition is frozen protectant displacement again.And embryo is specifically mainly placed in the glass freezing liquid of 15% ethylene glycol+15% dimethyl sulfoxide (DMSO) by glass freezing liquid cleaning step, cleaning 5-10 all over until in ovum or embryo's balancing run step the low concentration equilibrium liquid be wrapped in around embryo clean up, repeat no more here.
And carrier step is loaded for described embryo, mainly comprise following concrete steps in the present invention:
Step 11: the glass freezing liquid that the grid supporting body of ovum or embryo vitrification freezing carrier is placed in 15% ethylene glycol+15% dimethyl sulfoxide (DMSO) is infiltrated;
Step 12: the grid supporting body of the ovum or embryo vitrification freezing carrier of invading profit is completely placed in culture dish, during specific implementation, being preferably placed in culture dish by the grid supporting body of ovum or embryo vitrification freezing carrier is with bottom the most bottom surface laminating culture dish of the radian of grid supporting body;
Step 13: the embryo after the cleaning in glass freezing operating procedure is sucked glass pipette, transfers on the grid supporting body of ovum or embryo vitrification freezing carrier, and remaining liqs all in glass pipette are are all blown and beaten grid supporting body surface;
Step 14: the drawbar dragging ovum or embryo vitrification freezing carrier, moves the grid supporting body of this ovum or embryo vitrification freezing carrier, makes the freezing liquid on grid supporting body around embryo transfer to bottom culture dish along drawing direction bottom culture dish;
Step 15: after having dragged, is placed in liquid nitrogen by the grid supporting body of ovum or embryo vitrification freezing carrier, completes refrigeration operation.
Although preferred embodiment of the present invention has disclosed as above, the present invention has been not limited to above-described embodiment, any those skilled in the art, not departing from the scope disclosed by the present invention, when doing a little change and adjustment.
Claims (10)
1. realize embryo and load the system of freezing carrier, it is characterized in that, comprising: culture dish and ovum or embryo vitrification freezing carrier, wherein this ovum or embryo vitrification freezing carrier comprise:
Carrier body, the drawbar of the described carrier body that is integrally connected with described carrier body, draws, wherein said carrier body is grid supporting body, and described grid supporting body has radian on longitudinal section;
Bottom the most bottom surface laminating culture dish of the radian of the grid supporting body of described ovum or embryo vitrification freezing carrier, by the drawbar of ovum or embryo vitrification freezing carrier, the grid supporting body of this ovum or embryo vitrification freezing carrier with drawbar bottom culture dish mobile make to carry in grid supporting body ovum or embryo around freezing liquid transfer to bottom culture dish along drawing direction.
2. system according to claim 1, is characterized in that, described radian is semi arch.
3. system according to claim 1, is characterized in that, the grid aperture of grid supporting body is 80-100um.
4. ovum or an embryo vitrification freezing carrier, is characterized in that, comprising:
Carrier body, the drawbar of the described carrier body that is integrally connected with described carrier body, draws, wherein said carrier body is grid supporting body, and described grid supporting body has radian on longitudinal section.
5. ovum according to claim 4 or embryo vitrification freezing carrier, is characterized in that, described radian is semi arch.
6. ovum according to claim 4 or embryo vitrification freezing carrier, is characterized in that, the grid aperture of grid supporting body is 80-100um.
7. ovum or an embryo vitrifying freeze store method, comprising: ovum or embryo's balancing run step, glass freezing liquid cleaning step, embryo's loading carrier step, and it is characterized in that, described embryo loads carrier step and includes:
Step 11: the glass freezing liquid that the grid supporting body of ovum or embryo vitrification freezing carrier is placed in 15% ethylene glycol+15% dimethyl sulfoxide (DMSO) is infiltrated;
Step 12: the grid supporting body of the ovum or embryo vitrification freezing carrier of invading profit is completely placed in culture dish;
Step 13: the embryo after the cleaning in glass freezing operating procedure is sucked glass pipette, transfers on the grid supporting body of ovum or embryo vitrification freezing carrier, and remaining liqs all in glass pipette are are all blown and beaten grid supporting body surface;
Step 14: the drawbar dragging ovum or embryo vitrification freezing carrier, moves the grid supporting body of this ovum or embryo vitrification freezing carrier, makes the freezing liquid on grid supporting body around embryo transfer to bottom culture dish along drawing direction bottom culture dish;
Step 15: after having dragged, is placed in liquid nitrogen by the grid supporting body of ovum or embryo vitrification freezing carrier, completes refrigeration operation.
8. method according to claim 7, is characterized in that, being placed in culture dish by the grid supporting body of ovum or embryo vitrification freezing carrier in step 12 is with bottom the most bottom surface laminating culture dish of the radian of grid supporting body.
9. method according to claim 7, is characterized in that, ovum or embryo's balancing run step are specially:
Culture fluid embryo being placed in 7.5% ethylene glycol+7.5% dimethyl sulfoxide (DMSO) is placed about 10 minutes; Period embryo first experiences the dehydration of shrinkage; Experience the process that the hydrone returning to life size from collapsed condition is frozen protectant displacement again.
10. method according to claim 7, it is characterized in that, glass freezing liquid cleaning step is specially: glass freezing liquid embryo being placed in 15% ethylene glycol+15% dimethyl sulfoxide (DMSO), cleaning 5-10 all over until in ovum or embryo's balancing run step the low concentration equilibrium liquid be wrapped in around embryo clean up.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 true CN104855371A (en) | 2015-08-26 |
| CN104855371B CN104855371B (en) | 2019-05-21 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| CN104855371B (en) | 2019-05-21 |
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