CN109775148B - Method for transporting embryos by using dry ice and transporting device - Google Patents
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Abstract
The invention discloses a method for transporting embryos by using dry ice and a transporting device. The method comprises the following steps: s1, placing the obtained embryo in a balancing solution, then taking out and placing in a preservation solution; s2, sequentially putting a sucrose culture solution, an air spacer column, a preservation solution mixed with embryos, the air spacer column and the sucrose culture solution into a sterile straw; s3, packaging the sterilized straw subjected to sealing treatment in a heat transfer protective container, then placing the heat transfer protective container in a sterile incubator, and finally filling dry ice between the sterile incubator and the heat transfer protective container to enable the dry ice to coat the heat transfer protective container until the dry ice is transported to a destination. The invention can keep the embryos in a vitrification stable state at the temperature of dry ice by improving the preservation solution and the balancing solution, thereby completing the temporary preservation and transportation process of the whole embryos by using the dry ice with relatively low price, and effectively solving a series of problems caused by adopting liquid nitrogen or other freezing media for temporary preservation and transportation.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for transporting embryos by using dry ice and a transporting device.
Background
The assisted reproduction technology is a comprehensive technology integrating embryo segmentation, chimerism, transplantation, in vitro fertilization, transgenosis and other technologies, is not only an important technical revolution in the field of animal reproduction, but also an important technical means for treating human infertility, can fully excavate the inheritance and reproductive capacity of animals, increase the number of offspring of pure livestock and excellent individuals, shorten the improvement period of livestock and store embryos and genes with excellent inheritance characteristics by utilizing the technology, and is widely researched and applied in the fields of biology and medicine.
The embryo transplantation technology (also called fertilized egg transplantation technology) is an indispensable link in the whole assisted reproduction technology, and mainly refers to a new technology for transplanting embryos obtained by various technologies (such as natural embryos, in vitro fertilized embryos, cloned embryos and the like) into the bodies of homologous recipient females through surgical operation, developing at a later stage and obtaining offspring. In practice, embryo harvesting is usually performed in a laboratory, and embryo transfer is usually performed in a farm (or other dedicated laboratory). Therefore, during the period from the embryo production to the embryo transfer, there is a temporary embryo storage and transportation process, which is often a crucial influence on the embryo development and pregnancy rate in the recipient animal.
At present, the main mode adopted when the embryo is temporarily stored and transported in the industry is liquid nitrogen storage and transportation, namely: storing and transporting the obtained embryos by putting the embryos in a liquid nitrogen transport tank; however, this method has the following disadvantages: 1. the preservation of embryos by using liquid nitrogen is essentially to make the embryos in a vitrified state, which generally remains stable at the temperature of liquid nitrogen (i.e.: 196 ℃ below zero), but when the preservation temperature is higher than 130 ℃ below zero, the vitrified state of the embryos cannot be kept stable or shows an unstable condition, which easily causes the ice crystals in the cells inside the embryos to be formed again (i.e.: recrystallized), thereby damaging the embryo cells. 2. The liquid nitrogen transport tank is expensive, embryos must be placed in liquid nitrogen during the transportation process, the empty liquid nitrogen transport tank needs to be returned after the embryos are transported to a destination, the operation is very complicated, and the transportation and storage costs are very high.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, it is a first object of the present invention to provide a method for transporting embryos using dry ice; a second object of the invention is to provide a transport device suitable for this method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of transporting embryos using dry ice comprising the steps of:
s1, embryo pretreatment: treating the obtained embryo in a balancing solution for at least 2min, taking out and placing in a preservation solution, wherein the balancing solution is formed by mixing 11.0mol/kg of ethylene glycol, 0.7mol/kg of sucrose and 0.0062mol/kg of polysucrose by taking an HTF culture medium as a basic solution according to mass molar concentration; according to the mass molar concentration, the preservation solution is formed by mixing 24.6mol/kg of ethylene glycol, 3.1mol/kg of sucrose and 0.0062mol/kg of polysucrose by taking an HTF culture medium as a basic solution;
s2, embryo tube insertion: sequentially placing sucrose culture solution, spaced air columns, preservation solution mixed with embryos, spaced air columns and sucrose culture solution into a sterile straw according to a certain length, and then sealing two ports of the sterile straw;
s3, boxing and transportation: firstly, packaging a sterilized straw subjected to sealing treatment in a heat transfer protection container, then placing the heat transfer protection container in a sterile incubator, finally filling dry ice between the sterile incubator and the heat transfer protection container, and keeping the dry ice to cover the heat transfer protection container all the time until the heat transfer protection container is transported to a destination;
s4, recovering embryos: taking out the sterile straw and carrying out room temperature water bath treatment on the sterile straw, thus recovering the embryo.
Preferably, the sterile straw is placed in liquid nitrogen for temporary storage during the period after step 2 is completed and before step 3 is performed.
Preferably, the sterile straw is taken out and placed in liquid nitrogen for temporary preservation processing during a period after step S3 is completed and before step S4 is performed.
Preferably, in step S3, the sterile incubator is transported to the destination within 48 h; then taking out the sterile straws and carrying out temporary preservation treatment or embryo resuscitation treatment on the sterile straws.
Preferably, in step S2, the straw with a volume of 0.25ML is sterilized by ultraviolet irradiation to obtain a sterile straw, and the length of the preservation solution mixed with the embryo is 2 cm.
Preferably, in step S3, the heat-insulating box and the heat transfer protective container are sterilized by ultraviolet irradiation.
A transportation device for transporting embryos by using dry ice comprises a heat preservation box body, a heat transfer protection container arranged in the heat preservation box body, a dry ice fragment layer arranged between the inner wall of the heat preservation box body and the outer wall of the heat transfer protection container, and a sterile straw which is packaged in the heat transfer protection container and is filled with embryos, wherein two ports of the sterile straw are sealed; the sterile straw is internally provided with a nutrient solution section formed by filling a sucrose culture solution, an interval air section formed by filling air and a preservation solution section formed by filling a preservation solution and used for mixing embryos, wherein the preservation solution section is positioned between two adjacent interval air sections, and the nutrient solution section is adjacent to the interval air section and positioned at least at one end of the sterile straw.
Preferably, the heat preservation box body is made of polyethylene foam materials, a vacuum heat preservation space is formed between the heat preservation box body and the heat transfer protection container, and the dry ice fragment layer is located in the vacuum heat preservation space.
Preferably, the heat transfer protection container is a cylindrical structure body which is made of an aluminum alloy material and has hollow holes on the side wall.
By adopting the scheme, the embryo can keep a vitrification stable state at the temperature of dry ice by improving the preservation solution and the balance solution, so that the whole process of temporary preservation and transportation of the embryo can be completed by using the dry ice with relatively low price, and the series problems of high transportation cost, complex operation, unstable embryo vitrification state and the like caused by temporary preservation and transportation by adopting liquid nitrogen or other freezing media are effectively solved.
Drawings
FIG. 1 is a schematic view showing the distribution of the contents filled in a sterile barley tube according to an embodiment of the present invention;
fig. 2 is a schematic cross-sectional structure diagram of a transportation device according to an embodiment of the present invention.
Detailed Description
The embodiments of the invention will be described in detail below with reference to the drawings, but the invention can be implemented in many different ways as defined and covered by the claims.
The method for transporting embryos by using dry ice provided by the embodiment comprises the following steps:
s1, embryo pretreatment: treating the obtained embryo (including but not limited to natural embryo, in vitro fertilized embryo, cloned embryo, etc.) in balancing solution at room temperature for at least 2min (preferably 2-10min), and taking out the embryo from the balancing solution and placing in preservation solution; wherein, the balancing solution is formed by mixing 11.0mol/kg of ethylene glycol, 0.7mol/kg of sucrose and 0.0062mol/kg of polysucrose by taking an HTF culture medium as a basic solution according to the mass molar concentration; according to the mass molar concentration, the preservation solution is formed by mixing 24.6mol/kg of ethylene glycol, 3.1mol/kg of sucrose and 0.0062mol/kg of polysucrose by taking an HTF culture medium as a basic solution;
s2, embryo tube insertion: as shown in FIG. 1, a sucrose culture solution a1 (the basic components of which are mainly peptone, sodium chloride, dipotassium hydrogen phosphate, bromothymol blue aqueous solution, agar, distilled water, etc.), an interval air column b1 (i.e., an air section having a certain length distance formed by filling clean air into a sterile straw), a preservation solution c in which embryos are mixed, an interval air column b2 and a sucrose culture solution a2 are sequentially placed into a sterile straw at a certain length, then sealing two ports of the sterile straw (in specific implementation, one end of the sterile straw sucked by the material can be plugged by a sealing plug, and the other end of the sterile straw can be heated by alcohol and the like and then is subjected to heat pressure sealing treatment), so that the isolation of the embryo can be realized by utilizing the air and the sucrose culture solution positioned at two sides of the embryo, and the embryo is ensured to have sufficient nutrition and gas supply in the transportation process;
s3, boxing and transportation: firstly, packaging a sterilized straw subjected to sealing treatment in a heat transfer protection container (which can be made of a material with good heat conductivity according to actual conditions), then placing the heat transfer protection container in a sterile incubator, finally filling dry ice between the sterile incubator and the heat transfer protection container, and keeping the dry ice to cover the heat transfer protection container all the time until the dry ice is transported to a destination;
s4, recovering embryos: taking out the sterile straw and carrying out room-temperature water bath treatment on the sterile straw to recover the embryo; after the embryos are released in the culture dish, the preparation can be made for the subsequent embryo transplantation.
Therefore, the method is based on the current situations that the embryo transportation cost is high and the operation is complicated because the cryopreservation treatment can be carried out only by liquid nitrogen (only the temperature of the liquid nitrogen can ensure the vitrification stable state of the embryo) in the current embryo preservation and transportation process, and the problem that the cell is damaged because the recrystallization phenomenon of the embryo is easily caused if the preservation medium is replaced (namely, other media are adopted to replace the liquid nitrogen); from the viewpoint of solving the stability of frozen embryos, the embodiment of the invention provides an improved scheme (namely, the composition and the component proportion of the balancing liquid and the preserving liquid are optimized) for the existing preserving liquid and the balancing liquid, so that the embryos can keep a vitrification stable state at the temperature of dry ice (generally 79 ℃), the temporary preservation and transportation process of the whole embryos can be completed by using the dry ice with relatively low price, and the series problems caused by adopting liquid nitrogen or other freezing media are effectively solved; the method comprises the following steps of (1) preprocessing embryos by using a balancing solution, wherein the embryos can be fully permeated into the embryos to form internal protection of the embryos within a long processing time by using the characteristic of low toxicity of ethylene glycol at low concentration, and the moisture content in the embryos can be reduced by using the effect of promoting embryo dehydration of sucrose and ficoll in the balancing solution; by utilizing the characteristic of high-concentration freezing protection components of the preservation solution, the ethylene glycol can quickly permeate into the embryo and promote the water in the embryo to quickly seep out in a short time, and the high-concentration preservation solution can ensure that ice crystals are not formed inside and outside the embryo after the embryo is frozen to form a stable vitrification freezing state, thereby achieving the purpose of fully protecting the embryo and creating conditions for transporting the embryo by using dry ice.
In order to meet the requirement of the embryo in the actual processing process (such as the stages of obtaining, preprocessing, transporting, recovering, transplanting and the like), the sterile straw processed by the embryo tube-entering process can be placed in liquid nitrogen for temporary storage treatment (namely, the sterile straw is temporarily stored in a liquid nitrogen storage container in a laboratory at the starting place) in the period after the step 2 is completed and before the step 3 is executed. Accordingly, after step S3 is completed and before step S4 is executed, the sterile straws may be removed from the sterile incubator and the heat transfer protective container and placed in liquid nitrogen for temporary storage (i.e., the sterile straws may be temporarily stored in a liquid nitrogen storage container in the destination laboratory before the embryos are revived or transplanted).
In order to ensure that the frozen embryo does not lose survival rate and development potential after thawing and reviving to the maximum extent, in step S3, the sterile incubator is preferably transported to the destination within 48 h; then taking out the sterile straws and carrying out temporary preservation treatment or embryo resuscitation treatment on the sterile straws.
Preferably, in step S2, the sterile straw is obtained by sterilizing straw with a volume of 0.25ML by ultraviolet irradiation, and the length of the preservation solution containing the mixed embryo is preferably 2 cm. Therefore, abundant space can be provided for the sucrose culture solution and the spaced air columns to be implanted into the straws, and enough dosage is ensured, so that the embryos can obtain sufficient nutrients and gases in the transportation process. Of course, in specific implementation, the length of the preservation solution mixed with the embryo in the straw can be selected according to the above parameter ratio and the inverse ratio according to the specific volume of the straw.
In addition, in order to ensure the sterility of the sterile incubator and the heat transfer protective container, in step S3, the incubator and the heat transfer protective container may be sterilized by ultraviolet irradiation in advance to form the sterile incubator and the sterile heat transfer protective container, respectively, and then the sterile straw may be placed in the heat transfer protective container.
In order to fully verify the feasibility of the implementation of the method and fully embody the beneficial effects of the method, a mouse embryo with relatively high temperature sensitivity is selected (a mouse 2 cell (namely, a mouse 2 cell stage embryo) is selected as an experimental object (mainly based on the characteristic that the mouse embryo has higher temperature sensitivity in the temporary storage and transportation process than the embryo of a wild animal), the mouse embryo is taken as a comparison object in a fresh state, a frozen state after the preservation liquid and the balancing liquid of the embodiment are used for pretreatment and the temporary storage and transportation are carried out by using dry ice, and the survival rate of the mouse embryo and the survival rate of the transplantation are shown in the table I.
From the results shown in Table I, the mouse embryos treated with the equilibration and freezing solutions and transported on dry ice according to the present example did not suffer from a reduction in morphological and developmental potential, and the number born after embryo transfer was not significantly reduced, as compared to fresh mouse embryos. Based on this, it can be determined that: the method of the embodiment can achieve the same effect when the liquid nitrogen is used for embryo storage and transportation; compared with liquid nitrogen, the dry ice has the advantages that the cost for temporarily storing and transporting embryos can be effectively reduced, and the implementation mode is simple, convenient and fast.
Based on the transportation method (specifically, based on the characteristics of the preservation solution and the balancing solution), as shown in fig. 2 and combined with fig. 1, the embodiment of the invention also provides a transportation device for transporting embryos by using dry ice, which comprises a thermal insulation box body (mainly comprising a body 1 and a box cover 2), a heat transfer protection container 3 arranged in the thermal insulation box body, a dry ice block layer 4 arranged between the inner wall of the thermal insulation box body and the outer wall of the heat transfer protection container 3, and a sterile straw 5 which is sealed in the heat transfer protection container 3 and filled with embryos, wherein two ports of the sterile straw 5 are subjected to sealing treatment. Therefore, the incubator body is used as a carrier, the heat transfer protection container 3 is used as a packaging tool of the sterile straw 5 and an isolation part between the dry ice and the sterile straw 5 so as to avoid influence on embryos due to uneven cooled temperature of the sterile straw 5; in specific implementation, after the embryo is pretreated and put into the tube according to the method, the sterile straw 5 is placed in the heat transfer protection container 3 which is placed in the heat preservation box in advance, and dry ice fragments are filled between the heat preservation box and the heat transfer protection container 3 to form comprehensive coating on the heat transfer protection container 3, so that the cold energy generated by the dry ice fragments is uniformly radiated to the periphery of the sterile straw 5 by utilizing the excellent heat conduction performance of the heat transfer protection container 3 to form a freezing stable layer; and then the whole device is transported to the destination within 48h, so that the subsequent work of temporarily freezing, recovering, transplanting and the like of the embryo is carried out. The utilization of dry ice as the freezing medium can effectively solve a series of problems caused by the adoption of liquid nitrogen or other freezing media.
Preferably, in order to reduce the cost of the whole device to the maximum and ensure the stability of the embryo, the thermal insulation box of the embodiment is preferably a box structure made of polyethylene foam material with relatively low price and excellent thermal insulation performance, a vacuum thermal insulation space is formed between the thermal insulation box and the heat transfer protection container 3, and the dry ice block layer 4 is positioned in the vacuum thermal insulation space. In specific implementation, after the sterile straw 5 is packaged in the heat transfer protection container 3, the heat transfer protection container 3 is packaged in the heat preservation box and filled with the dry ice fragments, and the space between the heat preservation box and the heat transfer protection container 3 is vacuumized to form a vacuum heat preservation space, so that the inside of the whole transportation device can be ensured to be in a sterile state, and the stability of the heat transfer protection container 3 placed in the heat preservation box is facilitated to finally form a stable low-temperature protection layer around the sterile straw 5.
Preferably, in order to fully utilize the cold energy generated by the dry ice and ensure that the sterile straw 5 can be uniformly cooled, the heat transfer protection container 3 of the embodiment is preferably a cylindrical structure body which is made of an aluminum alloy material with excellent heat conductivity and has hollow holes on the side wall.
Based on the preservation solution and shown in fig. 1, a nutrient solution section a1 formed by filling the sucrose culture solution, a spaced air section (b1, b2) formed by filling air, and a preservation solution section c formed by filling the preservation solution for mixing embryos are formed in the sterile straw 5 of the embodiment, wherein the preservation solution section c is positioned between two adjacent spaced air sections (b1, b2), and the nutrient solution section a1 is adjacent to the spaced air section (b1 or b2) and positioned at least at one end of the sterile straw 5; therefore, by optimally selecting the internal components of the sterile straw 5, a relatively complete nutrition and gas supply system can be created for the embryo, and conditions are created for temporary preservation and transportation of the embryo.
In addition, it should be noted that: the HTF base Fluid mentioned in this example refers to Human oviduct Fluid (HTF) Medium (Human Tubal Fluid (HTF) Medium), which can be prepared as shown in table two.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. A method of transporting embryos using dry ice, comprising: it comprises the following steps:
s1, embryo pretreatment: treating the obtained embryo in a balancing solution for at least 2min, taking out and placing in a preservation solution, wherein the balancing solution is formed by mixing 11.0mol/kg of ethylene glycol, 0.7mol/kg of sucrose and 0.0062mol/kg of polysucrose by taking an HTF culture medium as a basic solution according to mass molar concentration; according to the mass molar concentration, the preservation solution is formed by mixing 24.6mol/kg of ethylene glycol, 3.1mol/kg of sucrose and 0.0062mol/kg of polysucrose by taking an HTF culture medium as a basic solution;
s2, embryo tube insertion: sequentially placing sucrose culture solution, spaced air columns, preservation solution mixed with embryos, spaced air columns and sucrose culture solution into a sterile straw according to a certain length, and then sealing two ports of the sterile straw;
s3, boxing and transportation: firstly, packaging a sterilized straw subjected to sealing treatment in a heat transfer protection container, then placing the heat transfer protection container in a sterile incubator, finally filling dry ice between the sterile incubator and the heat transfer protection container, and keeping the dry ice to cover the heat transfer protection container all the time until the heat transfer protection container is transported to a destination;
s4, recovering embryos: taking out the sterile straw and carrying out room temperature water bath treatment on the sterile straw, thus recovering the embryo.
2. A method of transporting embryos using dry ice as claimed in claim 1, wherein: after completion of step 2 and before execution of step 3, the sterile straws are placed in liquid nitrogen for temporary storage.
3. A method of transporting embryos using dry ice as claimed in claim 1, wherein: after step S3 is completed and before step S4 is executed, the sterile straw is taken out and put in liquid nitrogen for temporary storage processing.
4. A method of transporting embryos using dry ice as claimed in claim 1, wherein: in step S3, the sterile incubator is transported to the destination within 48 h; then taking out the sterile straws and carrying out temporary preservation treatment or embryo resuscitation treatment on the sterile straws.
5. A method of transporting embryos using dry ice as claimed in claim 1, wherein: in step S2, the straw having a volume of 0.25ML was sterilized by ultraviolet irradiation to obtain a sterile straw, and the length of the storage solution containing the embryos mixed therein was 2 cm.
6. A method of transporting embryos using dry ice as claimed in claim 1, wherein: in step S3, the heat-insulating box and the heat transfer protective container are sterilized by ultraviolet irradiation.
7. A transportation device for transporting embryos by using dry ice is characterized in that: the device comprises a heat preservation box body, a heat transfer protection container arranged in the heat preservation box body, a dry ice fragment layer arranged between the inner wall of the heat preservation box body and the outer wall of the heat transfer protection container, and a sterile straw which is packaged in the heat transfer protection container and is filled with embryos, wherein two ports of the sterile straw are subjected to sealing treatment; the aseptic straw is internally provided with a nutrient solution section formed by filling a sucrose culture solution, an interval air section formed by filling air and a preservation solution section formed by filling preservation solution for mixing embryos, wherein the preservation solution section is positioned between two adjacent interval air sections, and the nutrient solution section is adjacent to the interval air section and positioned at least one end of the aseptic straw.
8. A transport device for embryos using dry ice as claimed in claim 7, wherein: the heat preservation box body is made of polyethylene foam materials, a vacuum heat preservation space is formed between the heat preservation box body and the heat transfer protection container, and the dry ice fragment layer is located in the vacuum heat preservation space.
9. A transport device for embryos using dry ice as claimed in claim 7, wherein: the heat transfer protection container is a cylindrical structure body which is made of an aluminum alloy material and is provided with hollow hole positions on the side wall.
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| CN202081111U (en) * | 2011-06-22 | 2011-12-21 | 南京医科大学附属南京第一医院 | Large-scale hepatocyte culturing and freezing device |
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| CN102172331A (en) * | 2011-01-27 | 2011-09-07 | 安徽农业大学 | Method and device for transporting embryos |
| CN202081111U (en) * | 2011-06-22 | 2011-12-21 | 南京医科大学附属南京第一医院 | Large-scale hepatocyte culturing and freezing device |
| CN104488853A (en) * | 2015-01-12 | 2015-04-08 | 中国农业大学 | Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method |
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