JP4362633B2 - Culture container and culture apparatus - Google Patents

Description

The present invention is a culture vessel and the culture equipment, and more particularly, to a culture to transplantation capable embryo culture vessel and the culture device to produce an oocyte or fertilized embryos of livestock.

  Conventionally, in order to create an environment for cultivating in vitro fertilized embryos of livestock, generally, a pressure-resistant container having airtightness is used as a culture container, and nitrogen gas in which oxygen gas concentration and carbon dioxide gas concentration are adjusted in this container A method of connecting a cylinder filled with a mixed gas of oxygen gas and carbon dioxide gas, supplying the mixed gas into the pressure vessel and forcibly replacing the inside of the pressure vessel with the mixed gas has been performed.

  In addition, in order to culture immature eggs or in-vivo fertilized embryos, a thermostatic chamber with a carbon dioxide concentration adjustment function is generally used. This thermostatic chamber is connected to a cylinder filled with carbon dioxide, and carbon dioxide is supplied into the thermostatic chamber. There has been a way to do it.

  On the other hand, as a simple method, there has been proposed a method of injecting a culture atmosphere adjusting agent, for example, carbon dioxide powder as described in Patent Document 1, into a sealed accommodation chamber.

Japanese Patent No. 3082912

  The conventional method of forcibly substituting with a mixed gas using a hermetic pressure-resistant container requires a large-scale device, and is problematic in that it is cumbersome and troublesome to handle and requires maintenance and management of the device. In particular, when a change in the state of the cultured embryo is captured over time, the container is opened each time, and it is difficult to culture without destroying the environmental conditions.

  The culture of immature eggs or in-vivo fertilized embryos also has a problem that the gas environment in the thermostat is broken every time the thermostat is opened, and the culture of the in-vivo fertilized embryo is adversely affected.

  On the other hand, in the case of carbon dioxide powder, which is a culture atmosphere adjusting agent, a reaction with water is necessary when using this powder, and a special container with a hole is used. A method of injecting carbon dioxide powder into the tube is used. This method has a problem in that the amount of carbon dioxide generated varies depending on how the carbon dioxide powder is dissolved in water, and the desired carbon dioxide concentration may not be achieved, and handling is difficult. Further, carbon dioxide powder has a problem that even if the concentration of carbon dioxide gas can be adjusted, the concentration of oxygen gas cannot be adjusted, and fertilized embryos that can use the oxygen gas concentration are limited.

The present invention has been made in view of such problems, it is possible to make simple the culture environment suitable for culturing immature oocytes or fertilized embryos of livestock, and easy to handle culture vessel and culturing instrumentation The purpose is to provide a device.

In order to solve such a problem, the culture container of the present invention is portable with a sealed lid for storing a petri dish that accommodates an immature ovum or in-vivo fertilized embryo taken from the ovary of a livestock together with a culture solution. A culture container containing a gas concentration adjusting agent in a container body, the environment of which is set to a predetermined carbon dioxide gas concentration suitable for the growth of the immature ovum or the in-vivo fertilized embryo in the petri dish. When the lid is attached to the container main body, the lid is disposed at a position close to the petri dish housed in the container main body, and the lid and the bottom of the container main body can be seen through the container main body from the outside. It is characterized by being formed of a transparent material capable of irradiating light to immature ova or in vivo fertilized embryos in a petri dish .

According to the present invention, the environment inside the container body is immature ovum in a portable container body having a sealed lid for storing a petri dish containing the immature ovum or in-vivo fertilized embryo together with a culture solution. Or by storing a gas concentration adjusting agent to a predetermined carbon dioxide gas concentration suitable for the development of in-vivo fertilized embryos, the petri dish containing immature eggs or in-vivo fertilized embryos is stored in a sealed state in the container body, The inside of the container body is adjusted to a predetermined carbon dioxide gas concentration by a gas concentration adjusting agent. For this reason, the culture environment suitable for culture | cultivation of an immature ovum or an in-vivo fertilized embryo can be made simply, and handling of a culture container can be made easy by using a gas concentration regulator. In addition, since the container body is hermetically sealed, the culture environment surrounding the immature ovum or in vivo fertilized embryo changes greatly even if the container body is moved to another environment in order to observe the immature ovum or in vivo fertilized embryo. The immature ovum or in-vivo fertilized embryo can be cultured without destroying the environmental conditions. Further, when the lid is attached to the container body, the lid body is disposed in the vicinity of the petri dish in the container body, and the bottom part of the lid body and the container body is formed of a transparent material, so that the immature egg or fertilization in the petri dish is formed. When observing an embryo under a microscope, the objective lens can be brought close to an immature ovum or a fertilized embryo, and light can be reliably applied to the observation object, so that the focus adjustment becomes easy and the immature ovum or the fertilized embryo Observation can be facilitated.

In addition, the culture container of the present invention includes a portable container body having a sealed lid for storing a petri dish that contains in vitro fertilized embryos of livestock together with a culture solution so that the petri dish can be put in and out. A culture container that contains a gas concentration adjusting agent that has a predetermined oxygen gas concentration and carbon dioxide gas concentration suitable for the development of in vitro fertilized embryos, and when the lid is attached to the container body, The lid and the bottom of the container body are transparent materials that can be seen through the container body from the outside and can shine light on in vitro fertilized embryos in the dish. It is formed .

According to the present invention, the environment in the container body is suitable for the development of the in vitro fertilized embryo in the portable container body having a sealed lid for storing the petri dish for storing the in vitro fertilized embryo together with the culture medium. The petri dish containing the in vitro fertilized embryo is stored in a sealed state in the container body by storing the gas concentration adjusting agent that makes the predetermined oxygen gas concentration and carbon dioxide gas concentration, and the inside of the container body is the gas concentration adjusting agent To a predetermined oxygen gas concentration and carbon dioxide gas concentration. For this reason, the culture environment suitable for culture | cultivation of an in vitro fertilized embryo can be made simply, and handling of a culture container can be made easy by using a gas concentration regulator. In addition, since the inside of the container body is sealed, even if the container body is moved to another environment in order to observe the in vitro fertilized embryo, the culture environment surrounding the in vitro fertilized embryo does not change significantly, In vitro fertilized embryos can be cultured without breaking. Further, when the lid is mounted on the container body, the lid body and the petri dish inside the container body are arranged close to each other, and the bottom of the lid body and the container body is formed of a transparent material, so that the fertilized embryo in the petri dish is observed with a microscope. In this case, the objective lens can be brought close to the fertilized embryo, and light can be reliably applied to the observation object, so that the focus adjustment is facilitated and the observation of the fertilized embryo can be facilitated.

  Further, the present invention is characterized in that storage means (for example, the IC tag 35 in the embodiment) capable of reading and writing individual information relating to immature eggs or fertilized embryos is provided on the side surface of the container body.

  According to this invention, by providing storage means that can freely read and write individual information relating to immature ova or fertilized embryos on the side surface of the container body, individual management of immature ovum or fertilized embryos is facilitated and handled. Can be prevented from being misunderstood. Further, since the storage means can be freely read and written, the container body can be reused. In addition, since the storage means is provided on the side surface of the container body, when observing immature eggs or fertilized embryos with a microscope, the storage means does not disturb the ease of observing immature eggs and the like. There is no need to do.

  Furthermore, the present invention provides a peripheral wall portion having a diameter slightly larger than the diameter of the petri dish at the bottom of the container main body, and positions the petri dish with respect to the container main body by installing the petri dish inside the peripheral wall portion. Features.

  According to this invention, by providing the peripheral wall portion for positioning the petri dish at the bottom of the container main body, there is no possibility that the petri dish will shift with respect to the container main body. For this reason, an immature ovum or a fertilized embryo can be observed more accurately.

  Further, the gas concentration adjusting agent of the present invention is characterized in that the inside of the container body is set to a negative pressure.

  According to this invention, the gas concentration adjusting agent can further enhance the development ability of immature eggs or fertilized embryos housed in the petri dish by making the inside of the container body have a negative pressure.

  Furthermore, the culture container and gas concentration adjusting agent of the present invention are characterized by being sterilized.

  According to this invention, since the sterilized container and the gas concentration adjusting agent are used, it is possible to suppress a situation in which immature eggs or fertilized embryos are contaminated with bacteria during the culturing process. It is possible to prevent a decrease in development of a mature egg or a fertilized embryo.

  Moreover, the culture apparatus of the present invention is used for the growth of immature eggs or fertilized embryos containing the culture container according to any one of claims 1 to 5 and containing the culture container and containing the environment in the culture container in a petri dish. A thermostat bath that can be set to a suitable predetermined temperature is provided.

  According to the present invention, the culture apparatus includes a culture container and a thermostatic bath, and thus creates an environment suitable for the growth of immature eggs or fertilized embryos housed in the petri dish by placing the culture vessel in the thermostatic bath. And the developmental ability of immature eggs or fertilized embryos can be enhanced.

According to the culture vessel and the culture equipment according to the present invention, immature oocytes livestock, the internal fertilization embryo or IVF embryos were housed in a Petri dish with culture medium, a predetermined suitable for development of such immature oocytes in the dish The gas concentration adjusting agent for adjusting the oxygen gas concentration and the carbon dioxide gas concentration is stored in a portable container body having a sealed lid for storing the petri dish so that it can be taken in and out, and the container body is housed in a thermostat and stored in the petri dish. by a predetermined temperature suitable for growth of such immature oocytes temperature can be made to simplify the culture environment suitable for culturing such immature oocytes livestock, and easy to handle culture vessel and culturing instrumentation it is possible to provide a location.

Hereinafter, a preferred embodiment of the culture vessel and culture equipment according to the present invention will be described with reference to FIGS. Na us, the present embodiment, showing an example of the present invention, but is not limited thereto.

  As shown in FIG. 1 (perspective view) and FIG. 2 (cross-sectional view), the culture apparatus 1 is an immature ovum R taken from the ovaries of livestock or fertilized embryos of livestock (in vitro fertilized embryo H, in vivo fertilized embryo H ′). A culture vessel 10 configured to store the gas concentration adjusting agent 40 in a container body 20 that stores the petri dish 11 that can store the culture solution A together with the culture solution A, and a thermostatic chamber 50 that stores the culture vessel 10. .

  The container main body 20 of the culture container 10 has a depth that can accommodate the petri dish 11, and an upper portion that opens to form a bottomed container, and an upper opening of the storage barrel 21. And a lid 30 that is hermetically sealed and detachably attached. The container body 20 has a gas barrier property and is formed of a transparent material so that immature eggs or fertilized embryos in the petri dish 11 accommodated therein can be confirmed. When installed, the inside of the container body 20 can be kept airtight.

  The storage barrel 21 has a size capable of storing the petri dish 11 and the gas concentration adjusting agent 40, and is formed in a rectangular shape in plan view. In addition, the accommodation trunk | drum 21 should just accommodate the petri dish 11 and the gas concentration regulator 40, and the shape is not restricted to a rectangular shape. In general, the petri dish 11 used for embryo culture is often a plastic petri dish having a diameter of 35 mm. Therefore, the size of the accommodating body 21 has a size corresponding to the number of accommodating the petri dishes 11. .

Further, a concave groove 21 a that forms a peripheral wall portion 21 a ₁ having a diameter slightly larger than the diameter of the petri dish 11 is provided on the bottom surface of the housing body portion 21. The petri dish 11 is accommodated in the concave groove 21 a and positioned with respect to the accommodating body 21. For this reason, the petri dish 11 accommodated in the container main body 20 is stably accommodated. Incidentally, it formed so as to project the peripheral wall portion 21a ₁ from the bottom surface of the housing body portion 21 upward may accommodate Petri dish 11 to the inside. A locking projection 22 that protrudes outward is formed on the outer surface of the housing barrel 21.

  The lid body 30 that seals the upper opening of the housing body 21 includes a top plate portion 31 that is disposed so as to cover the upper opening of the housing body portion 21, and a side wall portion 32 that extends downward from the peripheral end of the top plate portion 31. It is comprised. When the lid 30 is mounted on the upper portion of the housing body 21, the upper portion of the housing body 21 is fitted inside the side wall portion 32, and the top plate 31 is placed on the upper surface of the petri dish 11 housed in the housing body. It is arranged at a close position. In addition, a packing member 23 is annularly attached to the lower portion of the top plate portion 31 in order to improve the hermeticity of the lid body 30 with respect to the housing body portion 21.

  On the outside of the side wall portion 32, a fixing plate 33 that fixes the lid body 30 to the accommodation barrel portion 21 with the lid 30 attached to the accommodation barrel portion 21, and causes the packing member 23 to be in close contact with the upper portion of the accommodation barrel portion 21. Is attached so as to be rotatable in the vertical direction. On the inner side of the distal end of the fixing plate 33, an engaging convex portion 34 that can be engaged with the locking projection portion 22 provided on the accommodating body portion 21 is formed. For this reason, when the lid body 30 is placed on the upper portion of the housing body 21 and the fixing plate 33 is rotated downward to engage the engaging projection 34 with the locking projection 22, the lid body 30 is accommodated. While being able to attach to the trunk | drum 21 in the sealed state, the inside of the container main body 20 can be made into an airtight state.

  Individual information regarding the immature ovum R and fertilized embryos H and H ′ housed in the petri dish 11 on the outer surface of the container body 20, that is, the outer surface of the housing body 21 or the outer surface of the side wall 32 of the lid 30. A readable / writable IC tag 35 is attached. In the IC tag 35, information on immature ovum R and fertilized embryos H and H ′, environmental conditions for culturing, and the like are recorded. The IC tag 35 has an IC chip embedded therein, and individual information recorded on the IC tag 35 can be read and written by an IC reader / writer. This facilitates the individual management of the immature ovum R or the fertilized embryos H and H ′, and prevents mistakes by those who handle them. Moreover, since the IC tag 35 can be freely read and written, the container body 20 can be reused.

  The gas concentration adjusting agent 40 is used as a package packaged with a breathable packaging material, and there are two types having different oxygen absorption capacity and carbon dioxide gas generation capacity. When the immature ovum R or the in-vivo fertilized embryo H ′ is accommodated in the container body 20, it has an oxygen absorption capacity and a carbon dioxide gas generation capacity with a carbon dioxide gas concentration of 5% and an oxygen gas concentration of 15%. Is used, and when the in vitro fertilized embryo H is accommodated in the container body 20, the one having an oxygen absorption capacity and a carbon dioxide generation capacity that makes the carbon dioxide gas concentration 5% and the oxygen gas concentration 5% is used. It is done. The gas concentration adjusting agent 40 that exhibits these functions is composed of a composition made of ascorbic acids, metal salts, porous pores, alkaline earth metal hydroxides, and the like. In addition, the gas concentration adjusting agent 40 has an absorption ability that makes the inside of the container body 20 have a negative pressure.

  The culture container 1 and the gas concentration adjusting agent 40 described above are sterilized. This is for preventing the immature ovum and the like from being contaminated by bacteria and the like in the process of culturing the immature ovum R and the fertilized embryos H and H ′, and preventing the growth of the immature ovum and the like from decreasing.

  There are four types of culture solutions A for culturing immature ova and fertilized embryos, depending on the application, those that mature immature eggs, those that are used at the time of fertilization, those that are used 7 days after fertilization, and in-vivo fertilization Some cultivate embryos. Further, in the culture container 1, a gauze 43 moistened with water is placed in order to cultivate immature eggs and the like with the humidity inside the container saturated.

  In order to culture the immature ovum R or the in-vivo fertilized embryo H ′ extracted from the ovary of livestock by the culture apparatus 1 configured as described above, the extracted immature ovum R or the in-vivo fertilized embryo H ′ together with the culture medium A is used. A gas concentration adjusting agent 40 which is housed in the petri dish 11 and accommodated in the container body 20 and has a predetermined carbon dioxide gas concentration (5%) suitable for the growth of immature ovum R or in-vivo fertilized embryo H ′. Is stored in the storage barrel 21, and the culture vessel 10 with the lid 30 attached to the storage barrel 21 is stored in the thermostat 50 to warm the immature ovum R or the in-vivo fertilized embryo H ′ at a predetermined temperature.

  In order to culture the in vitro fertilized embryo H, the in vitro fertilized embryo H is housed in a petri dish 11 together with a culture solution, and the petri dish 11 is housed in the container body 20 and at a predetermined oxygen suitable for the development of the in vitro fertilized embryo H. The culture container 10 in which the gas concentration adjusting agent 40 for adjusting the gas concentration (5%) and the carbon dioxide gas concentration (5%) is stored in the storage barrel 21 and the lid 30 is attached to the storage barrel 21 is placed in the thermostat 50. The in vitro fertilized embryo H is housed and warmed at a predetermined temperature.

  As described above, the culture container 10 of the present invention is a portable container body having a sealed lid 30 that accommodates the petri dish 11 that accommodates the immature egg R or the fertilized embryos H and H ′ together with the culture medium A so as to be put in and out. 20 contains a gas concentration adjusting agent 40 that adjusts the environment in the container body 20 to a predetermined oxygen gas concentration and carbon dioxide gas concentration suitable for the growth of a fertilized embryo or the like, thereby culturing a culture suitable for culturing a fertilized embryo or the like. The environment can be easily created, and handling of the culture vessel 10 can be facilitated by using the gas concentration adjusting agent 40. In addition, since the container body 20 is in a sealed state, even if the container body 20 is taken out of the thermostatic chamber 50 in order to observe the fertilized embryo or the like, the culture environment surrounding the fertilized embryo or the like does not change greatly, Fertilized embryos can be cultured without breaking. Moreover, the gas concentration adjusting agent 40 can further enhance the development ability of fertilized embryos and the like housed in the petri dish 11 by making the inside of the container body 20 have a negative pressure.

  Further, when the lid 30 of the culture vessel 10 is attached to the storage barrel 21, the lid 30 and the bottom of the storage barrel 21 are made of a transparent material. Therefore, when the fertilized embryo or the like in the petri dish 11 is observed with a microscope, the focus adjustment is facilitated and the fertilized embryo or the like can be easily observed.

  As described above, the culture container 10 of the present invention does not need to prepare an expensive and large-sized device in creating an ovum or fertilized embryo culture environment, and can be placed in an incubator or a constant temperature bath in a normal office. The egg or fertilized embryo can be cultured simply by putting the medium containing the egg or fertilized embryo and the gas concentration adjusting agent 40 into the culture vessel 10. In addition, a large number of eggs and fertilized embryos can be cultured individually at the same time, so that the environmental conditions can be stabilized and the working time can be shortened. Thereby, in many domestic animal fertilized egg related organizations, the production of in vitro fertilization, the culture of in-vivo fertilized embryos and the like can be performed more simply and inexpensively.

〔Experimental result〕
(1) Tests aimed at producing in vitro fertilized embryos (5% oxygen gas concentration, 5% carbon dioxide concentration)
Table 1 shows the results of comparing in vitro blastocyst development rates by producing in vitro fertilized embryos using the conventional method (forced replacement of mixed gas into a hermetic pressure-resistant container) and the method of the present invention (culture with a gas concentration regulator). Shown in

(2) Tests assuming in vitro fertilized embryo culture (5% oxygen gas concentration, 95% air)
Table 2 shows the results of comparison of hatching rates after 24-hour culture using 7-day-old in vitro fertilized embryos by the conventional method (CO ₂ incubator) and the method of the present invention (culture with a gas concentration adjusting agent).

  As described above, in the test assuming the production of in vitro fertilized embryos and the culture of in vivo fertilized embryos, it was confirmed that the method of the present invention improved the blastocyst generation rate and the hatching rate as compared with the conventional method.

The perspective view of the culture apparatus concerning one embodiment of the present invention is shown. Sectional drawing of the culture container which is a part of this culture apparatus is shown.

DESCRIPTION OF SYMBOLS 1 Culture apparatus 10 Culture container 11 Petri dish 20 Container main body 21a ₁ Surrounding wall part 30 Cover body 35 IC tag (memory | storage means)
40 Gas concentration regulator 50 Thermostatic bath A Culture medium H In vitro fertilized embryo H 'In vivo fertilized embryo R Immature egg

Claims (7)

A portable container body having a sealed lid for storing a petri dish for storing immature eggs or in-vivo fertilized embryos taken from the ovaries of livestock together with a culture solution in a removable manner, and the environment in the container body in the petri dish A culture container containing a gas concentration adjusting agent for making a predetermined carbon dioxide gas concentration suitable for development of the immature ovum or the in-vivo fertilized embryo ,
When the lid is attached to the container main body, the lid is disposed at a position close to the petri dish stored in the container main body,
The lid and the bottom of the container body are formed of a transparent material that can be seen through the container body from the outside and can shine light on the immature ovum or the in-vivo fertilized embryo in the petri dish. culture vessel, wherein it is. In a portable container body having a sealed lid for storing a petri dish that contains in vitro fertilized embryos of livestock together with a culture solution in a removable manner, the environment in the container body is used for the growth of the in vitro fertilized embryo in the petri dish A culture vessel containing a gas concentration adjusting agent for making a suitable predetermined oxygen gas concentration and carbon dioxide gas concentration ,
When the lid is attached to the container main body, the lid is disposed at a position close to the petri dish stored in the container main body,
The lid and the bottom of the container body are formed of a transparent material that can be seen through the container body from the outside and can shine light on the in vitro fertilized embryo in the petri dish. Culture container. The culture container according to claim 1 or 2 , wherein storage means capable of reading and writing individual information relating to the immature egg or fertilized embryo is provided on a side surface of the container body. A bottom wall portion of the container body is provided with a peripheral wall portion having a diameter slightly larger than the diameter of the petri dish, and the petri dish is positioned inside the peripheral wall portion to position the petri dish with respect to the container body. The culture container according to any one of claims 1 to 3 . The culture container according to any one of claims 1 to 4 , wherein the gas concentration adjusting agent creates a negative pressure in the container body. The culture vessel according to any one of claims 1 to 5 , wherein the culture vessel and the gas concentration adjusting agent are sterilized. The culture container according to any one of claims 1 to 6 , and a predetermined temperature suitable for the development of immature ovum or fertilized embryo that contains the culture container and contains the environment in the culture container in the petri dish A culture apparatus comprising: a thermostatic chamber that can be set to 1).

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