CN109775148A - A kind of method and transport device using dry ice transport embryo - Google Patents
A kind of method and transport device using dry ice transport embryo Download PDFInfo
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- CN109775148A CN109775148A CN201910096927.2A CN201910096927A CN109775148A CN 109775148 A CN109775148 A CN 109775148A CN 201910096927 A CN201910096927 A CN 201910096927A CN 109775148 A CN109775148 A CN 109775148A
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Abstract
The invention discloses a kind of methods and transport device using dry ice transport embryo.Wherein, method includes: S1, the embryo that will acquire is placed in equilibrium liquid, then takes out to be placed in and saves in liquid；S2, the preservation liquid for the sucrose culture solution being sequentially placed in into sterile straw, being spaced air column, being mixed with embryo, interval air column and sucrose culture solution；S3, the sterile straw of sealed processing is packaged in heat transfer protection container; heat transfer protection container is placed in sterile incubator again; finally dry ice is filled between sterile incubator and heat transfer protection container and makes dry ice cladding heat transfer protection container, until being transported to destination.The present invention passes through the improvement to liquid and equilibrium liquid is saved, embryo is set to keep vitrifying stable state under dry-ice temperature, to which the interim process saved and transport of entire embryo can be completed in the dry ice using relative low price, the series of problems generated due to temporarily being saved and being transported using liquid nitrogen or other refrigerants is efficiently solved.
Description
Technical field
It is especially a kind of to be filled using the method and transport of dry ice transport embryo the present invention relates to technical field of bioengineering
It sets.
Background technique
Assisted reproductive technology is a kind of to integrate the technologies such as embryo separating, chimeric, transplanting, in vitro fertilization, transgenosis
Integrated technology, the not still important technology revolution in animal reproduction field, and the important technology hand that the treatment mankind are infertile
Section can sufficiently excavate the heredity and fertility, the rear algebra for increasing purebred animal and excellent individual of animal using this technology
Amount, the improvement period for shortening domestic animal and preservation have embryo and the gene of excellent hereditary capacity, in biology and medical domain
Be extensively studied and apply.
Wherein, embryo transfer technology (also known as zygote transplation technology) is indispensable one in entire assisted reproductive technology
A link, so-called embryo transfer technology is primarily referred to as will be by embryo that various technologies obtain (such as natural embryo, in vitro fertilization
Embryo, clone embryos etc.), underwent operative operation is transplanted in recipient female animal body of the same race, after the later period develops and can obtain
The new technology in generation.During practical application, what the acquisition work of embryo usually carried out in laboratory, and embryo transfer
Operation is then usually to carry out in farm (or in other Special experimental rooms).Therefore, after Embryo Production to before transplanting
In this section of period, then there will necessarily be a process that embryo is temporarily saved and conveyed, and this process is often to embryo
Tire plays vital influence in the intracorporal development of receptor and pregnancy rate.
Currently, be mainly Liquid nitrogen storage transport by the way of when embryo is temporarily saved and transported in the industry,
That is: it is placed in liquid nitrogen transfer cask by the embryo that will acquire and completes storage and transport；However, but there is following lack in such mode
Fall into: 1, carrying out Embryo storage using liquid nitrogen is substantially that embryo is made to be in glassy state, and this state is generally in liquid nitrogen temperature
(that is: -196 degrees Celsius) can keep stable, but when storage temperature is higher than -130 degrees Celsius, the glassy state of embryo just can not
It keeps stablizing or shows unstable situation, the ice crystal for being easy to cause inside embryo intracellular forms (that is: tie again again
It is brilliant), to injure embryonic cell.2, liquid nitrogen transfer cask itself is expensive, and transportational process embryo must be placed at liquid nitrogen
In, also need to return vacant liquid nitrogen transfer cask after embryo transports to destination, not only operate it is extremely cumbersome, and transport and
The expense of preservation is extremely high.
Summary of the invention
In view of the deficiency of the prior art, the first purpose of this invention is to provide a kind of utilization dry ice transport
The method of embryo；Second object of the present invention is to provide a kind of transport device suitable for the method.
To achieve the goals above, the present invention adopts the following technical scheme:
A method of using dry ice transport embryo, it the following steps are included:
S1, embryo's pretreatment: the embryo that will acquire, which is placed in equilibrium liquid, handles at least 2min, and taking-up is placed in preservation liquid
In, wherein based on molality, equilibrium liquid is basic liquid by 11.0mol/kg ethylene glycol, 0.7mol/ with HTF culture medium
Kg sucrose and 0.0062mol/kg ficoll mix；Based on molality, saving liquid with HTF culture medium is basic liquid
It is mixed by 24.6mol/kg ethylene glycol, 3.1mol/kg sucrose and 0.0062mol/kg ficoll；
S2, embryo enter pipe: sucrose culture solution, the interval air being sequentially placed in into sterile straw respectively by certain length
Column, the preservation liquid for being mixed with embryo, interval air column and sucrose culture solution, then make two ports of sterile straw at sealing
Reason；
S3, vanning transport: the sterile straw Jing Guo encapsulation process is packaged in heat transfer protection container first, then again will
Heat transfer protection container is placed in sterile incubator, and finally dry ice is filled between sterile incubator and heat transfer protection container, and
Dry ice is kept to coat heat transfer protection container always until being transported to destination；
S4, embryo's recovery: taking out sterile straw and make room-temperature water bath processing to sterile straw, can recovery embryo.
Preferably, in a period of after completing step 2 and before executing step 3, sterile straw is placed in and makees to face in liquid nitrogen
Shi Baocun processing.
Preferably, in a period of after completing step S3 and before executing step S4, sterile straw is taken out and by sterile wheat
Pipe, which is placed in liquid nitrogen, makees interim preservation processing.
Preferably, in step s3, sterile incubator is transported to destination in 48h；Then take out sterile straw simultaneously
Interim preservation processing or embryo's recovery processing are made to sterile straw.
Preferably, in step s 2, to volume be 0.25ML straw make ultraviolet radiation disinfection processing it is sterile to obtain
The length of straw, the preservation liquid for being mixed with embryo of merging is 2cm.
Preferably, in step s3, ultraviolet radiation disinfection processing is made respectively to incubator and heat transfer protection container.
A kind of transport device using dry ice transport embryo, it includes thermal-insulating body, is placed in the intracorporal heat transfer guarantor of incubator
It protects container, the dry ice fragment layer being placed between the inner wall of thermal-insulating body and the outer wall for the protection container that conducts heat and is packaged in heat transfer and protect
It protects in container and inside is filled with the sterile straw of embryo, encapsulation process is made in two ports of the sterile straw.
Preferably, the thermal-insulating body is made of polyethylene foam material, the thermal-insulating body and heat transfer protection container it
Between be formed with vacuum heat-preserving space, the dry ice fragment layer is located in vacuum heat-preserving space.
Preferably, the heat transfer protection container is to be made of aluminum alloy materials and on side wall with the cylindrical shape of hollow out hole location
Structural body.
Preferably, it is formed in sterile straw and is filled through sucrose culture solution and is formed by nutrient solution section, is filled through air institute
The interval air section of formation and be filled through save liquid formed with the preservation liquid section for mixing embryo, the preservation liquid section
Between two adjacent interval air sections, and the nutrient solution section is adjacent with interval air section and is located at sterile straw at least
One end.
As the above scheme is adopted, the present invention enables embryo in dry ice by the improvement to liquid and equilibrium liquid is saved
At a temperature of keep vitrifying stable state, so that the interim preservation of entire embryo can be completed using the dry ice of relative low price
And the process of transport, it efficiently solves all because using liquid nitrogen or other refrigerants to generate due to temporarily saved and transported
Such as transportation cost is high, cumbersome, embryo vitrifying state labile series of problems.
Detailed description of the invention
Fig. 1 is the distribution schematic diagram of filled content in the sterile straw of the embodiment of the present invention；
Fig. 2 is the cross section structure schematic diagram of the transport device of the embodiment of the present invention.
Specific embodiment
The embodiment of the present invention is described in detail below in conjunction with attached drawing, but the present invention can be defined by the claims
Implement with the multitude of different ways of covering.
A kind of method using dry ice transport embryo provided in this embodiment, comprising the following steps:
S1, embryo's pretreatment: under room temperature state, the embryo that will acquire (including but not limited to such as natural embryo, in vitro by
Smart embryo, clone embryos etc.) be placed in equilibrium liquid handle at least 2min after (preferably 2-10min), then by embryo from balance
It takes out to be placed in liquid and save in liquid；Wherein, based on molality, equilibrium liquid with HTF culture medium be basic liquid by
11.0mol/kg ethylene glycol, 0.7mol/kg sucrose and 0.0062mol/kg ficoll mix；Based on molality,
It is basic liquid by 24.6mol/kg ethylene glycol, 3.1mol/kg sucrose and 0.0062mol/kg ficoll that liquid, which is saved, with HTF culture medium
It mixes；
S2, embryo enter pipe: as shown in Figure 1, the sucrose culture being sequentially placed in into sterile straw respectively by certain length
(its basic ingredient mainly has peptone, sodium chloride, dipotassium hydrogen phosphate, bromothymol blue aqueous solution, agar, distilled water to liquid a1
Deng), interval air column b1 (that is: the air with certain length distance is formed by after being filled in sterile straw by clean air
Section), the preservation liquid c that is mixed with embryo, interval air column b2 and sucrose culture solution a2, then two ports of sterile straw are made
Encapsulation process (in the specific implementation, can be by one end that above-mentioned material sucks is clogged with sealing-plug, opposite end is then using alcohol etc.
Make hot-press sealed processing after heating), so that the air and sucrose culture solution that not only can use positioned at embryo two sides are realized to embryo
The isolation of tire, and guarantee that embryo has sufficient nutrition and gas supply during transportation；
S3, vanning transport: the sterile straw Jing Guo encapsulation process is packaged in heat transfer protection container first, and (it can be according to reality
Border situation uses the material with Thermal conductivity to be made) in, heat transfer protection container is then placed in sterile heat preservation again
In case, finally dry ice is filled between sterile incubator and heat transfer protection container, and dry ice is kept to coat heat transfer protection always
Container is until be transported to destination；
S4, embryo's recovery: taking out sterile straw and make room-temperature water bath processing to sterile straw, can recovery embryo；It is inciting somebody to action
Embryo can be ready work after being released in culture dish for subsequent embryo transfer.
It is based in current Embryo storage and transportational process to carry out freezen protective processing as a result, by liquid nitrogen (because only
Have liquid nitrogen temperature just and can guarantee the stable state of embryo vitrifying) and it is easy to cause that embryo's transportation cost is high, cumbersome shows
Shape, and if replacement save medium (replacing liquid nitrogen using other media) if be easy to cause embryo generation recrystallization phenomenon and
The problem of damaging cells；From the angle for solving frozen embryo stability, the embodiment of the present invention passes through to existing preservation liquid
Improvement project (that is: optimization equilibrium liquid and save the constituent and composition proportion of liquid) is proposed with equilibrium liquid, enables embryo
Vitrifying stable state is kept under dry-ice temperature (generally 79 degrees Celsius), to utilize the dry ice of relative low price
The interim process saved and transport for completing entire embryo, is efficiently solved and is generated due to using liquid nitrogen or other refrigerants
Series of problems；Wherein, embryo is pre-processed using equilibrium liquid, using hypotoxicity of the ethylene glycol in low concentration
Characteristic, within the longer processing time keep ethylene glycol fully penetrated to inside embryo to be formed the internal protection to embryo, and
Using the effect for promoting embryo's dehydration of sucrose and ficoll played in equilibrium liquid, contain to reduce the moisture of inside embryo
Amount；The characteristics of using high concentration cryoprotection ingredient possessed by liquid is saved, not only ethylene glycol can be made in a short time rapid
It infiltrates into inside embryo and the moisture of inside embryo is promoted quickly to ooze out, while can also be to embryo using the preservation liquid of high concentration
So that the inside and outside of embryo is not will form ice crystal after being freezed, forms a stable glass freezing state, filled to reach
Code insurance protects the purpose of embryo, to create conditions to carry out transport to embryo using dry ice.
To meet need of the embryo in actual process (such as from acquisition, pretreatment, transport, recovery, transplanting stage)
It asks, in a period of after completing step 2 and before executing step 3, pipe can will be entered through embryo treated that sterile straw is placed in liquid
Make interim preservation processing (that is: being equivalent in the indoor Liquid nitrogen storage container of experiment for being temporarily stored into departure place) in nitrogen.Accordingly
Ground then can protect sterile straw from sterile incubator and heat transfer in a period of after completing step S3 and before executing step S4
Taken out in shield container and sterile straw is placed in liquid nitrogen make interim preservation processing (that is: be equivalent to recovering to embryo or
Before transplanting, in the indoor Liquid nitrogen storage container of the experiment that sterile straw first can be temporarily stored into destination).
Survival rate and potentiality of development are lost to guarantee that frozen embryo is no after recovery of thawing to the maximum extent, in step S3
In, preferably guarantee to transport sterile incubator to destination in 48h；It then takes out sterile straw and sterile straw is made interim
Save processing or embryo's recovery processing.
It preferably, in step s 2, can be by making at ultraviolet radiation disinfection to the straw that volume is 0.25ML
Reason is to obtain sterile straw, at this point, the length of the preservation liquid for being mixed with embryo of merging is preferably 2cm.It as a result, can be sucrose
It provides an ample space in culture solution and interval air column implantation straw and guarantees enough dosages, to guarantee that embryo is transporting
Sufficient nutrient and gas can be obtained in defeated process.Certainly, in the specific implementation, the preservation liquid of embryo is mixed in straw
Interior length can make inverse ratio selection processing according to the specific volume according to straw in above-mentioned parameter ratio.
In addition, to ensure that the aseptic performance of sterile incubator and heat transfer protection container in step s3 can be in advance to guarantor
Incubator and heat transfer protection container make ultraviolet radiation disinfection processing respectively, are held with forming sterile incubator and sterile heat transfer protection
Device, then in sterile straw to be placed in heat transfer protection container.
Its advantages, selection pair are sufficiently verified and fully demonstrated for the method progress exploitativeness to the present embodiment
The embryo of the relatively high mouse of temperature sensitivity (selects 2 cell of mouse (that is: 2 cell stage embryo of mouse) to be used as experimental subjects
(be mainly based upon mice embryonic than the embryo of wild animal it is interim save and during transport to the sensibility of temperature more
High feature), pre-process and utilize doing under fresh state and using the preservation liquid and equilibrium liquid of the present embodiment
It is comparison object that ice, which carries out mice embryonic under the interim freezing state saved after transporting,；The survival rate of mice embryonic and transplanting
Live birth rate is detailed in table one.
From the point of view of the result of implementation shown by the table one, compared with fresh mice embryonic, using the equilibrium liquid of the present embodiment
It is reduced with the mice embryonic after being transported after freezing liquid processing and using dry ice there is no generation form and potentiality of development
Problem, and after embryo transfer, litter size does not also reduce significantly.Based on this, can determine: the method for the present embodiment can
The identical effect when obtaining using liquid nitrogen progress Embryo storage and transport；And relative to liquid nitrogen, performance possessed by dry ice can
The cost that embryo temporarily saves and transports is effectively reduced, and embodiment is simple, convenient, fast.
Based on above-mentioned transportation resources (specifically: based on the characteristic for saving liquid and equilibrium liquid), such as Fig. 2 simultaneously combines Fig. 1 institute
Show, the embodiment of the invention also provides it is a kind of using dry ice transport embryo transport device, it include thermal-insulating body (its mainly by
Ontology 1 and case lid 2 are constituted), be placed in the intracorporal heat transfer protection container 3 of incubator, be placed in the inner wall of thermal-insulating body and heat transfer is protected
It dry ice fragment layer 4 between the outer wall of container 3 and is packaged in heat transfer protection container 3 and the internal sterile wheat for being filled with embryo
Make encapsulation process in two ports of pipe 5, sterile straw 5.As a result, using thermal-insulating body as delivery vehicle, protected using heat transfer
Container 3 is as the isolated part between the encapsulation tool and dry ice and sterile straw 5 of sterile straw 5 to avoid because of sterile straw
5 cold temperature is uneven and influences embryo；In the specific implementation, embryo can be pre-processed and is entered according to the above method
After pipe processing, sterile straw 5 is placed in and is pre-placed in the intracorporal heat transfer protection container 3 of incubator, and utilizes dry ice fragment
It is filled in form comprehensive cladding to heat transfer protection container 3 between thermal-insulating body and heat transfer protection container 3, to utilize heat transfer
The excellent heating conduction of protection container 3 by cooling capacity caused by dry ice fragment equably radiate to around sterile straw 5 with shape
At the steady layer of freezing；Then whole device is transported in 48h destination, temporarily freezes, recovers, moves to carries out subsequent embryo
The work such as plant.Due to can effectively solve to generate due to using liquid nitrogen or other refrigerants as refrigerant using dry ice
Series of problems.
It preferably, is the cost of reduction whole device to the maximum extent and the stability of guarantee embryo, this reality
The thermal-insulating body for applying example is preferably the cabinet knot made of the polyethylene foam material of relative low price and excellent thermal insulation performance
Structure, and vacuum heat-preserving space is formed between thermal-insulating body and heat transfer protection container 3, dry ice fragment layer 4 is located at vacuum guarantor
In warm space.In the specific implementation, after being packaged in sterile straw 5 in heat transfer protection container 3, then the protection container 3 that will conduct heat
It is packaged in thermal-insulating body and after filling dry ice fragment, the space between thermal-insulating body and heat transfer protection container 3 is vacuumized
Processing is to form vacuum heat-preserving space, thus can not only guarantee that the inside of entire transport device is in sterile state, and
Be conducive to the stability placed in thermal-insulating body of heat transfer protection container 3 formed around sterile straw 5 with finally it is stable
Low-temperature protection layer.
Preferably, to make full use of cooling capacity caused by dry ice, guarantee that sterile straw 5 can uniformly be cooled, originally
The heat transfer protection container 3 of embodiment is preferably made of the aluminum alloy materials with excellent heat conductivity performance and has hollow out on side wall
The cylinder-like structure body of hole location.
Based on preservation liquid above-mentioned and as shown in connection with fig. 1, it is formed in the sterile straw 5 of the present embodiment and is filled through sucrose training
Nutrient solution be formed by nutrient solution section a1, be filled through air be formed by interval air section (b1, b2) and be filled through save liquid institute
Formed with the preservation liquid section c for mixing embryo, wherein save liquid section c be located at two adjacent interval air sections (b1, b2) it
Between, and nutrient solution section a1 is adjacent with interval air section (b1 or b2) and is located at least one end of sterile 5 pipe of wheat；To by nothing
The optimum choice of the internal component of bacterium straw 5 can create a relatively complete nutrition and gas supply system for embryo, in turn
It creates conditions for the interim preservation and transport of embryo.
In addition, it is necessary to be pointed out that: the HTF basal liquid that the present embodiment is addressed refers to human tubal fluid (HTF) culture medium
(English be Human Tubal Fluid (HTF) Medium) can be found in table two and be made distribution and set.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
Equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content is applied directly or indirectly in other correlations
Technical field, be included within the scope of the present invention.
Claims (10)
1. it is a kind of using dry ice transport embryo method, it is characterised in that: it the following steps are included:
S1, embryo's pretreatment: the embryo that will acquire, which is placed in equilibrium liquid, handles at least 2min, and taking-up, which is placed in, to be saved in liquid,
In, based on molality, equilibrium liquid is basic liquid by 11.0mol/kg ethylene glycol, 0.7mol/kg sucrose with HTF culture medium
It is mixed with 0.0062mol/kg ficoll；Based on molality, save liquid with HTF culture medium be basic liquid by
24.6mol/kg ethylene glycol, 3.1mol/kg sucrose and 0.0062mol/kg ficoll mix；
S2, embryo enter pipe: the sucrose culture solution that is sequentially placed in into sterile straw respectively by certain length, interval air column,
It is mixed with the preservation liquid, interval air column and sucrose culture solution of embryo, encapsulation process is then made into two ports of sterile straw；
Sterile straw Jing Guo encapsulation process: being packaged in heat transfer protection container by S3, vanning transport first, then again will heat transfer
Protection container is placed in sterile incubator, and finally dry ice is filled between sterile incubator and heat transfer protection container, and keeps
Dry ice coats heat transfer protection container until being transported to destination always；
S4, embryo's recovery: taking out sterile straw and make room-temperature water bath processing to sterile straw, can recovery embryo.
2. a kind of method using dry ice transport embryo as described in claim 1, it is characterised in that: after completing step 2 simultaneously
In a period of executing before step 3, sterile straw is placed in liquid nitrogen and makees interim preservation processing.
3. a kind of method using dry ice transport embryo as described in claim 1, it is characterised in that: after completing step S3 simultaneously
In a period of executing before step S4, takes out sterile straw and sterile straw is placed in liquid nitrogen and make interim preservation processing.
4. a kind of method using dry ice transport embryo as described in claim 1, it is characterised in that: in step s3, in 48h
It is interior to transport sterile incubator to destination；It then takes out sterile straw and the interim preservation processing of sterile straw work or embryo is answered
Soviet Union's processing.
5. a kind of method using dry ice transport embryo as described in claim 1, it is characterised in that: in step s 2, to appearance
The straw that product is 0.25ML is made ultraviolet radiation disinfection and is handled to obtain sterile straw, the preservation liquid for being mixed with embryo of merging
Length is 2cm.
6. a kind of method using dry ice transport embryo as described in claim 1, it is characterised in that: in step s3, to guarantor
Incubator and heat transfer protection container make ultraviolet radiation disinfection processing respectively.
7. a kind of transport device using dry ice transport embryo, it is characterised in that: it includes thermal-insulating body, is placed in thermal-insulating body
Heat transfer protection container, the dry ice fragment layer that is placed between the inner wall of thermal-insulating body and the outer wall of heat transfer protection container and encapsulation
It is filled with the sterile straw of embryo in heat transfer protection container and inside, encapsulation process is made in two ports of the sterile straw.
8. a kind of transport device using dry ice transport embryo as claimed in claim 7, it is characterised in that: the thermal-insulating body
It is made of polyethylene foam material, vacuum heat-preserving space is formed between the thermal-insulating body and heat transfer protection container, it is described dry
Ice fragment layer is located in vacuum heat-preserving space.
9. a kind of transport device using dry ice transport embryo as claimed in claim 7, it is characterised in that: the heat transfer protection
Container is to be made of aluminum alloy materials and on side wall with the cylinder-like structure body of hollow out hole location.
10. a kind of transport device using dry ice transport embryo as claimed in claim 7, it is characterised in that: the sterile wheat
It is formed in pipe and is filled through sucrose culture solution and is formed by nutrient solution section, is filled through air and is formed by interval air section and warp
Filling saves liquid and is formed with the preservation liquid section for mixing embryo, and the preservation liquid section is located at two adjacent interval air sections
Between, and the nutrient solution section is adjacent with interval air section and is located at least one end of sterile straw.
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| CN201910096927.2A CN109775148B (en) | 2019-01-31 | 2019-01-31 | Method for transporting embryos by using dry ice and transporting device |
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| CN201910096927.2A CN109775148B (en) | 2019-01-31 | 2019-01-31 | Method for transporting embryos by using dry ice and transporting device |
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Cited By (1)
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Effective date of registration: 20220315 Address after: 518000 workshop 803, building C, jinweiyuan industrial plant, No. 41, Qingsong Road, Zhukeng community, Longtian street, Pingshan District, Shenzhen, Guangdong Patentee after: INLEMS LIFE TECHNOLOGY (SHENZHEN) Co.,Ltd. Address before: 518000 c802, jinweiyuan Industrial Park, JuLongshan District, Pingshan street, Pingshan District, Shenzhen, Guangdong Patentee before: LIMENG LOW TEMPERATURE MEDICAL SCIENCE (SHENZHEN) Co.,Ltd. |