JPH119139A - Transportation of mammal embryo - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable long-distance transportation of mammal embryo without lowering survival ability and growing ability of embryo by attaching a cell capable of assisting growth of embryo to the inner wall lower than culture medium level in a vessel, putting mammal embryo into the vessel in a state dispersed in the culture medium, hermetically closing the vessel and transporting the vessel. SOLUTION: In a method for putting mammal embryo into a vessel in which a culture medium is put and transporting the embryo, a cell for assisting growth of embryo is attached to at least part of inner wall under culture medium level in the vessel and mammal embryo is put in the vessel in a state dispersed in the culture medium and the vessel is hermetically sealed and transported. The cell for assisting growth of mammal embryo is preferably at least one kind of cell selected from cumulus cell, granulosa cell, oviduct epithelial cell, trophoblast, endometrial cell, uterine fibroblast and testicular fibroblast.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for transporting mammalian embryos. More specifically, the present invention relates to a method for transporting embryos capable of long-term transport of mammalian embryos without reducing their viability and developmental ability.

[0002]

2. Description of the Related Art Conventionally, embryos of livestock such as cattle and other mammals have been transported by gently freezing the embryos and then freezing them in liquid nitrogen to transport the embryos. In general, a method of enclosing in a transport container, a straw or the like in a state of being dispersed in a liquid such as an added phosphate buffer and transporting the same is generally adopted. However, mammalian embryos, particularly mammalian in vitro fertilized embryos, sex-determining embryos, in vitro cultured embryos, and embryos of animal species that are difficult to cryopreserve (eg, pig embryos) are easily affected by environmental changes. In the case of the above-mentioned conventional freeze transport method or the method of dispersing in a fetal serum-added phosphate buffer solution and enclosing in a container and transporting the embryo, the viability and development ability of the embryo are significantly reduced during the transport. .

[0003] Therefore, even when embryos produced with sufficient care to maintain high viability and developmental ability are transported, embryos of good quality are selected especially from a large number of embryos and transported. However, even if it does, the embryo dies during or after transportation, or its viability and developmental ability is significantly reduced even if it does not die, so that valuable embryos can be transplanted after transportation, used for various research purposes, and for other uses. At present, it cannot be fully utilized. Furthermore, in the case of the freeze transport method, it is said that it is desirable to thaw the transported frozen embryo and determine its viability before use in order to increase the survival rate of the embryo in each use. However, there is a drawback in that determination of embryo viability takes time and effort.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for safely and easily transporting a mammalian embryo while preventing a decrease in its viability and developmental ability. An object of the present invention is to provide a method for transporting mammalian embryos, which can directly and effectively utilize the transported embryos as they are for each purpose without any trouble.

[0005]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors to achieve the above object, the present inventors have assisted the development of embryos in enclosing and transporting mammalian embryos in a transport container. Using a container with cells attached to it, placing the embryo in such a state that the embryo is dispersed in the culture solution, and transporting the container tightly, even if it is transported over long distances, the damage to the embryo Thus, the present invention was completed by finding that the viability and developmental ability of embryos were kept good and that they could be directly and effectively used for each purpose and use even after transportation.

That is, the present invention relates to a method for transporting a mammalian embryo by placing the embryo in a container containing a culture solution, which assists the development of the embryo on at least a part of the inner wall below the surface of the culture solution in the container. A method for transporting a mammalian embryo, comprising attaching the cells, placing the mammalian embryo in a state dispersed in a culture solution, placing the container in the container, and transporting the container with the container sealed.

[0007] The present invention relates to a method of adhering to the inner wall of the container,
Cumulus cells, as cells that support the development of mammalian embryos,
A preferred embodiment includes the above-described method for transporting a mammalian embryo using at least one selected from granulosa cells, fallopian tube epithelial cells, trophoblasts, endometrial cells, uterine fibroblasts and testicular fibroblasts. .

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. As the "mammalian embryo" in the present invention, cattle,
Domestic animals such as horses, goats, pigs, and sheep, pets such as cats and dogs, various mammalian embryos such as gorillas, orangutans, chimpanzees, pandas, giraffes, tigers, and lions,
Examples include human embryos (fertilized eggs). Among them, the transportation method of the present invention is applicable to livestock such as cattle and pigs, embryos such as various rare animals such as gorillas, orangutans, and pandas whose survival is reduced and need protection, animal species which are difficult to cryopreserve. Suitable for transporting embryos (eg pigs).

[0009] The term "embryo" as used in the present invention includes any egg that has been fertilized and can be transported. Thing, 4
Any of the cell stage, 8-cell stage, morula, blastocyst (blastocyst), escaped blastocyst and the like may be used. Among them, the method of the present invention is suitable for transporting embryos such as morula, blastocyst and the like. In addition, the transport method of the present invention is applicable to any embryos such as mammalian in vitro fertilized embryos, in vivo fertilized embryos, their in vitro cultured embryos, sex-determined embryos, and embryos subjected to operations such as nuclear transfer. It is possible. Among them, the transport method of the present invention is highly sensitive to environmental changes and is easily damaged by transport, in vitro fertilized embryos, in vitro cultured embryos, sex-determined embryos, embryos that have been subjected to operations such as nuclear transfer, and pigs. It is suitable for transporting embryos such as embryos that are easily damaged at low temperatures (cold-sensitive embryos).

In the present invention, the mammalian embryo as described above is transported in a container having cells supporting the development of the embryo attached to the inner wall. As used herein, the term “cells that assist embryo development” refers to cells that have the function of assisting the development (development) of embryos when they are cultured. Examples of the cell that assists the development of the embryo that can be used in the present invention (hereinafter referred to as “embryonic development assisting cell”) include, for example, cumulus cells (cumulus cecum).
ll), granulosa cells, fallopian tube epithelial cells,
Examples include trophoblasts (trophoblastic vesicles; differentiated cells of the blastocyst wall), endometrial cells, uterine fibroblasts, and testicular fibroblasts.

Among them, cumulus cells are preferably used in the present invention as embryonic development assisting cells. Cumulus cells are cells in which the granulosa cells surrounding the unfertilized egg after ovulation have proliferated and become thickened.In the present invention, cumulus cells collected from unfertilized eggs may be used, or commercially available ones may be used. . Granulosa cells, fallopian tube epithelial cells, trophoblasts, endometrial cells, uterine fibroblasts, testicular fibroblasts, and the like, which have been collected from the respective biological tissues, can also be used. As the embryo developmental assisting cells to be attached to the inner wall of the transport container, it is preferable to use cells obtained from a mammal of the same species as the embryo to be transported.

The material and shape of the transport container are not particularly limited as long as it can smoothly attach the embryonic development assisting cells to at least a part of the inner wall below the culture solution. Although not limited in any way, examples of the transport container that can be used in the present invention include a substance capable of promoting the attachment of embryonic development assisting cells, such as collagen, gelatin, fibronectin, laminin, polylysine, matrigel, and the like. Various plastic and glass containers and straws whose wall surfaces are coated, straws, containers made of polypropylene and other plastics containing additives that promote the attachment of embryonic growth assisting cells, so that embryonic growth assisting cells can adhere easily. In addition, plastic and glass containers and straws whose inner wall surfaces have been subjected to physical treatment such as electric discharge treatment or chemical treatment can be mentioned. More specifically, a polypropylene transport container sold by Nunc under the trade name of “cryotube” is preferably used.

In the present invention, one of the above-mentioned embryonic development assisting cells is
The seed or two or more kinds are used by being attached to at least a part of the inner wall below the culture solution surface of the container for transferring embryos. In that case,
It is preferable that the embryonic growth assisting cells be attached to 10% or more of the inner wall area below the culture solution surface of the container for transporting the embryo, since the viability and growth ability of the embryo can be kept higher. It is more preferable to adhere to 50% or more, more preferably to 80% or more of the inner wall area.
If the ratio of adhering embryonic growth assisting cells to the inner wall of the container for embryo transfer is less than 10% of the inner wall area below the culture solution, the embryo will be damaged during transport, and its viability and growth capacity will be reduced. It tends to be reduced. Further, the attachment of the embryonic development assisting cells to the inner wall below the culture solution surface of the embryo transporting container may be in a monolayer state. However, in some cases, the embryonic development assisting cells may be attached to the inner wall in a state where two or more layers overlap (layered state). Even when the embryo is transported in combination with the embryonic growth assisting cells together with the mammalian embryo, the embryonic growth assisting cells are dispersed in the liquid without adhering to the inner wall below the culture solution surface of the embryo transporting container. If the embryo is placed together with the mammalian embryo in a transport container in a transported state, the damage to the embryo during transport cannot be reduced, and the embryo's viability and development ability will be reduced.

There is no particular limitation on the method for attaching the embryonic development assisting cells to the inner wall of the transport container, and at least a part of the inner wall below the culture solution surface of the embryo transport container, preferably one of the inner wall area, is used.
Any method may be employed as long as it allows the embryonic growth assisting cells to adhere to 0% or more, more preferably 50% or more. Without limitation, for example,
The culture solution containing the embryonic growth assisting cells is placed in a transport container to which the embryonic growth assisting cells can adhere to the inner wall, and the embryonic growth assisting cells are cultured in the transport container for a predetermined time, and the embryo is placed on the inner wall of the transport container. A method of growing and attaching growth-promoting cells is preferably employed. In attaching the embryonic growth assisting cells to the inner wall of the transport container, the embryonic growth assisting cells may be separated from each other, placed in the transport container, and attached to the inner wall of the transport container. This is preferable in that the embryo growth assisting cells can be favorably and promptly attached to the inner wall of the cell. However, the present invention is not limited to this, and in some cases, embryonic development assisting cells may be placed in the form of colonies in a transport container and attached to the inner wall thereof.

After a layer of embryonic development assisting cells is formed on the inner wall of the transport container as described above, a gas phase suitable for embryo development (for example, a gas phase composed of 5% CO ₂ /95% air or the like)
It is preferable to equilibrate in the gas phase. At this time, it is preferable to discharge the culture solution used for culturing the embryonic development assisting cells from the transport container, and to add a fresh culture solution before performing the gas phase equilibrium described above.

Next, the embryo to be transported is transferred to a transport container in which the embryonic growth assisting cells are adhered to at least a part of the inner wall below the surface of the culture solution and which is preferably equilibrated in the gas phase. Put in a dispersed state. At this time, if the embryo is placed in a transport container in an isolated state without being dispersed in the culture solution, the damage to the embryo increases and the viability and developmental ability of the embryo are reduced, so care must be taken. In this case, the type of culture solution in which the embryo is dispersed is selected according to the type of embryo, the stage of embryo development, and the like. As the culture solution, a solution of the same kind as that used for culturing a mammalian embryo, or a solution after being used for culturing an embryo in a stage before transport is preferably used. Although not limited, for example, in transporting bovine embryos, a tissue culture solution (TCM: Tissue Culture Mediu
m) and the like are preferably used.

[0017] After placing a mammalian embryo to the shipping container in the above, preferably a gas phase portion remaining shipping container suitable for embryo development gas (e.g. 5% CO _2/9
(5% air, etc.), and then sealed, put it in a heat insulator or the like, and transport it to the destination while keeping the temperature as constant as possible. There is no particular limitation on the type of the heat retaining device used for transport, and any of the thermal retaining devices conventionally used for transporting mammalian embryos can be used. Further, as the heat retention temperature during transportation, a temperature suitable for survival and development of each embryo may be adopted according to the type of embryo (type of mammal), the stage of embryo development, and the like. In general, for transporting fertilized eggs of cattle, a warming temperature of 35 to 39 ° C. is preferably employed, and for transporting embryos of other mammals, the same temperature as described above is preferably employed.

According to the transport method of the present invention, even in the case of long-distance transport requiring more than 24 hours for transport, the decrease in the viability and developmental ability of the mammalian embryo is small. Since the cells can survive and develop well, and their survival and development ability is kept high even after transportation, precious embryos of mammals can be used very effectively for each purpose.

When the mammalian embryo is transported by employing the transport method of the present invention, it is not clear why the embryo receives less damage during transport and the embryo's viability and developmental ability are maintained well. No, but it is speculated as follows. That is,
The embryonic development assisting cells do not proliferate in a state of being dispersed in the culture solution, but proliferate when attached to the inner wall of the container. Therefore, it secretes a substance that assists the development of the embryo when attached to the inner wall of the container and proliferates, and has the effect of reducing the damage that the substance receives to the embryo at the time of transport. It is presumed to prevent a decrease in growth ability.

[0020]

EXAMPLES The present invention will be described below in more detail with reference to examples and the like, but the present invention is not limited thereto.

Example 1 (1) Preparation of embryo for transport: (i) Ovarian small follicles (1 to 7) of cows shipped to a slaughterhouse
mm), the eggs were collected in a syringe with an injection needle, and only eggs having good morphology were selected.
M HEPES buffer Earle type TCM-199 (SI
GMA Cat. M-2520) based on 10% (v /
v) Fetal calf serum (FCS, GIBCO, Cat. 26140-079) inactivated by heating at 56 ° C. for 30 minutes.
Lot, 38N8150) and antibiotics (penicillin and streptomycin) were added, and the culture was further matured at 38.5 ° C. for 24 hours at 38.5 ° C. under gas phase conditions saturated with steam in 5% CO ₂ /95% air. went. (Ii) Bacteria containing caffeine, heparin and bovine serum albumin were added to the eggs that had undergone maturation culture in (i) above. O.
Sperm concentration in the solution (Brackett and Olifant.,) Using frozen-thawed spermatozoa, 10 × 10 ⁶ / ml
The in vitro fertilized embryo obtained was heated at 56 ° C. for 30 minutes and inactivated by 5% calf serum (CS, GIBCO,
lOT. 22P-4457)
Using PES-buffered Earle-type TCM-199 as a culture medium, co-culture with cumulus cells at 38.5 ° C. for 7 days under a gas phase condition in which water vapor was saturated with 5% CO ₂ /95% air. To obtain blastocysts. Blastocysts having good morphology and less than 10% of degenerated cells were selected therefrom and subjected to a transport test.

(2) Preparation of Container for Transport: (i) A colony-like cumulus cell mass collected from unfertilized eggs of cow contains 0.5% of heated dispase (manufactured by Sanko Junyaku Co., Ltd.). In addition to the sample tube, a suction / discharge treatment with a pipette was performed to make it easier to separate the cell mass into individual cells, and then heated at 37 ° C. for 30 minutes to separate the cell mass into individual cells. Then, after centrifugation was performed to remove the supernatant, the residue was added with 5 parts of TCM-199.
Calf serum (CS, GI) inactivated by heating at 6 ° C for 30 minutes
BCO, IOT. 22P-4457) was added thereto at a ratio of 1%, and the individually separated cells collected at the bottom were suspended in the culture solution. The operation after the centrifugation was repeated once to wash the cells. (Ii) The number of cumulus cells in the culture solution obtained in (i) above was counted using a toma-type hemocytometer, and
It was adjusted so that cumulus cells were contained at a rate of 5 × 10 ⁵ cells / ml. (Iii) 1 ml of the culture solution containing the cumulus cells prepared in (ii) above (that is, 5 × 10 ⁵ cumulus cells) was placed in a transport container (“Cryotube” manufactured by Nunc: capacity: 1.8 ml).
1 ml of TCM-199 culture medium containing
Cumulus cells were cultured and propagated for 48 hours in a shipping container under the condition of ° C., and attached to the inner wall of the shipping container. (Iv) After draining the culture solution used for culturing cumulus cells from the transport container with cumulus cells attached to the inner wall obtained in (iii) above, replace it with fresh culture solution, and incubate for 6 hours in an incubator. and gas phase equilibrium (5% CO _2/95% air).

(3) Introducing and transporting the embryo into the transport container: The transfer container prepared in (iv) of (2) above, in which the cumulus cells adhere to the inner wall and are equilibrated in the gas phase, are placed at 56 ° C. At 30
TCM-19 supplemented with 5% fetal calf serum inactivated by heating for 1 minute
The blastocysts 39 obtained in the above (1) (ii) were added to 1 ml of the 9 solutions.
After putting the liquid obtained by dispersing the individual pieces and closing the stopper, put it in a fertilized egg transporter (Fujidaira “Cell culture transporter”),
Long-distance transport for 20 hours while maintaining the temperature at 39 ° C (15
From time to 11:00 the next day).

(4) Investigation of transport results: (i) Immediately after arriving at the destination, the embryo is taken out of the transport container, the state of development of the embryo is observed with a microscope, and the embryo that has developed during the transport is examined. Counted the number. (Ii) Next, the embryos taken out of the transport
25 mM HEPES buffered Earle type TCM-1 containing 5% fetal calf serum inactivated by heating at 30 ° C. for 30 minutes.
Using 99 as a culture solution, 5% CO ₂ /95%
38.5 ° C. under gas-phase conditions in which air is saturated with water vapor
The number of embryos that were co-cultured with cumulus cells for 48 hours and were able to develop into escaped blastocysts were counted 24 hours and 48 hours after culture, respectively.
Was as shown in FIG.

Comparative Example 1 (1) Gas phase equilibrium (5%
CO _2/95% air) and comprising, 56 5% fetal bovine serum was inactivated by heating 30 minutes at ℃ added TCM-199 solution 1m
1 shows the blastocyst 36 obtained in Example 1 (1) (ii).
The cells were dispersed, and the resulting dispersion was placed in a transport container (the same cryotube used in Example 1) in which the cumulus cells were not adhered to the inner wall.
Transported in the same manner as. (2) Immediately after arriving at the destination, the embryo was taken out of the transport container, the state of development of the embryo was observed with a microscope, and the number of embryos that had developed during transport was counted. (3) Next, the embryos taken out from the transport container were cultured in the same manner as in (ii) of (4) of Example 1, and the number of embryos that could develop to the escaped blastocysts was counted for 24 hours. The counts after and 48 hours later were as shown in Table 1 below.

<< Comparative Example 2 >> (1) Blastocyst 3 obtained in (1)-(ii) of Example 1
Five and 5 × 10 ⁵ cumulus cells separated into individual cells by performing the same operation as (i) and (ii) in (2) of Example 1
Each of them was previously subjected to gas phase equilibrium (5% CO ₂ /
Dispersed in 1 ml of inactivated 5% fetal bovine serum-added TCM-199 solution by heating at 56 ° C. for 30 minutes and dispersing the dispersion, and dispersing the dispersion (same as used in Example 1). Example 1
Transport was carried out in exactly the same way. (2) Immediately after arriving at the destination, the embryos were taken out of the transport container, the state of development of the embryos was observed with a microscope, and the number of embryos that had developed during the transport was counted. (3) Next, the embryos taken out from the transport container were cultured in the same manner as in (ii) of (4) of Example 1, and the number of embryos that could develop to the escaped blastocysts was counted for 24 hours. The counts after and 48 hours later were as shown in Table 1 below.

[0027]

[Table 1] Number of blastocysts escaped in culture after transportation at the time of transportation Number of blastocysts Number of developing embryos 24 hours after culture 48 hours after culture Example 1 39 20 (51%) 13 (33%) 24 (62%) Comparative Example 1 36 12 (33%) 6 (17%) 14 (39%) Comparative Example 2 35 12 pieces (34%) 7 pieces (20%) 15 pieces (43%)

From the results in Table 1 above, it is found that the embryonic development assisting cells (cumulus cells) are attached to at least a part of the inner wall below the surface of the culture solution in the container, and the embryo is dispersed in the culture solution in the container. Comparative Example 1 in which only the embryo and the culture solution were placed in the container and transported without using the embryo development auxiliary cells, and in the case of Example 1 in which Is used, but compared to Comparative Example 2 in which the embryo is dispersed in a culture solution together with the embryo without being attached to the inner wall of the transport container, the embryo is less damaged during transport, the viability of the embryo and It can be seen that the growth ability is maintained well, and that the blastocyst can be satisfactorily grown after transportation.

[0029]

According to the transportation method of the present invention, the damage to the mammalian embryo during transportation is reduced, and the mammalian embryo can be safely and securely protected while preventing the decrease in its viability and growth ability. It can be easily transported. And
The mammalian embryo transported by the method of the present invention can be effectively used as it is for transplantation and other uses without any trouble such as thawing.

Claims (2)

[Claims] 1. A method for transporting a mammalian embryo by placing the embryo in a container containing a culture solution, wherein cells for supporting the growth of the embryo are attached to at least a part of the inner wall below the culture solution level in the container. A method of transporting a mammalian embryo, wherein the mammalian embryo is dispersed in a culture solution, placed in the container, and the container is sealed and transported. 2. A cell that supports the development of a mammalian embryo,
2. The transport method according to claim 1, wherein the transport method is at least one selected from cumulus cells, granulosa cells, fallopian tube epithelial cells, trophoblasts, endometrial cells, uterine fibroblasts, and testicular fibroblasts.