JP6793305B1 - Culturing methods to improve in vivo viability of mammalian embryos - Google Patents

Culturing methods to improve in vivo viability of mammalian embryos Download PDF

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JP6793305B1
JP6793305B1 JP2019228790A JP2019228790A JP6793305B1 JP 6793305 B1 JP6793305 B1 JP 6793305B1 JP 2019228790 A JP2019228790 A JP 2019228790A JP 2019228790 A JP2019228790 A JP 2019228790A JP 6793305 B1 JP6793305 B1 JP 6793305B1
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邱寒峰
呉▲てぃん▼
徐雅萍
董信陽
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Abstract

【課題】哺乳動物胚のインビボ生存率を向上する培養方法の提供。【解決手段】受精後のヒツジ胚をＴＣＭ１９９培養液にて繰返してフラッシュ洗い、その後ヒツジ胚のインビボ培養液中に置き、そしてインキュベータ中に置いて胚のインビボ培養を行い、該インキュベータ内の温度を３８〜３９℃に維持し、気体濃度を５％ＣＯ２に維持する。ただし、前記インキュベータは、培養室と、ドアと、該培養室と連通される気体濃度調節室３とを含み、前記培養室内に複数個の培養ビンが備えられ、複数個の前記培養ビン内にそれぞれ培地セッターと隔離手段が設けられること。本発明のインキュベータ内環境条件が安定であることで、胚の生存率を保証する。【選択図】図１PROBLEM TO BE SOLVED: To provide a culture method for improving an in vivo survival rate of a mammalian embryo. After fertilization, sheep embryos are repeatedly flush-washed with TCM199 culture medium, then placed in in vivo culture medium of sheep embryos, and placed in an incubator for in vivo culture of embryos, and the temperature in the incubator is adjusted. The temperature is maintained at 38 to 39 ° C., and the gas concentration is maintained at 5% CO2. However, the incubator includes a culture chamber, a door, and a gas concentration control chamber 3 communicating with the culture chamber, and a plurality of culture bottles are provided in the culture chamber, and the culture chambers are provided with a plurality of culture bottles. A medium setter and isolation means shall be provided for each. Stable environmental conditions in the incubator of the present invention guarantee embryo viability. [Selection diagram] Fig. 1

Description

本発明はヒツジ胚のインビボ（ｉｎ ｖｉｔｒｏ）培養技術分野に属するもので、特に
哺乳動物胚のインビボ生存率を向上する培養方法に関する。 The present invention belongs to the field of in vivo culturing of sheep embryos, and particularly relates to a culturing method for improving the in vivo viability of mammalian embryos.

胎のインビボ培養とは、胚をインキュベータ（培養箱）中に置き、インキュベータ中で
一定の温度と一定濃度の二酸化炭素を維持し、子宮の生理環境をシミュレートし、培地に
おける胚と相互に作用および物質交換で、胚の生長を実現する。従来の培養方法では、胚
をインキュベータ内に入れた後、操作者により時々胚を観察するや操作する必要がある。
このとき、インキュベータのドアを開きなければならない。この過程では、インキュベー
タ内の雰囲気が外部空気と気体交換をすることで、インキュベータ内の雰囲気に影響を及
ぼし、変化が発生する。これにより、インキュベータ内の安定的な温度と雰囲気環境を常
に保証することが難しく、胚の損傷や死亡などに至ることもあり、胚の生存率が低い。 In vivo culture of the womb involves placing the embryo in an incubator, maintaining a constant temperature and concentration of carbon dioxide in the incubator, simulating the physiological environment of the uterus, and interacting with the embryo in the medium. And by exchanging substances, embryo growth is achieved. In the conventional culture method, after the embryo is placed in the incubator, it is necessary for the operator to occasionally observe or manipulate the embryo.
At this time, the incubator door must be opened. In this process, the atmosphere inside the incubator exchanges gas with the outside air, which affects the atmosphere inside the incubator and causes changes. As a result, it is difficult to always guarantee a stable temperature and atmospheric environment in the incubator, which may lead to embryo damage or death, resulting in a low embryo survival rate.

本発明は、従来の技術における欠陥を克服するために、生存率が高く、哺乳動物胚のイ
ンビボ生存率を向上した培養方法を提供する。 The present invention provides a culture method with high viability and improved in vivo viability of mammalian embryos in order to overcome defects in conventional techniques.

上記の目的を実現するために、本発明にかかる哺乳動物胚のインビボ生存率を向上する
培養方法は、以下の工程を含む。
（１） 雌ヒツジからヒツジ卵母細胞を取り、成熟培養液内に置き、インキュベータ内で
２０〜２４時間培養し、成熟したヒツジ卵母細胞を得ること、
（２） 成熟したヒツジ卵母細胞を***とともに受精培養液中に置き、インキュベータ内
で１８〜２４時間培養し、受精させ、ヒツジ初期胚を得ること、
（３） 受精後のヒツジ胚をＴＣＭ１９９培養液にて繰返してフラッシュ洗い、その後ヒ
ツジ胚のインビボ培養液中に置き、そしてインキュベータ中に置いて胚のインビボ培養を
行い、該インキュベータ内の温度を３８〜３９℃に維持し、気体濃度を５％ＣＯ２に維持
すること。 In order to achieve the above object, the culture method for improving the in vivo viability of a mammalian embryo according to the present invention includes the following steps.
(1) Take sheep oocytes from female sheep, place them in a mature culture medium, and incubate them in an incubator for 20 to 24 hours to obtain mature sheep oocytes.
(2) A mature sheep oocyte is placed in a fertilization culture medium together with sperm, cultured in an incubator for 18 to 24 hours, and fertilized to obtain an early sheep embryo.
(3) The fertilized sheep embryos were repeatedly flush-washed with TCM199 culture medium, then placed in the incubator of the sheep embryos, and then placed in the incubator for in vivo culture of the embryos, and the temperature in the incubator was 38. Maintain at ~ 39 ° C. and maintain gas concentration at 5% CO2.

ただし、前記インキュベータは、培養室と、該培養室と嵌合されるドアと、該培養室と連
通される気体濃度調節室とを含む。前記培養室内に複数個の培養ビンが備えられる。複数
個の前記培養ビン内にそれぞれ培地セッターと隔離手段が設けられ、前記培地セッターが
培養ビン内まで挿入可能で、且つ挿入後に培地セッター（４）と培養ビン（１１）の入口
が封止嵌合となる。前記培養の過程では、インキュベータ内の各位置の胚を観察する場合
、操作者により培地セッターを独立の培養ビン中から抽出することができ、且つ前記培地
セッターが抽出される過程では、前記隔離手段により相応の培養ビンと外部との連通を隔
離する。 However, the incubator includes a culture chamber, a door fitted with the culture chamber, and a gas concentration control chamber communicating with the culture chamber. A plurality of culture bottles are provided in the culture chamber. A medium setter and an isolation means are provided in each of the plurality of culture bins, the medium setter can be inserted into the culture bin, and the inlets of the medium setter (4) and the culture bin (11) are sealed and fitted after the insertion. It becomes a match. In the process of culturing, when observing embryos at each position in the incubator, the medium setter can be extracted from an independent culture bottle by the operator, and in the process of extracting the medium setter, the isolation means. Isolate the communication between the appropriate culture bottle and the outside.

本発明におけるインキュベータ内の各培養ビンが、独立に配置したもので、且つ培養ビン
内のセッターが外へ抽出されるときに、隔離手段で培養ビンと外部の連通を常に隔離する
ことができる。それで、胚の観察際は、抽出されたセッター上の培地のみが、しばらく外
部空気と接触するのである。他の位置における培地がいずれも外部空気と接触しない。そ
れに、隔離手段により隔離を実施するとすれば、開いた培養ビンから外部空気が気体濃度
調節室の全体内へ進入するおそれがない。これによっても、他位置の培養ビンが外部環境
にインフルエンスされることを回避する。このため、インキュベータ内の環境の安定性を
向上するとともに、胚の生存率を確保する。また、複数個の培養ビンがいずれも気体濃度
調節室と相互に連通するので、気体濃度調節室内における調節済みの気体を直接に培養ビ
ン内へ送ることができ、培養ビンごとに気体濃度検測器や進気管道を備えておく必要がな
く、構成が簡単で、気体の調節もより快速となる。 Each culture bin in the incubator in the present invention is arranged independently, and when the setter in the culture bin is extracted to the outside, the culture bin and the external communication can always be isolated by the isolation means. Therefore, when observing embryos, only the medium on the extracted setter is in contact with the outside air for a while. None of the media at other locations comes into contact with outside air. Moreover, if isolation is performed by isolation means, there is no risk of external air entering the entire gas concentration control chamber from the open culture bottle. This also prevents the culture bottles at other positions from being influenced by the external environment. Therefore, the stability of the environment in the incubator is improved and the viability of the embryo is ensured. In addition, since a plurality of culture bins communicate with each other with the gas concentration control chamber, the adjusted gas in the gas concentration control chamber can be directly sent into the culture bins, and the gas concentration is measured for each culture bin. There is no need to have a vessel or an air duct, the configuration is simple, and the gas can be adjusted more quickly.

工程（１）では、インキュベータ内の温度を３８〜３９℃に維持し、気体濃度を５％ＣＯ
２に維持する。この温度と気体濃度の条件では、胚が最適な製造環境にあり、生長速度が
はやく、生存率が高い。 In step (1), the temperature in the incubator is maintained at 38 to 39 ° C., and the gas concentration is 5% CO.
Keep at 2. Under these temperature and gas concentration conditions, the embryos are in an optimal production environment, have a fast growth rate and a high viability.

工程（２）における受精培養液は３％のヒツジ血清を含むＴＣＭ１９９培養液とする。
好ましくは、前記隔離手段は、前記培養ビン内に前後に動作可能に設けられる移動板と、
前記移動板の外側壁に設けられ、前記培養ビンの内壁に封止嵌合される封止リングと、前
記移動板に設けられる一方向出気部材および一方向進気部材、導通部材と、および培地セ
ッターと移動板の間の接続嵌合を実現するための接続構成とを含む。 The fertilization culture medium in step (2) is a TCM199 culture medium containing 3% sheep serum.
Preferably, the isolation means comprises a moving plate operably provided in the culture bin back and forth.
A sealing ring provided on the outer wall of the moving plate and sealed and fitted to the inner wall of the culture bottle, a one-way air outlet member, a one-way air advancing member, and a conduction member provided on the moving plate, and Includes a connection configuration to achieve a connection fit between the medium setter and the moving plate.

前記培地セッターが培養ビン内に挿入すると、前記培地セッターが移動板に抵触しながら
後向きに移動するとともに、前記一方向進気部材により気体濃度調節室内の気体を一方向
に培養ビン内まで進入するように制御する。 When the medium setter is inserted into the culture bottle, the medium setter moves backward while in contact with the moving plate, and the gas in the gas concentration control chamber enters the culture bottle in one direction by the unidirectional air advance member. To control.

前記移動板が最後端位置まで移動すると、前記接続構成により培地セッターと移動板の間
の接続嵌合を実現するとともに、前記導通部材により培養ビンと気体濃度調節室の直接連
通を実現する。 When the moving plate moves to the rearmost position, the connection configuration realizes a connection fitting between the medium setter and the moving plate, and the conduction member realizes direct communication between the culture bottle and the gas concentration control chamber.

前記培養ビン内の培養状況を観察するや取得を実施する場合、前記培地セッターを外へ引
き取り、前記接続構成により培地セッターが封止部材と培養ビンの間の摩擦力に抵抗しな
がら、移動板が連動して前向きに移動するとともに、前記一方向出気部材により培養ビン
内の気体を一方向に気体濃度調節室内まで進入するように制御する。 When observing or acquiring the culture status in the culture bottle, the medium setter is taken out, and the medium setter resists the frictional force between the sealing member and the culture bottle due to the connection configuration, and the moving plate. Are interlocked and move forward, and the one-way air outlet member controls the gas in the culture bottle to enter the gas concentration control chamber in one direction.

前記移動板が最前端位置まで移動すると、前記培地セッターが培養ビンの外へ延出し、且
つ前記移動板により常に培養ビンの内部と外部との連通を隔離する。 When the moving plate moves to the foremost position, the medium setter extends out of the culture bottle, and the moving plate always isolates communication between the inside and the outside of the culture bottle.

このような構成において、セッターが外へ抽出される時は、一方向出気部材により培養ビ
ン内の気体をインキュベータ内までに進入させるよう制御することとなるので、セッター
の抽出過程では、培養ビンと外部が連通状態となるのに、培養ビン内の気体を優先的にイ
ンキュベータ内へ返送して、混入した外部空気もわずかで、大きい程度で、培養ビンを開
くときに外部気体がインキュベータ内に進入する可能性を減少し、さらにインキュベータ
内の環境安定性に及ぼす影響を低減させる。 In such a configuration, when the setter is extracted to the outside, the gas in the culture bin is controlled to enter the incubator by the one-way air outlet member. Therefore, in the process of extracting the setter, the culture bin is controlled. The gas in the culture bottle is preferentially returned to the incubator, and the amount of external air mixed in is small and large, and the external gas enters the incubator when the culture bottle is opened. It reduces the possibility of entry and further reduces the impact on environmental stability in the incubator.

一方、観察または操作が終了してから、セッターを培養ビン内へ戻り押す過程では、イン
キュベータ内の気体が、押出により気圧が大きくなり、インキュベータ内から培養ビン内
へ噴出するようになり、培養ビン内に存在した元の外部空気を外へ押し出す。これにより
、セッターがまったく培養ビン内へ戻ったら、培養ビン内に残る外部空気がわずかである
。インキュベータ内の環境安定に及ぼす影響をさらに減少し、気体濃度調節室にて気体濃
度を調整する時間を短縮し、インキュベータ内の環境がインフルエンスされる時間を減少
し、胚の生存率を向上する。 On the other hand, in the process of pushing the setter back into the culture bin after the observation or operation is completed, the air pressure in the incubator increases due to extrusion, and the gas in the incubator is ejected from the incubator into the culture bin. It pushes out the original external air that was inside. This leaves little external air in the culture bin once the setter has returned completely into the culture bin. Further reduce the effect on environmental stability in the incubator, shorten the time to adjust the gas concentration in the gas concentration control room, reduce the time that the environment in the incubator is influenced, and improve the survival rate of embryos. ..

前記一方向出気部材は、前記移動板に設けられる両円形開口と、前記円形開口から移動板
の後端面へ延伸して形成された第１コネクターと、および前記第１コネクターに設けられ
る一方向弁とを含む。 The one-way air outlet member includes both circular openings provided in the moving plate, a first connector formed by extending from the circular opening to the rear end surface of the moving plate, and one direction provided in the first connector. Including valves.

前記一方向進気部材は、前記移動板に設けられる両貫通穴と、前記貫通穴から移動板の前
端面へ延伸して形成された第２コネクターと、および前記第２コネクターに設けられる一
方向弁とを含む。
また、上記のような構成は、簡単で、実施されやすく、加工効率も高い。 The unidirectional air advance member includes both through holes provided in the moving plate, a second connector formed by extending from the through holes to the front end surface of the moving plate, and one direction provided in the second connector. Including valves.
Further, the above configuration is simple, easy to carry out, and has high processing efficiency.

よりに好ましくは、前記培地セッターは、横断面がＵ型状とするフレームと、前記フレー
ムの一端に固定接続される蓋板と、および前記蓋板に設けられる引手とを含む。 More preferably, the medium setter includes a frame having a U-shaped cross section, a lid plate fixedly connected to one end of the frame, and a pull tab provided on the lid plate.

前記接続構成は、前記培養ビンの入口端に設けられる固定座と、前記固定座に設けられ、
前記フレームが貫通する方形開口と、前記移動板に設けられ、前記フレームの先端が挿入
するＵ型溝と、前記フレームの先端に設けられ、間隔的に均一に配布される複数個のＵ型
凸片と、前記Ｕ型溝内に設けられ、前記凸片が挿入する開口溝と、前記フレームに固定設
置されるＬ型の挿接部材と、前記挿接部材の先端に設けられる三角形の挿接ヘッドと、前
記移動板に設けられ、前記挿接ヘッドが挿入する挿接溝と、前記挿接溝内に左右に移動可
能に設けられる限位ブロックと、前記限位ブロックの側壁と挿接溝の内壁の間に設けられ
るバネと、および前記限位ブロック上に設けられ、前記挿接ヘッドと嵌合される第１傾斜
面および第２傾斜面とを含む。 The connection configuration is provided in a fixed seat provided at the inlet end of the culture bottle and in the fixed seat.
A square opening through which the frame penetrates, a U-shaped groove provided in the moving plate into which the tip of the frame is inserted, and a plurality of U-shaped protrusions provided in the tip of the frame and uniformly distributed at intervals. A piece, an opening groove provided in the U-shaped groove into which the convex piece is inserted, an L-shaped insertion member fixedly installed in the frame, and a triangular insertion provided at the tip of the insertion member. A head, an insertion groove provided on the moving plate and into which the insertion head is inserted, a limiting block provided so as to be movable left and right in the insertion groove, and a side wall and an insertion groove of the limiting block. Includes a spring provided between the inner walls of the frame and a first inclined surface and a second inclined surface provided on the limiting block and fitted with the insertion head.

前記バネはその一端が限位ブロックに固定接続され、その他端が挿接溝の内壁に固定接続
される。 One end of the spring is fixedly connected to the limiting block, and the other end is fixedly connected to the inner wall of the insertion groove.

前記第１傾斜面が限位ブロックの前端面に位置され、前記第２傾斜面が限位ブロックの後
端面に位置され、且つ該第１傾斜面の傾斜角度が第２傾斜面の傾斜角度より大きくされる
。 The first inclined surface is located on the front end surface of the limiting block, the second inclined surface is located on the rear end surface of the limiting block, and the inclination angle of the first inclined surface is larger than the inclination angle of the second inclined surface. Be enlarged.

上記のような構成において、フレームが培養ビン内に挿入すると、フレームの端部がＵ型
溝内まで挿入するとともに、移動板を後向きに推移する。それに連動しては、移動板をも
っと後向きに動けない位置まで移動させると、フレームによる後向きの推力により、フレ
ームの端部を駆動してより裏側にＵ型溝内まで挿入させる。これにより凸片を開口溝内ま
で挿入させ、挿接ヘッドが第１傾斜面に沿って挿接溝内へ滑る。バネの弾性力で限位ブロ
ックが後方向に押される。これにより挿接ヘッドを挿接溝内まで挿入させ、その後限位ブ
ロックがバネの作用で元位置へ跳ね返される。 In the above configuration, when the frame is inserted into the culture bin, the end of the frame is inserted into the U-shaped groove and the moving plate is moved backward. In conjunction with this, when the moving plate is moved to a position where it cannot move backward, the rearward thrust of the frame drives the end of the frame to insert it further into the U-shaped groove on the back side. As a result, the convex piece is inserted into the opening groove, and the insertion head slides into the insertion groove along the first inclined surface. The elastic force of the spring pushes the limiting block backward. As a result, the insertion head is inserted into the insertion groove, and then the limiting block is bounced back to its original position by the action of the spring.

そして、フレームが外へ抽出されると、カムと開口溝の間、および挿接ヘッドと限位ブロ
ックの間によりフレームと移動板の間の禁固接続を回避することにより、移動板上の封止
部材と培養ビンの内壁の間は封止嵌合により大きい摩擦力を有する場合でも、フレームと
移動板とが分離しないように確保でき、フレームに連動して移動板を外へ移動させること
を保証する。 Then, when the frame is extracted to the outside, the sealing member on the moving plate is avoided by avoiding the imprisoned connection between the frame and the moving plate between the cam and the opening groove and between the insertion head and the limiting block. It is possible to ensure that the frame and the moving plate are not separated from each other even when the inner wall of the culture bottle has a larger frictional force for the sealing fitting, and it is guaranteed that the moving plate is moved to the outside in conjunction with the frame.

また、第２傾斜面の斜度が第１傾斜面の斜度より小さくすることにより、一般的に引かれ
るとすれば、挿接ヘッドが第２傾斜面に沿って滑り、限位ブロックを押して後方向に移動
させるのが、第１傾斜面の場合よりかなり難しい。これで、第２傾斜面により、挿接ヘッ
ドを快速に挿接溝内に挿入することができるとともに、挿接溝内から容易に抜き出されな
いように確保する上で、挿接ヘッドが限位ブロックを押し移動させることを確保できる。
それによってさらに挿接ヘッドが自動的に外へ抜き出されることも実現できる。別途に限
位ブロックを移動駆動して挿接ヘッドを外へ抜き出す可能な構成を設ける必要がない。 Further, if the inclination of the second inclined surface is made smaller than the inclination of the first inclined surface, and if it is generally pulled, the insertion head slides along the second inclined surface and pushes the limiting block. It is much more difficult to move backwards than in the case of the first inclined surface. Now, the second inclined surface allows the insertion head to be quickly inserted into the insertion groove, and the insertion head is limited in order to ensure that it is not easily pulled out from the insertion groove. You can ensure that the block is pushed and moved.
As a result, the insertion head can be automatically pulled out. It is not necessary to separately provide a configuration capable of moving and driving the limiting block to pull out the insertion head.

また固定座を設けて、フレームの挿入過程では、フレームに対する支持機能を発揮する。
挿入は、フレームの先端もすでに直接に一部がＵ型溝内まで挿入したのを意味するため、
移動板と固定座によりフレームに対する両点の支持をなす。これにより、フレームが安定
的に培養ビン内まで挿入することができ、培養ビンの内壁に案内軌道や滑り軌道などの構
成を設けてフレームを位置付けて案内する必要がなく、培養ビンの内壁の平坦度を確保し
、移動板と培養ビンの間のより適宜な封止嵌合を保証する。 In addition, a fixed seat is provided to exert a support function for the frame in the process of inserting the frame.
Insertion means that the tip of the frame has already been partially inserted directly into the U-shaped groove.
A moving plate and a fixed seat support both points with respect to the frame. As a result, the frame can be stably inserted into the culture bottle, and it is not necessary to provide a configuration such as a guide trajectory or a sliding track on the inner wall of the culture bottle to position and guide the frame, and the inner wall of the culture bottle is flat. Ensure a degree and ensure a more adequate sealing fit between the moving plate and the culture bin.

さらに、固定座を設けることにより、フレームを培養ビン内に挿入した後、架空状態と維
持できるので、フレームの各部位における環境状態がより均一的かつ安定的になり、フレ
ーム上の胚の保存により有益となる。 Furthermore, by providing a fixed locus, the frame can be maintained in a fictitious state after being inserted into the culture bin, so that the environmental state at each part of the frame becomes more uniform and stable, and the preservation of embryos on the frame makes it possible. It will be beneficial.

前記フレームが培養ビン内まで挿入すると、該フレームの下表面と培養ビンの底面の間に
間隔が残り、該フレームに間隔的に複数個の導気穴が均一に配布される。導気穴により、
フレームに仕切られる上下のチャンバーを相互に導通させることができるように確保でき
、且つ気体の流れがはやくなり、培養ビン内の環境がはやめに安定状態となることを確保
でき、調節の効率が高い。 When the frame is inserted into the culture bottle, a gap remains between the lower surface of the frame and the bottom surface of the culture bottle, and a plurality of air guide holes are uniformly distributed in the frame at intervals. Due to the air guide hole
It is possible to secure the upper and lower chambers partitioned by the frame so that they can be electrically connected to each other, and it is possible to secure that the gas flow becomes fast and the environment in the culture bottle becomes stable, and the adjustment efficiency is high. ..

より好ましくは、前記導通部材は、前記移動板に長さ方向に沿って間隔的に配布される複
数個の長尺状開口と、前記移動板の後側に設けられ且つ移動板に対して往復移動可能な移
動部材と、前記移動部材に設けられ、移動板に長さ方向に沿って間隔的に配布される複数
個の長尺状導通口と、前記移動板に設けられ、移動部材が移動する滑り軌道と、前記移動
部材と嵌合される復位ユニットと、および前記培養ビン内に固定設置される駆動部とを含
む。 More preferably, the conductive member is provided on the rear side of the moving plate and reciprocates with respect to the moving plate and a plurality of elongated openings distributed to the moving plate at intervals along the length direction. A movable moving member, a plurality of elongated conduction ports provided on the moving member and distributed to the moving plate at intervals along the length direction, and a plurality of elongated conduction ports provided on the moving plate to move the moving member. Includes a sliding track, a repositioning unit fitted with the moving member, and a drive unit fixedly installed in the culture bin.

前記フレームが移動板を推移して最後端位置まで移動させると、前記駆動部が移動部材を
駆動部から離れる位置へ移動させるように駆動することにより、前記長尺状開口と長尺状
導通口の間の連通を実現する。 When the frame moves the moving plate to the rearmost position, the driving unit drives the moving member to move away from the driving unit, thereby causing the elongated opening and the elongated conduction port. Achieve communication between.

このような構成においては、フレームを駆動部が位置する位置まで後向きに推移させると
、駆動部によりフレームの後向き移動付勢を制限する。さらに、フレームに加えた作用力
で移動部材を駆動件の傾斜面に沿って滑動させるとともに、復位ユニットの作用に抵抗し
て後向きに移動させる。これにより、長尺状開口と長尺状導通口を相互に重ねるようにな
り、培養ビンと気体濃度調節室の連通を実現して、自動的な連通を実現する。効率が高く
て、培養ビン内の環境調節がはやくなる。 In such a configuration, when the frame is moved backward to the position where the drive unit is located, the drive unit limits the backward movement bias of the frame. Further, the acting force applied to the frame causes the moving member to slide along the inclined surface of the driving subject, and moves backward against the action of the repositioning unit. As a result, the long opening and the long conduction port are overlapped with each other, and the culture bottle and the gas concentration control chamber are communicated with each other to realize automatic communication. The efficiency is high and the environment in the culture bottle can be adjusted quickly.

そして、フレームが外へ移動する過程では、移動部材が復位部材の作用で快速復位を実現
する。これにより、移動板の隔離効果を保証する。 Then, in the process of moving the frame to the outside, the moving member realizes rapid repositioning by the action of the repositioning member. This guarantees the isolation effect of the moving plate.

また、移動板に設けられるのが、複数個の長尺状導通口となるので、導通後の面積が大き
く、培養ビン内と気体濃度調節室内の気体の混合がはやくなり、さらに培養ビン内におけ
る気体濃度を所定状態に向上させる速度を向上し、規格を満たさない環境での胚の滞在時
間を減少し、さらに胚の生存率を向上する。 In addition, since a plurality of long conduction ports are provided on the moving plate, the area after conduction is large, the gas in the culture bottle and the gas in the gas concentration control chamber are mixed quickly, and further in the culture bottle. It improves the rate at which the gas concentration is improved to a predetermined state, reduces the residence time of embryos in a non-standard environment, and further improves the survival rate of embryos.

前記気体濃度調節室内に気体均一度を調節するための循環手段が設けられ、該循環手段は
、前記気体濃度調節室内に設けられる第１仕切板および第２仕切板と、前記第１仕切板に
設けられる複数個の扇風機と、および前記第２仕切板に設けられ、気流を培養ビン内へ案
内するドレイン部材とを含み、前記第１仕切板と第２仕切板が相互に接続され、該第１仕
切板と第２仕切板により気体濃度調節室を第１チャンバーと第２チャンバーに仕切り、該
第１チャンバーが前記培養ビンに連通する。 A circulation means for adjusting the gas uniformity is provided in the gas concentration adjusting chamber, and the circulation means is provided in the first partition plate and the second partition plate provided in the gas concentration adjusting chamber, and the first partition plate. The first partition plate and the second partition plate are connected to each other and include a plurality of electric fans provided and a drain member provided on the second partition plate to guide the air flow into the culture bin. The gas concentration control chamber is divided into a first chamber and a second chamber by a partition plate and a second partition plate, and the first chamber communicates with the culture bin.

このような構成においては、はやめにインキュベータ内の気体濃度が、安定的で均一な状
態になることができるように保証する。調整が快速である。 In such a configuration, it is ensured that the gas concentration in the incubator can be stable and uniform. The adjustment is fast.

またドレイン部材の案内により、培養ビン内の気体の快速な混合をはやめに実現できる。
培養ビン内ではキャビティが小いためにその内の気体が流れ難い状況を回避する。培養ビ
ン内の気体も気体濃度調節室内の気体と混合できるように保証する。これにより、全体な
環境を調節する。 In addition, by guiding the drain member, it is possible to quickly mix the gas in the culture bottle.
Avoid the situation where the gas in the culture bottle is difficult to flow due to the small cavity. Guarantee that the gas in the culture bottle can also be mixed with the gas in the gas concentration control chamber. This regulates the overall environment.

本発明にかかる培養過程におけるインキュベータ内の各培養ビンが、独立に配置したもの
で、且つ培養ビン内のセッターが外へ抽出されるときに、隔離手段で隔離されると、開い
た培養ビンから外部空気が気体濃度調節室の全体内へ進入するおそれがない。これによっ
ても、他位置の培養ビンが外部環境にインフルエンスされることを回避する。このため、
インキュベータ内の環境の安定性を向上するとともに、胚の生存率を確保する。 When each culture bottle in the incubator in the culture process according to the present invention is independently arranged and the setter in the culture bottle is extracted to the outside, if it is isolated by the isolation means, the open culture bottle is released. There is no risk of outside air entering the entire gas concentration control chamber. This also prevents the culture bottles at other positions from being influenced by the external environment. For this reason,
Improve the stability of the environment in the incubator and ensure embryo viability.

図１は本発明の構成模式図である。FIG. 1 is a schematic configuration diagram of the present invention. 図２は本発明の正面図である。FIG. 2 is a front view of the present invention. 図３は同図２におけるＡ-Ａ線に沿う断面図である。FIG. 3 is a cross-sectional view taken along the line AA in FIG. 図４は本発明にかかるセッターと隔離手段の一の嵌合模式図である。FIG. 4 is a schematic view of fitting one of the setter and the isolation means according to the present invention. 図５は本発明にかかるセッターと隔離手段の二の嵌合模式図であるｙ。FIG. 5 is a schematic view of fitting the setter and the isolation means according to the present invention. 図６は同図５の正面図である。FIG. 6 is a front view of FIG. 5. 図７は同図６におけるＢ‐Ｂ線に沿う断面図である。FIG. 7 is a cross-sectional view taken along the line BB in FIG. 図８は同図７における部分拡大図である。FIG. 8 is a partially enlarged view of FIG. 7. 図９は同図８における拡大図である。FIG. 9 is an enlarged view of FIG. 図１０は本発明にかかる気体濃度調節室の縦方向断面模式図である。FIG. 10 is a schematic vertical cross-sectional view of the gas concentration adjusting chamber according to the present invention. 図１１は本発明にかかる気体濃度調節室の横方向断面模式図である。FIG. 11 is a schematic cross-sectional view of the gas concentration adjusting chamber according to the present invention.

実施例１
哺乳動物胚のインビボ生存率を向上する培養方法を提供する。以下の工程を含む。
（１） 雌ヒツジからヒツジ卵母細胞を取り、成熟培養液内に置き、インキュベータ内で
２０〜２４時間培養し、成熟したヒツジ卵母細胞を得ること。ただし、成熟培養液は従来
の技術であり、実際の要求により選択され得るもので、ここで詳細な紹介を略す。
（２） 成熟したヒツジ卵母細胞を***とともに受精培養液中に置き、インキュベータ内
で１８〜２４時間培養し、受精させ、ヒツジ初期胚を得ること。ただし、受精培養液は３
％のヒツジ血清を含むＴＣＭ１９９培養液とする。
（３） 受精後のヒツジ胚をＴＣＭ１９９培養液にて繰返してフラッシュ洗い、その後ヒ
ツジ胚のインビボ培養液中に置き、そしてインキュベータ中に置いて胚のインビボ培養を
行い、該インキュベータ内の温度を３８〜３９℃に維持し、気体濃度を５％ＣＯ２に維持
すること。ただし、前記培養液はＴＣＭ１９９培養液とする。 Example 1
Provided is a culture method for improving the in vivo viability of mammalian embryos. The following steps are included.
(1) Take sheep oocytes from female sheep, place them in a mature culture medium, and incubate them in an incubator for 20 to 24 hours to obtain mature sheep oocytes. However, the mature culture medium is a conventional technique and can be selected according to actual requirements, and detailed introduction is omitted here.
(2) Mature sheep oocytes are placed in a fertilization culture medium together with sperm, cultured in an incubator for 18 to 24 hours, and fertilized to obtain early sheep embryos. However, the fertilization culture solution is 3
A TCM199 culture medium containing% sheep serum.
(3) The fertilized sheep embryos were repeatedly flush-washed with TCM199 culture medium, then placed in the incubator of the sheep embryos, and then placed in the incubator for in vivo culture of the embryos, and the temperature in the incubator was 38. Maintain at ~ 39 ° C. and maintain gas concentration at 5% CO2. However, the culture solution is TCM199 culture solution.

図１〜１１に示すように、前記各工程に記載されるインキュベータは、同種類のインキュ
ベータである。該インキュベータは、培養室１と、該培養室と嵌合されるドア２と、該培
養室と連通される気体濃度調節室３とを含む。
ドア２が培養室に対して枢動でき、好ましくは、前記ドア２に複数個のビンドア２１が配
布され、複数個のビンドア２１の位置がそれぞれ複数個の培養ビン１１に対応して設置さ
れる。これにより単一の培養ビン内の胚だけを観察または操作する際には、対応位置のビ
ンドア２１を開ければよく、ドア２全体を開く必要がなく、インキュベータ内部の熱エネ
ルギーの過度損失を避ける。 As shown in FIGS. 1 to 11, the incubators described in each of the steps are the same type of incubator. The incubator includes a culture chamber 1, a door 2 fitted with the culture chamber, and a gas concentration control chamber 3 communicating with the culture chamber.
The door 2 can be pivoted with respect to the culture chamber, preferably a plurality of bin doors 21 are distributed to the door 2, and the positions of the plurality of bin doors 21 are installed corresponding to the plurality of culture bins 11, respectively. .. As a result, when observing or manipulating only embryos in a single culture bottle, it is sufficient to open the bin door 21 at the corresponding position, and it is not necessary to open the entire door 2 to avoid excessive loss of heat energy inside the incubator.

前記培養室１内に複数個の培養ビン１１が備えられ、複数個の前記培養ビン内にそれぞれ
培地セッター４と隔離手段が設けられ、前記培地セッター４が培養ビン１１内まで挿入可
能で、且つ挿入後に培地セッター４と培養ビン１１の入口が封止嵌合となる。 A plurality of culture bins 11 are provided in the culture chamber 1, a medium setter 4 and an isolation means are provided in each of the plurality of culture bins, and the culture medium setter 4 can be inserted into the culture bin 11 and can be inserted into the culture bin 11. After insertion, the inlets of the medium setter 4 and the culture bin 11 are sealed and fitted.

前記培養の過程では、インキュベータ内の各位置の胚を観察する場合、操作者により培地
セッター４を独立の培養ビン１１中から抽出することができ、且つ前記培地セッター４が
抽出される過程では、前記隔離手段により相応の培養ビン１１と外部との連通を隔離する
。 In the process of culturing, when observing embryos at each position in the incubator, the medium setter 4 can be extracted from the independent culture bin 11 by the operator, and in the process of extracting the medium setter 4, the medium setter 4 is extracted. The isolation means isolates the communication between the corresponding culture bin 11 and the outside.

前記培養室１の内部は中空状に設置されて、前記培養ビンをまわって設けられる水浴チャ
ンバー１２を形成する。該水浴チャンバー内にお湯を充填して、培養ビンを保温する。 The inside of the culture chamber 1 is installed in a hollow shape to form a water bath chamber 12 provided around the culture bottle. The water bath chamber is filled with hot water to keep the culture bottle warm.

水浴チャンバー１２のお湯は、注入するとしてもよく、水浴チャンバー１２内の加熱管に
より加熱され取得するとしてもよい。その動作原理は従来技術のもので、ここで詳細な説
明を略す。好ましくは、培養室１外にさらに温度隔離カバー１３が覆われており、温度隔
離カバーと培養室１の間のキャビティは、バキュームまたは防温綿充填としてもよい。 The hot water in the water bath chamber 12 may be injected, or may be heated and obtained by a heating pipe in the water bath chamber 12. Its operating principle is that of the prior art, and detailed description is omitted here. Preferably, the temperature isolation cover 13 is further covered outside the culture chamber 1, and the cavity between the temperature isolation cover and the culture chamber 1 may be filled with vacuum or heat-insulating cotton.

具体的には、前記隔離手段は、前記培養ビン内に前後に動作可能に設けられる移動板５１
と、前記移動板の外側壁に設けられ、前記培養ビンの内壁に封止嵌合される封止リング５
２と、前記移動板に設けられる一方向出気部材および一方向進気部材、導通部材と、およ
び培地セッターと移動板の間の接続嵌合を実現するための接続構成とを含む。 Specifically, the isolation means is provided in the culture bin so as to be movable back and forth.
A sealing ring 5 provided on the outer wall of the moving plate and sealed and fitted to the inner wall of the culture bottle.
2. Includes a unidirectional air outlet member, a unidirectional air advancing member, a conductive member provided on the moving plate, and a connection configuration for realizing a connection fitting between the medium setter and the moving plate.

前記培地セッター４が培養ビン１１内に挿入すると、前記培地セッター４が移動板５１に
抵触しながら後向きに移動するとともに、前記一方向進気部材により気体濃度調節室内の
気体を一方向に培養ビン内まで進入するように制御する。 When the medium setter 4 is inserted into the culture bin 11, the medium setter 4 moves backward while colliding with the moving plate 51, and the gas in the gas concentration adjusting chamber is unidirectionally cultured by the unidirectional air advance member. Control to enter inside.

前記移動板５１が最後端位置まで移動すると、前記接続構成により培地セッター４と移動
板５１の間の接続嵌合を実現するとともに、前記導通部材により培養ビン１１と気体濃度
調節室３の直接連通を実現する。 When the moving plate 51 moves to the rearmost position, the connection configuration realizes connection fitting between the medium setter 4 and the moving plate 51, and the conduction member directly communicates the culture bin 11 with the gas concentration adjusting chamber 3. To realize.

前記培養ビン１１内の培養状況を観察するや取得を実施する場合、前記培地セッター４を
外へ引き取り、前記接続構成により培地セッター４が封止部材５２と培養ビン１１の間の
摩擦力に抵抗しながら、移動板５１が連動して前向きに移動するとともに、前記一方向出
気部材により培養ビン１１内の気体を一方向に気体濃度調節室３内まで進入するように制
御する。 When observing or acquiring the culture state in the culture bin 11, the medium setter 4 is taken out, and the medium setter 4 resists the frictional force between the sealing member 52 and the culture bin 11 due to the connection configuration. At the same time, the moving plate 51 moves forward in conjunction with the movement plate 51, and the unidirectional air outlet member controls the gas in the culture bin 11 to enter the gas concentration adjusting chamber 3 in one direction.

上記のように言及された封止部材は、ゴム製とし、移動板に嵌設けられる。言及された一
方向弁は、市販されるゴム製のアヒル口弁とし、その構成および動作原理の説明はここで
略される。 The sealing member mentioned above is made of rubber and is fitted to the moving plate. The one-way valve mentioned is a commercially available rubber duck mouth valve, and the description of its configuration and operating principle is omitted here.

前記培地セッター４は、横断面がＵ型状とするフレーム４１と、前記フレームの一端に固
定接続される蓋板４２と、および前記蓋板に設けられる引手４３とを含む。 The culture medium setter 4 includes a frame 41 having a U-shaped cross section, a lid plate 42 fixedly connected to one end of the frame, and a pull tab 43 provided on the lid plate.

上記のように言及された接続構成は、具体的に、前記培養ビンの入口端に設けられる固定
座７１と、前記固定座に設けられ、前記フレームが貫通する方形開口７２と、前記移動板
に設けられ、前記フレームの先端が挿入するＵ型溝７３と、前記フレームの先端に設けら
れ、間隔的に均一に配布される複数個のＵ型凸片７４と、前記Ｕ型溝内に設けられ、前記
凸片が挿入する開口溝と、前記フレームに固定設置されるＬ型の挿接部材７５と、前記挿
接部材の先端に設けられる三角形の挿接ヘッド７６と、前記移動板に設けられ、前記挿接
ヘッドが挿入する挿接溝７７と、前記挿接溝内に左右に移動可能に設けられる限位ブロッ
ク７８と、前記限位ブロックの側壁と挿接溝の内壁の間に設けられるバネ７９と、および
前記限位ブロック上に設けられ、前記挿接ヘッドと嵌合される第１傾斜面７６１および第
２傾斜面７６２とを含む。 Specifically, the connection configuration referred to as described above includes a fixed seat 71 provided at the inlet end of the culture bottle, a square opening 72 provided in the fixed seat through which the frame penetrates, and the moving plate. A U-shaped groove 73 provided and inserted into the tip of the frame, a plurality of U-shaped convex pieces 74 provided at the tip of the frame and evenly distributed at intervals, and provided in the U-shaped groove. , An opening groove into which the convex piece is inserted, an L-shaped insertion member 75 fixedly installed on the frame, a triangular insertion head 76 provided at the tip of the insertion member, and the moving plate. , The insertion groove 77 into which the insertion head is inserted, the limiting block 78 provided so as to be movable left and right in the insertion groove, and the side wall of the limiting block and the inner wall of the insertion groove. It includes a spring 79 and a first inclined surface 761 and a second inclined surface 762 provided on the limiting block and fitted with the insertion head.

前記バネはその一端が限位ブロックに固定接続され、その他端が挿接溝の内壁に固定接続
される。 One end of the spring is fixedly connected to the limiting block, and the other end is fixedly connected to the inner wall of the insertion groove.

前記第１傾斜面７６１が限位ブロックの前端面に位置され、前記第２傾斜面７６２が限位
ブロックの後端面に位置され、且つ該第１傾斜面７６１の傾斜角度が第２傾斜面７６２の
傾斜角度より大きくされる。上記のように言及された「前」、「後」はいずれもインキュ
ベータ全体の方向をもとに定義される。インキュベータにおいて、ドアの方向が前とし、
気体濃度調節室の方向が後とする。 The first inclined surface 761 is located on the front end surface of the limiting block, the second inclined surface 762 is located on the rear end surface of the limiting block, and the inclination angle of the first inclined surface 761 is the second inclined surface 762. Is made larger than the tilt angle of. Both "before" and "after" mentioned above are defined based on the direction of the entire incubator. In the incubator, the direction of the door is forward
The direction of the gas concentration control chamber is behind.

前記固定座が板体構成とする。前記フレーム４１が培養ビン１１内まで挿入すると、該フ
レーム４１の下表面と培養ビン１１の底面の間に間隔が残り、該フレーム４１に間隔的に
複数個の導気穴４１１が均一に配布される。 The fixed seat has a plate structure. When the frame 41 is inserted into the culture bin 11, an interval remains between the lower surface of the frame 41 and the bottom surface of the culture bin 11, and a plurality of air guide holes 411 are uniformly distributed to the frame 41 at intervals. To.

具体的には、前記導通部材は、前記移動板に長さ方向に沿って間隔的に配布される複数個
の長尺状開口８１と、前記移動板の後側に設けられ且つ移動板に対して往復移動可能な移
動部材８２と、前記移動部材に設けられ、移動板に長さ方向に沿って間隔的に配布される
複数個の長尺状導通口８３と、前記移動板に設けられ、移動部材が移動する滑り軌道と、
前記移動部材と嵌合される復位ユニットと、および前記培養ビン内に固定設置される駆動
部８５とを含む。 Specifically, the conductive member is provided on the moving plate with a plurality of elongated openings 81 periodically distributed along the length direction, and on the rear side of the moving plate and with respect to the moving plate. A moving member 82 that can be reciprocated, a plurality of elongated conduction ports 83 that are provided on the moving member and are distributed to the moving plate at intervals along the length direction, and a plurality of elongated conduction ports 83 that are provided on the moving plate. The sliding trajectory on which the moving member moves and
It includes a repositioning unit fitted with the moving member and a drive unit 85 fixedly installed in the culture bin.

前記フレーム４１が移動板５１を推移して最後端位置まで移動させると、前記駆動部８５
が移動部材８２を駆動部から離れる位置へ移動させるように駆動することにより、前記長
尺状開口８１と長尺状導通口８３の間の連通を実現する。 When the frame 41 moves the moving plate 51 to the rearmost end position, the driving unit 85
Drives the moving member 82 so as to move it away from the drive unit, thereby realizing communication between the elongated opening 81 and the elongated conduction port 83.

その中、当該移動部材は板体構成とする。その駆動部寄りの一端に、外へ延出する凸部が
設けられる。前記駆動部は培養室の内壁に固定設置される凸ブロックである。且つ、凸ブ
ロックの一側面が弧状構成とされる。前記移動部材の下端が滑り軌道内まで挿入し、滑り
軌道により移動部材の変位に対して位置付けおよび案内をする。前記復位ユニットは移動
部材の左右両端に接続されるバネであり、且つバネはいずれも移動板に相互に連続され、
移動部材が移動した後、をれを自動復位させる。 Among them, the moving member has a plate structure. A convex portion extending outward is provided at one end near the drive portion. The driving unit is a convex block fixedly installed on the inner wall of the culture chamber. Moreover, one side surface of the convex block has an arcuate structure. The lower end of the moving member is inserted into the sliding track, and the sliding track positions and guides the displacement of the moving member. The repositioning unit is a spring connected to both left and right ends of the moving member, and all the springs are continuously connected to the moving plate.
After the moving member has moved, it is automatically repositioned.

前記気体濃度調節室３内に気体均一度を調節するための循環手段が設けられ、該循環手段
は、前記気体濃度調節室内に設けられる第１仕切板９１および第２仕切板９２と、前記第
１仕切板に設けられる複数個の扇風機９３と、および前記第２仕切板に設けられ、気流を
培養ビン内へ案内するドレイン部材とを含み、前記第１仕切板９１と第２仕切板９２が相
互に接続され、該第１仕切板９１と第２仕切板９２により気体濃度調節室３を第１チャン
バー３１と第２チャンバー３２に仕切り、該第１チャンバー３１が前記培養ビン１１に連
通する。 A circulation means for adjusting the gas uniformity is provided in the gas concentration adjusting chamber 3, and the circulation means includes a first partition plate 91 and a second partition plate 92 provided in the gas concentration adjusting chamber, and the first partition plate 91. The first partition plate 91 and the second partition plate 92 include a plurality of fans 93 provided on the first partition plate and a drain member provided on the second partition plate to guide the air flow into the culture bin. The gas concentration adjusting chamber 3 is divided into the first chamber 31 and the second chamber 32 by the first partition plate 91 and the second partition plate 92, and the first chamber 31 communicates with the culture bin 11.

具体的には、複数セットのドレイン部材を設けている。それぞれが上下の両培養ビンの間
の位置に対応して設置される。該ドレイン部材は複数個のドレイン開口９４と、ドレイン
開口に対して上下枢動可能なドレイン板９５と、およびドレイン板内を貫通して設置され
る引きロッドまたは引き縄９６とを含む。前記培養ビン１１の先端位置に隔離板１１１が
設けられ、該隔離板１１１が培養ビン１１の中間レベルに位置される。その中、扇風機が
小型のモータに接続される。第１チャンバーの上部にプーリが設けられる。引き縄がプー
リに巻き付けられ、プーリがモータに接続され、モータに連動してプーリが所定方向に回
動すれば、引き縄を上向きまたは下向きに移動させることができ、ひいては、ドレイン板
が連動して角度変動する。その動作原理は、窓シャッターと同理であり、詳細な説明を略
させる。ドレイン板の位置が両隔離板の間に対応して位置されるので、ドレイン板を介し
て培養ビンへ流れる風が隔離板により阻害されることにより、培養ビンの一半部開口だけ
から培養ビン内に進入する。培養ビンに元々存在した気体が他半部開口から外へ排出され
ることにより、培養ビン内における気体を順調に流通させることを保証する。これにより
、気体濃度調節室内の気体全体が均一に混合する。ドレイン板が上下に枢動可能であるの
で、一つのドレイン板で上下両培養ビン内の気体流通を実現することができ、これにより
、構成がより簡単で、効果がより高い。 Specifically, a plurality of sets of drain members are provided. Each is installed corresponding to the position between the upper and lower culture bottles. The drain member includes a plurality of drain openings 94, a drain plate 95 that can be pivotally moved up and down with respect to the drain opening, and a pull rod or rope 96 that is installed through the drain plate. An isolation plate 111 is provided at the tip of the culture bin 11, and the isolation plate 111 is located at an intermediate level of the culture bin 11. Among them, the electric fan is connected to a small motor. A pulley is provided at the top of the first chamber. If the tow rope is wound around the pulley, the pulley is connected to the motor, and the pulley rotates in a predetermined direction in conjunction with the motor, the tow rope can be moved upward or downward, and the drain plate is interlocked. The angle fluctuates. Its operating principle is the same as that of a window shutter, and detailed explanation is omitted. Since the position of the drain plate is located between the two isolation plates, the wind flowing to the culture bin through the drain plate is blocked by the isolation plate, so that the culture bin enters the culture bottle only through a half opening of the culture bin. To do. By discharging the gas originally present in the culture bottle to the outside through the opening of the other half, the gas in the culture bottle is ensured to flow smoothly. As a result, the entire gas in the gas concentration adjusting chamber is uniformly mixed. Since the drain plate can be pivoted up and down, one drain plate can realize gas flow in both the upper and lower culture bottles, which makes the configuration simpler and more effective.

もちろん、気体濃度調節室内に熱伝導センサーが設けられ、さらに気体濃度調節室にその
内に二酸化炭素を充填するための充気管が接続される。具体的な感知原理および充気原理
はいずれも従来技術であり、伝統のインキュベータと同様で、ここでその詳細な説明を略
す。 Of course, a heat conduction sensor is provided in the gas concentration control chamber, and an air filling tube for filling carbon dioxide in the gas concentration control chamber is connected. The specific sensing principle and the filling principle are both conventional techniques and are similar to those of the traditional incubator, and detailed description thereof will be omitted here.

実施例２
哺乳動物胚のインビボ生存率を向上する培養方法を提供する。以下の工程を含む。
（１） 雌ヒツジからヒツジ卵母細胞を取り、成熟培養液内に置き、インキュベータ内で
２４時間培養し、成熟したヒツジ卵母細胞を得ること。ただし、成熟培養液は従来の技術
であり、実際の要求により選択され得るもので、ここで詳細な紹介を略す。
（２） 成熟したヒツジ卵母細胞を***とともに受精培養液中に置き、インキュベータ内
で２４時間培養し、受精させ、ヒツジ初期胚を得ること。該インキュベータ内の温度を３
９℃に維持し、気体濃度を５％ＣＯ２に維持する。ただし、受精培養液は３％のヒツジ血
清を含むＴＣＭ１９９培養液とする。
（３） 受精後のヒツジ胚をＴＣＭ１９９培養液にて繰返してフラッシュ洗い、その後ヒ
ツジ胚のインビボ培養液中に置き、そしてインキュベータ中に置いて胚のインビボ培養を
行い、該インキュベータ内の温度を３９℃に維持し、気体濃度を５％ＣＯ２に維持するこ
と。ただし、前記培養液はＴＣＭ１９９培養液とする。 Example 2
Provided is a culture method for improving the in vivo viability of mammalian embryos. The following steps are included.
(1) Take sheep oocytes from female sheep, place them in a mature culture medium, and incubate them in an incubator for 24 hours to obtain mature sheep oocytes. However, the mature culture medium is a conventional technique and can be selected according to actual requirements, and detailed introduction is omitted here.
(2) Mature sheep oocytes are placed in a fertilization culture medium together with sperm, cultured in an incubator for 24 hours, and fertilized to obtain early sheep embryos. The temperature in the incubator is 3
Maintain at 9 ° C. and maintain gas concentration at 5% CO2. However, the fertilization culture medium is a TCM199 culture medium containing 3% sheep serum.
(3) The fertilized sheep embryo was repeatedly flush-washed with TCM199 culture medium, then placed in the incubator of the sheep embryo, and then placed in the incubator for in vivo culture of the embryo, and the temperature in the incubator was set to 39. Maintain at ° C and maintain gas concentration at 5% CO2. However, the culture solution is TCM199 culture solution.

実施例３
哺乳動物胚のインビボ生存率を向上する培養方法を提供する。以下の工程を含む。
（１） 雌ヒツジからヒツジ卵母細胞を取り、成熟培養液内に置き、インキュベータ内で
２２時間培養し、成熟したヒツジ卵母細胞を得ること。ただし、成熟培養液は従来の技術
であり、実際の要求により選択され得るもので、ここで詳細な紹介を略す。
（２） 成熟したヒツジ卵母細胞を***とともに受精培養液中に置き、インキュベータ内
で２０時間培養し、受精させ、ヒツジ初期胚を得ること。該インキュベータ内の温度を３
８℃に維持し、気体濃度を５％ＣＯ２に維持する。ただし、受精培養液は３％のヒツジ血
清を含むＴＣＭ１９９培養液とする。
（３） 受精後のヒツジ胚をＴＣＭ１９９培養液にて繰返してフラッシュ洗い、その後ヒ
ツジ胚のインビボ培養液中に置き、そしてインキュベータ中に置いて胚のインビボ培養を
行い、該インキュベータ内の温度を３８．５℃に維持し、気体濃度を５％ＣＯ２に維持す
ること。ただし、前記培養液はＴＣＭ１９９培養液とする。 Example 3
Provided is a culture method for improving the in vivo viability of mammalian embryos. The following steps are included.
(1) Take sheep oocytes from female sheep, place them in a mature culture medium, and incubate them in an incubator for 22 hours to obtain mature sheep oocytes. However, the mature culture medium is a conventional technique and can be selected according to actual requirements, and detailed introduction is omitted here.
(2) Mature sheep oocytes are placed in a fertilization culture medium together with sperm, cultured in an incubator for 20 hours, and fertilized to obtain early sheep embryos. The temperature in the incubator is 3
Maintain at 8 ° C. and maintain gas concentration at 5% CO2. However, the fertilization culture medium is a TCM199 culture medium containing 3% sheep serum.
(3) The fertilized sheep embryos were repeatedly flush-washed with TCM199 culture medium, then placed in the incubator of the sheep embryos, and then placed in the incubator for in vivo culture of the embryos, and the temperature in the incubator was 38. Maintain at .5 ° C and maintain gas concentration at 5% CO2. However, the culture solution is TCM199 culture solution.

Claims (3)

（１） 雌ヒツジからヒツジ卵母細胞を取り、成熟培養液内に置き、インキュベータ内で
２０〜２４時間培養し、成熟したヒツジ卵母細胞を得ること、
（２） 成熟したヒツジ卵母細胞を***とともに受精培養液中に置き、インキュベータ内
で１８〜２４時間培養し、受精させ、ヒツジ初期胚を得ること、
（３） 受精後のヒツジ胚をＴＣＭ１９９培養液にて繰返してフラッシュ洗い、その後ヒ
ツジ胚のインビボ培養液中に置き、そしてインキュベータ中に置いて胚のインビボ培養を
行い、該インキュベータ内の温度を３８〜３９℃に維持し、気体濃度を５％ＣＯ２に維持
すること、
ただし、
前記インキュベータは、培養室（１）と、該培養室と嵌合されるドア（２）と、該培養室
と連通される気体濃度調節室（３）とを含み、前記培養室（１）内に複数個の培養ビン（
１１）が備えられ、複数個の前記培養ビン内にそれぞれ培地セッター（４）と隔離手段が
設けられ、前記培地セッター（４）が培養ビン（１１）内まで挿入可能で、且つ挿入後に
培地セッター（４）と培養ビン（１１）の入口が封止嵌合となること、
前記培養の過程では、インキュベータ内の各位置の胚を観察する場合、操作者により培地
セッター（４）を独立の培養ビン（１１）中から抽出することができ、且つ前記培地セッ
ター（４）が抽出される過程では、前記隔離手段により相応の培養ビン（１１）と外部と
の連通を隔離すること、
前記（１）では、インキュベータ内の温度を３８〜３９℃に維持し、気体濃度を５％ＣＯ
２に維持すること、
前記（２）における受精培養液は３％のヒツジ血清を含むＴＣＭ１９９培養液とすること
、
を含み、
前記隔離手段は、前記培養ビン内に前後に動作可能に設けられる移動板（５１）と、前記
移動板の外側壁に設けられ、前記培養ビンの内壁に封止嵌合される封止リング（５２）と
、前記移動板に設けられる一方向出気部材および一方向進気部材、導通部材と、および培
地セッターと移動板の間の接続嵌合を実現するための接続構成とを含むこと、
前記培地セッター（４）が培養ビン（１１）内に挿入すると、前記培地セッター（４）が
移動板（５１）に抵触しながら後向きに移動するとともに、前記一方向進気部材により気
体濃度調節室内の気体を一方向に培養ビン内まで進入するように制御すること、
前記移動板（５１）が最後端位置まで移動すると、前記接続構成により培地セッター（４
）と移動板（５１）の間の接続嵌合を実現するとともに、前記導通部材により培養ビン（
１１）と気体濃度調節室（３）の直接連通を実現すること、
前記培養ビン（１１）内の培養状況を観察するや取得を実施する場合、前記培地セッター
（４）を外へ引き取り、前記接続構成により培地セッター（４）が封止部材（５２）と培
養ビン（１１）の間の摩擦力に抵抗しながら、移動板（５１）が連動して前向きに移動す
るとともに、前記一方向出気部材により培養ビン（１１）内の気体を一方向に気体濃度調
節室（３）内まで進入するように制御すること、
前記移動板（５１）が最前端位置まで移動すると、前記培地セッター（４）が培養ビン（
１１）の外へ延出し、且つ前記移動板（５１）により常に培養ビン（１１）の内部と外部
との連通を隔離すること、
前記一方向出気部材は、前記移動板に設けられる両円形開口（６１）と、前記円形開口か
ら移動板の後端面へ延伸して形成された第１コネクターと、および前記第１コネクターに
設けられる一方向弁（６３）とを含むこと、
前記一方向進気部材は、前記移動板に設けられる両貫通穴（６４）と、前記貫通穴から移
動板の前端面へ延伸して形成された第２コネクターと、および前記第２コネクターに設け
られる一方向弁（６３）とを含むこと、
を含むことを特徴とする哺乳動物胚のインビボ生存率を向上する培養方法。 (1) Take sheep oocytes from female sheep, place them in a mature culture medium, and incubate them in an incubator for 20 to 24 hours to obtain mature sheep oocytes.
(2) A mature sheep oocyte is placed in a fertilization culture medium together with sperm, cultured in an incubator for 18 to 24 hours, and fertilized to obtain an early sheep embryo.
(3) The fertilized sheep embryos were repeatedly flush-washed with TCM199 culture medium, then placed in the incubator of the sheep embryos, and then placed in the incubator for in vivo culture of the embryos, and the temperature in the incubator was 38. Maintaining ~ 39 ° C. and maintaining gas concentration at 5% CO2,
However,
The incubator includes a culture chamber (1), a door (2) fitted with the culture chamber, and a gas concentration control chamber (3) communicating with the culture chamber, and is inside the culture chamber (1). Multiple culture bottles (
11) is provided, and a medium setter (4) and an isolation means are provided in each of the plurality of culture bins, and the medium setter (4) can be inserted into the culture bottle (11), and the medium setter after insertion. The inlets of (4) and the culture bottle (11) are sealed and fitted.
In the process of culturing, when observing embryos at each position in the incubator, the medium setter (4) can be extracted from the independent culture bottle (11) by the operator, and the medium setter (4) can be used. In the process of extraction, the communication between the appropriate culture bottle (11) and the outside is isolated by the isolation means.
In (1) above, the temperature in the incubator is maintained at 38 to 39 ° C., and the gas concentration is 5% CO.
Keeping at 2,
The fertilization culture medium in (2) above shall be a TCM199 culture medium containing 3% sheep serum.
Including
The isolation means are a moving plate (51) provided in the culture bottle so as to be movable back and forth, and a sealing ring provided on the outer wall of the moving plate and sealed and fitted to the inner wall of the culture bottle. 52), including a one-way air outlet member, a one-way air advancing member, a conduction member provided on the moving plate, and a connection configuration for realizing a connection fitting between the medium setter and the moving plate.
When the medium setter (4) is inserted into the culture bottle (11), the medium setter (4) moves backward while in contact with the moving plate (51), and the gas concentration control chamber is provided by the one-way air-advancing member. To control the gas to enter the culture bottle in one direction,
When the moving plate (51) moves to the rearmost position, the medium setter (4) has the connection configuration.
) And the moving plate (51), and the culture bin (
To realize direct communication between 11) and the gas concentration control chamber (3),
When observing or acquiring the culture status in the culture bin (11), the medium setter (4) is taken out, and the medium setter (4) is connected to the sealing member (52) and the culture bin according to the connection configuration. While resisting the frictional force between (11), the moving plate (51) moves forward in conjunction with the moving plate (51), and the gas concentration in the culture bottle (11) is adjusted in one direction by the one-way air outlet member. Control to enter the room (3),
When the moving plate (51) moves to the foremost position, the medium setter (4) moves to the culture bin (
Extending to the outside of 11) and always isolating the communication between the inside and the outside of the culture bottle (11) by the moving plate (51).
The one-way air outlet member is provided in both circular openings (61) provided in the moving plate, a first connector formed by extending from the circular opening to the rear end surface of the moving plate, and the first connector. Includes a one-way valve (63)
The unidirectional air advance member is provided in both through holes (64) provided in the moving plate, a second connector formed by extending from the through holes to the front end surface of the moving plate, and the second connector. Includes a one-way valve (63)
A method of culturing to improve the in vivo viability of mammalian embryos, which comprises. 前記培地セッター（４）は、横断面がＵ型状とするフレーム（４１）と、前記フレームの
一端に固定接続される蓋板（４２）と、および前記蓋板に設けられる引手（４３）とを含
むこと、
前記接続構成は、前記培養ビンの入口端に設けられる固定座（７１）と、前記固定座に設
けられ、前記フレームが貫通する方形開口（７２）と、前記移動板に設けられ、前記フレ
ームの先端が挿入するＵ型溝（７３）と、前記フレームの先端に設けられ、間隔的に均一
に配布される複数個のＵ型凸片（７４）と、前記Ｕ型溝内に設けられ、前記凸片が挿入す
る開口溝と、前記フレームに固定設置されるＬ型の挿接部材（７５）と、前記挿接部材の
先端に設けられる三角形の挿接ヘッド（７６）と、前記移動板に設けられ、前記挿接ヘッ
ドが挿入する挿接溝（７７）と、前記挿接溝内に左右に移動可能に設けられる限位ブロッ
ク（７８）と、前記限位ブロックの側壁と挿接溝の内壁の間に設けられるバネ（７９）と
、および前記限位ブロック上に設けられ、前記挿接ヘッドと嵌合される第１傾斜面（７６
１）および第２傾斜面（７６２）とを含むこと、
前記バネはその一端が限位ブロックに固定接続され、その他端が挿接溝の内壁に固定接続
されること、
前記第１傾斜面（７６１）が限位ブロックの前端面に位置され、前記第２傾斜面（７６２
）が限位ブロックの後端面に位置され、且つ該第１傾斜面（７６１）の傾斜角度が第２傾
斜面（７６２）の傾斜角度より大きいこと、
前記フレーム（４１）が培養ビン（１１）内まで挿入すると、該フレーム（４１）の下表
面と培養ビン（１１）の底面の間に間隔が残り、該フレーム（４１）に間隔的に複数個の
導気穴（４１１）が均一に配布されること、
を含むことを特徴とする請求項１に記載の哺乳動物胚のインビボ生存率を向上する培養
方法。 The medium setter (4) includes a frame (41) having a U-shaped cross section, a lid plate (42) fixedly connected to one end of the frame, and a pull tab (43) provided on the lid plate. Including,
The connection configuration includes a fixed seat (71) provided at the inlet end of the culture bottle, a square opening (72) provided in the fixed seat through which the frame penetrates, and a moving plate provided in the frame. A U-shaped groove (73) into which the tip is inserted, a plurality of U-shaped convex pieces (74) provided at the tip of the frame and evenly distributed at intervals, and a U-shaped groove provided in the U-shaped groove. An opening groove into which a convex piece is inserted, an L-shaped insertion member (75) fixedly installed on the frame, a triangular insertion head (76) provided at the tip of the insertion member, and the moving plate. An insertion groove (77) provided and inserted by the insertion head, a limiting block (78) provided so as to be movable left and right in the insertion groove, and a side wall and an insertion groove of the limiting block. A spring (79) provided between the inner walls and a first inclined surface (76) provided on the limiting block and fitted with the insertion head.
1) and a second inclined surface (762),
One end of the spring is fixedly connected to the limiting block, and the other end is fixedly connected to the inner wall of the insertion groove.
The first inclined surface (761) is located on the front end surface of the limiting block, and the second inclined surface (762) is located.
) Is located on the rear end surface of the limiting block, and the inclination angle of the first inclined surface (761) is larger than the inclination angle of the second inclined surface (762).
When the frame (41) is inserted into the culture bin (11), an interval remains between the lower surface of the frame (41) and the bottom surface of the culture bin (11), and a plurality of intervals remain in the frame (41). The air guide holes (411) are evenly distributed,
Culture method of improving the in vivo viability of mammalian embryo of claim 1, wherein it to contain. 前記導通部材は、前記移動板に長さ方向に沿って間隔的に配布される複数個の長尺状開
口（８１）と、前記移動板の後側に設けられ且つ移動板に対して往復移動可能な移動部材
（８２）と、前記移動部材に設けられ、移動板に長さ方向に沿って間隔的に配布される複
数個の長尺状導通口（８３）と、前記移動板に設けられ、移動部材が移動する滑り軌道と
、前記移動部材と嵌合される復位ユニットと、および前記培養ビン内に固定設置される駆
動部（８５）とを含むこと、
前記フレーム（４１）が移動板（５１）を推移して最後端位置まで移動させると、前記
駆動部（８５）が移動部材（８２）を駆動部から離れる位置へ移動させるように駆動する
ことにより、前記長尺状開口（８１）と長尺状導通口（８３）の間の連通を実現すること
、
前記気体濃度調節室（３）内に気体均一度を調節するための循環手段が設けられ、該循
環手段は、前記気体濃度調節室内に設けられる第１仕切板（９１）および第２仕切板（９
２）と、前記第１仕切板に設けられる複数個の扇風機（９３）と、および前記第２仕切板
に設けられ、気流を培養ビン内へ案内するドレイン部材とを含み、前記第１仕切板（９１
）と第２仕切板（９２）が相互に接続され、該第１仕切板（９１）と第２仕切板（９２）
により気体濃度調節室（３）を第１チャンバー（３１）と第２チャンバー（３２）に仕切
り、該第１チャンバー（３１）が前記培養ビン（１１）に連通すること、
を含むことを特徴とする請求項１に記載の哺乳動物胚のインビボ生存率を向上する培養
方法。 The conductive member has a plurality of elongated openings (81) distributed to the moving plate at intervals along the length direction, and is provided on the rear side of the moving plate and reciprocates with respect to the moving plate. A possible moving member (82), a plurality of elongated conduction ports (83) provided on the moving member and distributed to the moving plate at intervals along the length direction, and provided on the moving plate. Includes a sliding track on which the moving member moves, a repositioning unit fitted with the moving member, and a drive unit (85) fixedly installed in the culture bin.
When the frame (41) moves the moving plate (51) to the rearmost end position, the driving unit (85) drives the moving member (82) to move away from the driving unit. To realize communication between the long opening (81) and the long conduction port (83).
A circulation means for adjusting the gas uniformity is provided in the gas concentration adjusting chamber (3), and the circulation means includes a first partition plate (91) and a second partition plate (91) provided in the gas concentration adjusting chamber. 9
2), a plurality of fans (93) provided on the first partition plate, and a drain member provided on the second partition plate to guide the air flow into the culture bin, and the first partition plate. (91
) And the second partition plate (92) are connected to each other, and the first partition plate (91) and the second partition plate (92)
The gas concentration control chamber (3) is divided into a first chamber (31) and a second chamber (32), and the first chamber (31) communicates with the culture bin (11).
The culture method for improving the in vivo viability of a mammalian embryo according to claim 1, wherein the method comprises.
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