JP5776956B2 - Cell culture vessel - Google Patents

Cell culture vessel Download PDF

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JP5776956B2
JP5776956B2 JP2010227173A JP2010227173A JP5776956B2 JP 5776956 B2 JP5776956 B2 JP 5776956B2 JP 2010227173 A JP2010227173 A JP 2010227173A JP 2010227173 A JP2010227173 A JP 2010227173A JP 5776956 B2 JP5776956 B2 JP 5776956B2
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智紀 赤井
智紀 赤井
大西 康仁
康仁 大西
今井 敬
敬 今井
芳雄 相川
芳雄 相川
正樹 大竹
正樹 大竹
ソムファイ・タマス
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Dai Nippon Printing Co Ltd
National Livestock Breeding Center Inc Administrative Agency
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National Livestock Breeding Center Inc Administrative Agency
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    • C12M23/12Well or multiwell plates
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization

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Description

本発明は、受精卵などの個別管理が必要な細胞を培養するための培養容器、ならびに該培養容器を用いた培養細胞の判別方法に関する。   The present invention relates to a culture container for culturing cells that require individual management, such as a fertilized egg, and a method for distinguishing cultured cells using the culture container.

培養系で精子と卵子とを体外受精させて受精卵(接合子)を作製して、さらに受精卵を卵割、桑実胚、胚盤胞の段階を経て、透明帯から孵化した脱出胚盤胞の段階まで培養することが可能となり、この卵割から胚盤胞の段階にある受精卵を子宮に移植して産子を得る補助的生殖技術(ART)が、家畜領域のみならずヒトの不妊医療でも確立されている。   The in vitro fertilized egg (zygote) is produced by fertilizing sperm and ovum in a culture system, and the fertilized egg goes through the cleavage, morula, and blastocyst stages, and then emerges from the zona pellucida It is possible to culture up to the blastocyst stage. Assistive reproductive technology (ART) to transfer a fertilized egg from the cleavage to the blastocyst stage to the uterus to give birth to a baby is not limited to the livestock region. Established in infertility medicine.

しかし、体外受精による妊娠成功率は必ずしも高くはなく、たとえばヒトにおいては、その妊娠成功率は、依然として25〜35%程度に留まっている。その原因の一つとして、培養において子宮への移植に適した良質な受精卵を得られる確率が高くないことが挙げられる。培養された受精卵は、専門家が顕微鏡で個別に観察することにより、子宮への移植に適した良質な受精卵であるか否か判別されている。従って、その判別にはコスト及び時間がかかるという問題がある。そこで、子宮への移植に適した良質な受精卵を低コストかつ短時間で判別する技術が求められている。また、良質な受精卵の判断が個人により異なるという問題もあり、良質な受精卵だという基準や選別する技術が必要とされている。   However, the success rate of pregnancy by in vitro fertilization is not necessarily high. For example, in humans, the success rate of pregnancy is still about 25 to 35%. One of the causes is that the probability of obtaining a high-quality fertilized egg suitable for transplantation into the uterus in culture is not high. A cultured fertilized egg is individually observed with a microscope to determine whether it is a high-quality fertilized egg suitable for transplantation into the uterus. Therefore, there is a problem that the determination takes cost and time. Therefore, there is a need for a technique for discriminating high-quality fertilized eggs suitable for transplantation into the uterus at a low cost and in a short time. In addition, there is a problem that the judgment of a high-quality fertilized egg varies depending on the individual, so that a standard for selecting a high-quality fertilized egg and a screening technique are required.

従来、受精卵の培養は、培養容器上のウェル内に500μL程度の培養液を入れ、該培養液中で受精卵を培養する方法、培養容器上のウェル内に20μL程度の微小滴を載せ、該微小滴の表面をミネラルオイルで被覆し、その中に受精卵を入れる方法(非特許文献1)等により行われていた。また、非特許文献2及び3は、培養容器上に複数の窪みを設け、それぞれの窪みごとに受精卵を導入して培養することにより受精卵を個別に管理する方法を開示している。非特許文献4は、培養容器上にメッシュ体を配置し、メッシュの編目ごとに受精卵を導入して培養することにより受精卵を個別に管理する方法を開示している。   Conventionally, fertilized eggs are cultured by placing a culture solution of about 500 μL in a well on the culture vessel and culturing the fertilized egg in the culture solution, placing about 20 μL of microdroplets in the well on the culture vessel, This method has been performed by a method in which the surface of the microdroplet is coated with mineral oil and a fertilized egg is placed therein (Non-patent Document 1). Non-Patent Documents 2 and 3 disclose a method of individually managing fertilized eggs by providing a plurality of depressions on a culture container and introducing and culturing the fertilized eggs for each depression. Non-Patent Document 4 discloses a method of managing a fertilized egg individually by arranging a mesh body on a culture container and introducing and cultivating the fertilized egg for each mesh stitch.

さらに近年では、細胞治療・再生医療の研究開発が進み、細胞培養中の無菌環境を維持することや、細胞の品質確保、あるいは細胞を迅速且つ大量に培養し判別することについてのニーズが高まっている。しかし従来は、上記非特許文献1〜4に示すような培養容器を用い、手作業で培養細胞の状態を観察しているため、連続的な細胞状態の観察が困難であること、培養環境の変化や雑菌による汚染リスクなど細胞への影響が懸念されること、培養細胞の判別にコストと時間がかかり効率が悪いことなどが課題となっていた。したがって、培養細胞を自動的に判別する技術の開発が望まれていた。また、例えば畜産の分野においても、国により「家畜改良増殖目標」が策定され、この目標達成のため受精卵移植を活用した肉専用種の増頭の推進が図られている中、細胞の自動判別技術は非常に有用と考えられる。   In recent years, research and development of cell therapy and regenerative medicine has progressed, and there has been an increasing need for maintaining a sterile environment during cell culture, ensuring cell quality, or culturing and discriminating cells rapidly and in large quantities. Yes. However, conventionally, since the state of the cultured cells is manually observed using the culture vessels as shown in Non-Patent Documents 1 to 4 above, it is difficult to observe the continuous cell state. There are concerns about the effects on cells such as changes and risk of contamination by various bacteria, and the cost and time required for discrimination of cultured cells are inefficient. Therefore, development of a technique for automatically discriminating cultured cells has been desired. In the field of animal husbandry, for example, the “Livestock Improvement and Growth Target” has been formulated by the government. Discrimination technology is considered very useful.

菅原,尾川(編)、「生殖機能細胞の培養法」、日本、学会出版センター、1993年6月出版、第25〜153頁Sugawara, Ogawa (eds.), “Methods for culturing reproductive function cells”, Japan, Japan Society for Publishing Press, June 1993, pp. 25-153 M. Taka, et al. Journal of Reproduction and Development, 51, 533-537 (2005)M. Taka, et al. Journal of Reproduction and Development, 51, 533-537 (2005) Vajta G., et al. Molecular Reproduction and Development, 55, 256-264 (2000)Vajta G., et al. Molecular Reproduction and Development, 55, 256-264 (2000) P. J. Booth, et al. Biology of Reproduction, 77, 765-779 (2007)P. J. Booth, et al. Biology of Reproduction, 77, 765-779 (2007)

本発明者らは、上記従来の状況に鑑み、子宮への移植に適した良質な受精卵を低コストかつ短時間で判別すべく、受精卵の判別の自動化の可能性を検討した。そして、非特許文献1に記載される培養容器を用いて自動化を検討したところ、培養している複数の受精卵がプレート内や微小滴内で移動してしまい、それぞれ質の異なる受精卵を個別に管理することができず、受精卵の判別を自動で行うのには適してないことが明らかとなった。また、受精卵の自動判別においては、CCDカメラ等を用いて受精卵の画像を撮像することが考えられるが、従来の培養容器では受精卵を個別に撮影することが困難であることも明らかとなった。   In view of the above-described conventional situation, the present inventors examined the possibility of automating discrimination of fertilized eggs in order to discriminate high-quality fertilized eggs suitable for transplantation into the uterus at low cost and in a short time. And when automation was examined using the culture container described in Non-Patent Document 1, a plurality of fertilized eggs that have been cultured moved in a plate or in microdrops, and fertilized eggs having different qualities were individually obtained. It was revealed that it was not suitable for automatically discriminating fertilized eggs. In automatic discrimination of fertilized eggs, it is conceivable to take images of fertilized eggs using a CCD camera or the like, but it is also clear that it is difficult to individually photograph fertilized eggs with conventional culture containers. became.

そこで、非特許文献2〜4に記載される複数の窪みを設けた培養容器や、メッシュを配置した培養容器を用いて自動化を検討したところ、受精卵の密度も高く、受精卵を個別に管理することは可能であるが、窪み内やメッシュの編目内で受精卵が移動して窪みの側壁やメッシュに接触した場合には、取得した画像の輪郭抽出処理により受精卵の画像を抽出することが困難であることを見出した。   Therefore, when automation was examined using a culture vessel provided with a plurality of depressions described in Non-Patent Documents 2 to 4 and a culture vessel provided with a mesh, the density of fertilized eggs is high, and the fertilized eggs are individually managed. It is possible to extract the fertilized egg image by the contour extraction process of the acquired image when the fertilized egg moves in the hollow or mesh stitch and comes into contact with the hollow side wall or mesh. Found it difficult.

従って、本発明は、培養細胞の自動判別に適した培養容器を提供することを目的とする。   Therefore, an object of the present invention is to provide a culture container suitable for automatic discrimination of cultured cells.

本発明者らは、上記課題を解決すべく検討を行った結果、壁面が、最も低い位置から外縁に進むに従って高くなるような傾斜面を有する凹部が密に配置された細胞収容部を有する培養容器を用いて細胞を培養することにより、培養細胞の撮影画像における細胞の輪郭抽出処理が効率よく実施でき、従って、培養細胞の自動判別に適していることを見出し、本発明を完成した。   As a result of studies to solve the above-mentioned problems, the inventors of the present invention have a culture having a cell containing portion in which concave portions having inclined surfaces whose wall surfaces become higher from the lowest position to the outer edge are densely arranged. By culturing the cells using the container, it was found that the cell contour extraction process in the photographed image of the cultured cells can be efficiently performed, and therefore, it is suitable for automatic discrimination of the cultured cells, and the present invention has been completed.

すなわち、本発明は以下の発明を包含する。
(1)個別管理が必要とされる細胞を培養するための、底壁と側壁とを有する培養容器であって、
底壁に、凹部を有する細胞収容部が配置されており、
凹部が4個以上近接しており、
凹部の壁面が、凹部の最も低い位置から凹部の外縁に進むに従って高くなるような傾斜面を有し、
近接する凹部間のピッチが1mm以下である、
前記培養容器。
That is, the present invention includes the following inventions.
(1) A culture vessel having a bottom wall and a side wall for culturing cells that require individual management,
A cell housing portion having a recess is disposed on the bottom wall,
4 or more concave parts are close to each other,
The wall surface of the recess has an inclined surface that increases from the lowest position of the recess toward the outer edge of the recess,
The pitch between adjacent recesses is 1 mm or less,
The culture container.

(2)凹部の壁面が、直線部分を含む傾斜面を有する、(1)に記載の培養容器。
(3)傾斜面の表面粗さについて、最大高さRyが1.0μm未満である、(2)に記載の培養容器。
(4)凹部の開口部の開口幅が100μm〜300μmである、(1)〜(3)のいずれかに記載の培養容器。
(5)凹部の深さが50μm〜200μmである、(1)〜(4)のいずれかに記載の培養容器。
(2) The culture vessel according to (1), wherein the wall surface of the recess has an inclined surface including a straight portion.
(3) The culture container according to (2), wherein the maximum height Ry is less than 1.0 μm for the surface roughness of the inclined surface.
(4) The culture vessel according to any one of (1) to (3), wherein the opening width of the opening of the recess is 100 μm to 300 μm.
(5) The culture container according to any one of (1) to (4), wherein the depth of the recess is 50 μm to 200 μm.

(6)凹部の開口部が円形である、(1)〜(5)のいずれかに記載の培養容器。
(7)凹部の壁面が円錐状又は円錐台状の部分を有する、(6)に記載の培養容器。
(8)円錐又は円錐台の中心線と母線とのなす角度が89〜45°である、(7)に記載の培養容器。
(6) The culture container according to any one of (1) to (5), wherein the opening of the recess is circular.
(7) The culture vessel according to (6), wherein the wall surface of the recess has a conical or truncated cone portion.
(8) The culture vessel according to (7), wherein an angle formed between the center line of the cone or the truncated cone and the generatrix is 89 to 45 °.

(9)近接する凹部が、1mmあたり1個以上の密度で配置されている、(1)〜(8)のいずれかに記載の培養容器。
(10)近接する4個以上の凹部が正方格子状又は最密充填状に配置されている、請求項(1)〜(9)のいずれかに記載の培養容器。
(9) The culture vessel according to any one of (1) to (8), wherein the adjacent concave portions are arranged at a density of 1 or more per 1 mm 2 .
(10) The culture vessel according to any one of claims (1) to (9), wherein four or more adjacent concave portions are arranged in a square lattice shape or a close-packed shape.

(11)凹部が24個以上配置されている、(1)〜(10)のいずれかに記載の培養容器。
(12)細胞が、受精卵、卵細胞、ES細胞及びiPS細胞からなる群から選択される、(1)〜(11)のいずれかに記載の培養容器。
(13)細胞が、ウシの受精卵である、(12)に記載の培養容器。
(14)近接する4個以上の凹部が、それらを囲む内壁により、培養容器内のその他の部分と隔てられている、(1)〜(13)のいずれかに記載の培養容器。
(15)培養容器の外周部であって細胞収容部を有しない部分に液体収容部を有する、(14)に記載の培養容器。
(11) The culture vessel according to any one of (1) to (10), wherein 24 or more concave portions are arranged.
(12) The culture container according to any one of (1) to (11), wherein the cells are selected from the group consisting of fertilized eggs, egg cells, ES cells, and iPS cells.
(13) The culture container according to (12), wherein the cells are bovine fertilized eggs.
(14) The culture container according to any one of (1) to (13), wherein four or more adjacent recesses are separated from other parts in the culture container by an inner wall surrounding them.
(15) The culture container according to (14), wherein the culture container has a liquid container in a portion that is an outer peripheral part of the culture container and does not have a cell container.

(16)培養細胞の自動判別に使用するための、(1)〜(15)のいずれかに記載の培養容器。
(17)倍率4倍の対物レンズを用いて細胞収容部を顕微鏡観察したときに、観察視野内に4個以上の凹部が含まれる、(16)に記載の培養容器。
(18)培養細胞の判別方法であって、(1)〜(17)のいずれかに記載の培養容器の細胞収容部の凹部に、受精卵、卵細胞、ES細胞及びiPS細胞からなる群から選択される細胞をそれぞれ導入して培養し、顕微鏡により取得した培養細胞の画像を検出装置によって撮像し、得られた像を輪郭抽出処理に付すことを含む、前記判別方法。
(16) The culture container according to any one of (1) to (15) for use in automatic discrimination of cultured cells.
(17) The culture container according to (16), wherein when the cell container is observed with a microscope using an objective lens having a magnification of 4 times, four or more concave portions are included in the observation field.
(18) A method for discriminating cultured cells, which is selected from the group consisting of a fertilized egg, an egg cell, an ES cell, and an iPS cell in the recess of the cell container of the culture container according to any one of (1) to (17) Each of the cells to be cultured is introduced and cultured, and an image of the cultured cells obtained by a microscope is captured by a detection device, and the obtained image is subjected to contour extraction processing.

本発明により、培養細胞の自動判別に適した培養容器が提供される。   According to the present invention, a culture container suitable for automatic discrimination of cultured cells is provided.

本発明の実施形態を示す概略図である。It is the schematic which shows embodiment of this invention. 本発明の実施形態を示す概略図である。It is the schematic which shows embodiment of this invention. 本発明の実施形態である培養容器Aを示す概略図である。It is the schematic which shows the culture container A which is embodiment of this invention. 本発明の実施形態を示す概略図である。It is the schematic which shows embodiment of this invention. 近接した凹部を最密充填状に配置する場合の平面図である。It is a top view in the case of arrange | positioning the adjacent recessed part in the close-packed form. 比較例で用いた培養容器Bを示す。The culture container B used in the comparative example is shown. 本発明の培養容器Aで培養した受精卵の顕微鏡写真である。It is a microscope picture of the fertilized egg cultured with the culture container A of this invention. 図5の画像に対して閾値処理を行った結果を示す。The result of having performed threshold processing with respect to the image of FIG. 5 is shown. 比較例で用いた培養容器Bで培養した受精卵の顕微鏡写真である。It is a microscope picture of the fertilized egg cultured with the culture container B used by the comparative example. 図7の画像に対して閾値処理を行った結果を示す。The result of having performed the threshold process with respect to the image of FIG. 7 is shown. 比較例で用いた培養容器Bで培養した受精卵の顕微鏡写真である。It is a microscope picture of the fertilized egg cultured with the culture container B used by the comparative example. 図9の画像に対して閾値処理を行った結果を示す。The result of having performed the threshold process with respect to the image of FIG. 9 is shown. 本発明の実施形態である培養容器Cを示す平面図である。It is a top view which shows the culture container C which is embodiment of this invention. 図11のIIIa−IIIa断面図である。It is IIIa-IIIa sectional drawing of FIG. 図11における細胞収容部の部分拡大図である。It is the elements on larger scale of the cell accommodating part in FIG. 図13のIIIb−IIIb断面図である。It is IIIb-IIIb sectional drawing of FIG. 図13における凹部の部分拡大図である。It is the elements on larger scale of the recessed part in FIG. 図15のIIIc−IIIc断面図である。It is IIIc-IIIc sectional drawing of FIG. 培養容器Cの外観写真である。It is an external appearance photograph of the culture container C. 培養容器Cの細胞収容部の断面形状計測結果を示すグラフである。It is a graph which shows the cross-sectional shape measurement result of the cell accommodating part of the culture container C. 培養容器Cの凹部の断面形状計測結果を示すグラフである。It is a graph which shows the cross-sectional shape measurement result of the recessed part of the culture container C. FIG. 図19の右側傾斜面に対して線形フィッティングを行い、傾きを補正した図である。It is the figure which performed linear fitting with respect to the right side inclined surface of FIG. 19, and corrected the inclination. 培養容器Dの凹部の断面形状計測結果を示すグラフである。It is a graph which shows the cross-sectional shape measurement result of the recessed part of the culture container D. FIG. 図21の右側傾斜面に対して線形フィッティングを行い、傾きを補正した図である。It is the figure which performed linear fitting with respect to the right side inclined surface of FIG. 21, and correct | amended the inclination. 本発明の実施形態である培養容器Eを示す平面図である。It is a top view which shows the culture container E which is embodiment of this invention. 図23のIVa−IVa断面図である。It is IVa-IVa sectional drawing of FIG. 図23における細胞収容部の部分拡大図である。It is the elements on larger scale of the cell accommodating part in FIG. 図25のIVb−IVb断面図である。It is IVb-IVb sectional drawing of FIG. 図25における凹部の部分拡大図である。It is the elements on larger scale of the recessed part in FIG. 図27のIVc−IVc断面図である。It is IVc-IVc sectional drawing of FIG. 培養容器Eの凹部の断面形状計測結果を示すグラフである。4 is a graph showing a cross-sectional shape measurement result of a recess of a culture vessel E. 図29の右側傾斜面に対して線形フィッティングを行い、傾きを補正した図である。It is the figure which performed linear fitting with respect to the right side inclined surface of FIG. 29, and correct | amended the inclination. 本発明の培養容器Cで培養したマウス受精卵の顕微鏡写真である。It is a microscope picture of the mouse | mouth fertilized egg cultured with the culture container C of this invention. 図31の画像に対して閾値処理を行った結果を示す。The result of having performed threshold processing with respect to the image of FIG. 31 is shown. 本発明の培養容器Cで培養したウシ受精卵の顕微鏡写真である。It is a microscope picture of the cow fertilized egg cultured with the culture container C of this invention. 図33の画像に対して閾値処理を行った結果を示す。The result of having performed the threshold process with respect to the image of FIG. 33 is shown.

本発明は、個別管理が必要とされる細胞を培養するための、底壁と側壁とを有する培養容器であって、
底壁に、凹部を有する細胞収容部が配置されており、
凹部が4個以上近接しており、
凹部の壁面が、凹部の最も低い位置から凹部の外縁に進むに従って高くなるような傾斜面を有し、
近接する凹部間のピッチが1mm以下である、
前記培養容器に関する。
The present invention is a culture vessel having a bottom wall and a side wall for culturing cells that require individual management,
A cell housing portion having a recess is disposed on the bottom wall,
4 or more concave parts are close to each other,
The wall surface of the recess has an inclined surface that increases from the lowest position of the recess toward the outer edge of the recess,
The pitch between adjacent recesses is 1 mm or less,
It relates to the culture vessel.

個別管理が必要とされる細胞とは、培養中及び培養後において個々の細胞を特定する必要があるものをさし、複数の細胞を培養している培養容器において互いに混同してしまうことが望ましくない細胞をさす。個別管理が必要とされる細胞としては、例えば、受精卵、卵細胞、ES細胞(胚性幹細胞)及びiPS細胞(人工多能性幹細胞)が挙げられる。卵細胞は、未受精の卵細胞をさし、未成熟卵母細胞及び成熟卵母細胞が含まれる。受精卵は、受精後、卵割により2細胞期、4細胞期、8細胞期と細胞数が増えていき、桑実胚を経て、胚盤胞へと発生する。受精卵には、2細胞胚、4細胞胚及び8細胞胚などの初期胚、桑実胚、胚盤胞(初期胚盤胞、拡張胚盤胞及び脱出胚盤胞を含む)が含まれる。胚盤胞は、胎盤を形成する潜在能力がある外部細胞と胚を形成する潜在能力がある内部細胞塊からなる胚を意味する。ES細胞は胚盤胞の内部細胞塊から得られる未分化な多能性又は全能性細胞をさす。iPS細胞は、体細胞(主に線維芽細胞)へ数種類の遺伝子(転写因子)を導入することにより、ES細胞に似た分化万能性を持たせた細胞をさす。すなわち、本発明において培養の対象となる細胞には、受精卵や胚盤胞のように複数の細胞の集合体も包含される。本発明の培養容器は、好ましくは哺乳動物及び鳥類の細胞、特に哺乳動物の細胞の培養に好適である。哺乳動物は、温血脊椎動物をさし、例えば、ヒト及びサルなどの霊長類、マウス、ラット及びウサギなどの齧歯類、イヌ及びネコなどの愛玩動物、ならびにウシ、ウマ及びブタなどの家畜が挙げられる。本発明の培養容器は、ウシの受精卵の培養に特に好適である。   The cells that need to be individually managed are those that need to identify individual cells during and after culture, and should be confused with each other in a culture vessel in which multiple cells are cultured. Refers to no cells. Examples of cells that require individual management include fertilized eggs, egg cells, ES cells (embryonic stem cells), and iPS cells (artificial pluripotent stem cells). An egg cell refers to an unfertilized egg cell, and includes an immature oocyte and a mature oocyte. After fertilization, the fertilized egg increases in number of cells from the 2 cell stage, the 4 cell stage, and the 8 cell stage by cleavage, and develops into a blastocyst through a morula. Fertilized eggs include early embryos such as 2-cell embryos, 4-cell embryos and 8-cell embryos, morulas, blastocysts (including early blastocysts, expanded blastocysts and escaped blastocysts). A blastocyst means an embryo composed of external cells with the potential to form the placenta and internal cell masses with the potential to form embryos. ES cells refer to undifferentiated pluripotent or totipotent cells obtained from the inner cell mass of a blastocyst. An iPS cell refers to a cell having a pluripotency similar to that of an ES cell by introducing several types of genes (transcription factors) into somatic cells (mainly fibroblasts). That is, in the present invention, the cells to be cultured include a collection of a plurality of cells such as fertilized eggs and blastocysts. The culture vessel of the present invention is preferably suitable for culturing mammalian and avian cells, particularly mammalian cells. Mammals refer to warm-blooded vertebrates, for example, primates such as humans and monkeys, rodents such as mice, rats and rabbits, pets such as dogs and cats, and domestic animals such as cattle, horses and pigs. Is mentioned. The culture container of the present invention is particularly suitable for culturing bovine fertilized eggs.

本発明の培養容器は、底壁と側壁とを有し、底壁と側壁とから形成される空間に液体を収容可能である。底壁の形状は特に制限されず、三角形及び四角形等の多角形の形状でもよく、円(円形及び楕円形を含む)の形状でもよく、側壁は底壁の外縁を囲うように形成される。本発明の培養容器において、通常、底壁と反対側は開口している。なお、本発明の培養容器は、通常のシャーレと同様に蓋を有していてもよい。本発明の培養容器の底壁と側壁は、好ましくは慣用のシャーレと同様の形状を形成する。そして、培養容器の底壁には、凹部を有する細胞収容部が形成されている。細胞収容部の凹部は、培養容器の底壁に直接窪みとして設けられた凹部でもよいし(図1a)、底壁から突出した部材により形成される凹部でもよい(図1b)。   The culture container of the present invention has a bottom wall and a side wall, and can store a liquid in a space formed by the bottom wall and the side wall. The shape of the bottom wall is not particularly limited, and may be a polygonal shape such as a triangle and a quadrangle, may be a circle (including a circle and an ellipse), and the side wall is formed so as to surround the outer edge of the bottom wall. In the culture container of the present invention, the side opposite to the bottom wall is usually open. In addition, the culture container of this invention may have a lid | cover similarly to a normal petri dish. The bottom wall and side wall of the culture vessel of the present invention preferably form the same shape as a conventional petri dish. And the cell storage part which has a recessed part is formed in the bottom wall of a culture container. The concave part of the cell storage part may be a concave part provided directly as a depression in the bottom wall of the culture vessel (FIG. 1a) or a concave part formed by a member protruding from the bottom wall (FIG. 1b).

凹部は、4個以上、好ましくは6個以上、より好ましくは8個以上が、近接して形成されている。凹部は、少なくとも4個が近接して形成されていればよく、さらに近接していない凹部が別途形成されていてもよい。また、4個以上近接して形成された凹部の群が、複数群配置されていてもよく、それらの群は互いに近接していなくてもよい。   Four or more, preferably six or more, more preferably eight or more recesses are formed close to each other. It is sufficient that at least four recesses are formed close to each other, and further recesses that are not close to each other may be formed separately. Further, a plurality of groups of recesses formed close to four or more may be arranged, and these groups do not have to be close to each other.

凹部は、壁面及び開口部を有し、開口部の外縁の形状は特に制限されないが、好ましくは円形(円形及び楕円形を含む)である。   The recess has a wall surface and an opening, and the shape of the outer edge of the opening is not particularly limited, but is preferably a circle (including a circle and an ellipse).

近接する凹部間のピッチは1mm以下である。ただし、上記ピッチは収容する細胞の種類に依存して異なる。   The pitch between adjacent recesses is 1 mm or less. However, the pitch varies depending on the type of cells to be accommodated.

例えば、ウシ受精卵を収容する場合、近接する凹部間のピッチは通常1mm以下、好ましくは0.7mm以下、さらに好ましくは0.45mm以下である。観察装置として、1/2インチのCCD素子、4、10、20倍の対物レンズを備えたものがよく用いられる。このような観察装置で、4倍の対物レンズを選択した場合の観察可能な視野はおよそ1.6mm×1.2mmであり、この観察視野内に4個以上の凹部が含まれるように設計することが好ましい。凹部の直径がウシ受精卵の直径(0.25mm)より大きく、0.3mmだとすると、ピッチが0.9mm以下の場合、1.6mm×1.2mmの顕微鏡視野内に2×2=4個の受精卵を観察することができ、ピッチが0.65mm以下の場合、1.6mm×1.2mmの顕微鏡視野内に3×2=6個の受精卵を観察することができ、ピッチが0.43mm以下の場合、1.6mm×1.2mmの顕微鏡視野内に4×3=12個の受精卵を観察することができる。観察視野内に4個以上の凹部が含まれるように設計することで、例えば培養細胞の自動判別を行う場合に、一度に多数個の細胞を処理でき、また視野内の一つの受精卵と他の受精卵との比較判別が容易になるため、判別操作をより効率的に行うことができる。   For example, when a bovine fertilized egg is accommodated, the pitch between adjacent concave portions is usually 1 mm or less, preferably 0.7 mm or less, more preferably 0.45 mm or less. As an observation device, a device equipped with a 1/2 inch CCD element, 4, 10, and 20 times objective lens is often used. In such an observation apparatus, when a 4 × objective lens is selected, an observable visual field is approximately 1.6 mm × 1.2 mm, and the observation visual field is designed to include four or more concave portions. It is preferable. If the diameter of the recess is larger than the diameter of the bovine fertilized egg (0.25 mm) and is 0.3 mm, 2 × 2 = 4 pieces in a 1.6 mm × 1.2 mm microscope field when the pitch is 0.9 mm or less. A fertilized egg can be observed. When the pitch is 0.65 mm or less, 3 × 2 = 6 fertilized eggs can be observed within a 1.6 mm × 1.2 mm microscope field, and the pitch is 0. In the case of 43 mm or less, 4 × 3 = 12 fertilized eggs can be observed in a 1.6 mm × 1.2 mm microscope field. Designed to include four or more recesses in the observation field, for example, when automatically culturing cultured cells, a large number of cells can be processed at the same time. Since the comparison with the fertilized egg becomes easy, the discrimination operation can be performed more efficiently.

凹部間のピッチは近接する凹部の中心間の距離である(図1のa)。凹部の中心は、凹部の開口部の外縁が形成する図形の重心位置とし、外縁が円形であればその円の中心をさす。上記凹部間のピッチは通常平均ピッチをさし、平均ピッチは、ある凹部に関しては、近接する全ての凹部とのピッチから平均値を算出したものをさす。凹部間のピッチは、凹部の開口部の外縁の寸法より大きい。凹部の開口部の外縁の寸法は、開口部の外縁が円形であればその直径をさし、そうでなければ開口部の外縁が形成する図形の最大径と最短径の平均値とする。換言すれば、近接する凹部は、1mmあたり1個以上、好ましくは4個以上の密度で配置されている。近接する4個以上の凹部は、正方格子状又は最密充填状に配置されていることが好ましい。例えば、25個の凹部を5×5の正方格子状に配置する場合を挙げることができる。正方格子状又は最密充填状に配置することにより、培養容器の底壁における各凹部の位置の特定が容易になり、自動化処理に適用しやすい。また、凹部は、培養容器1個あたり、合計で好ましくは24個以上、より好ましくは50個以上配置されている。培養容器1個あたりに多くの凹部を設けることにより、一定数の細胞を培養するのに必要な培養容器の数を低減することができ、経済的に有利である。 The pitch between the recesses is the distance between the centers of the adjacent recesses (a in FIG. 1). The center of the recess is the center of gravity of the figure formed by the outer edge of the opening of the recess. The pitch between the recesses usually refers to an average pitch, and the average pitch refers to a value obtained by calculating an average value from pitches with all adjacent recesses for a certain recess. The pitch between the recesses is larger than the dimension of the outer edge of the opening of the recess. The dimension of the outer edge of the opening of the concave portion is the diameter if the outer edge of the opening is circular, and otherwise the average value of the maximum diameter and the shortest diameter of the figure formed by the outer edge of the opening. In other words, the adjacent recesses are arranged at a density of 1 or more, preferably 4 or more per 1 mm 2 . It is preferable that four or more adjacent recesses are arranged in a square lattice shape or a close-packed shape. For example, a case where 25 concave portions are arranged in a 5 × 5 square lattice shape can be mentioned. By arranging in a square lattice shape or a close-packed shape, it becomes easy to specify the position of each concave portion on the bottom wall of the culture vessel, and it is easy to apply to an automated process. In addition, the total number of recesses is preferably 24 or more, more preferably 50 or more per culture container. By providing a large number of recesses per culture container, the number of culture containers necessary for culturing a certain number of cells can be reduced, which is economically advantageous.

凹部の開口部の外縁が円形である場合、近接する凹部間のピッチは、X+m+n(ここで、Xは細胞の最大径を表し、mは凹部の開口部の外縁が形成する円の直径から細胞の最大径を引いた長さを表し、nは凹部間の仕切りの長さを表す)と規定することもできる。凹部間の仕切りとは、近接する凹部間の外縁と外縁の最短距離をさす。ここでmは通常0.1mm以下、好ましくは0.07mm以下、さらに好ましくは0.05mm以下であり、nは通常0.6mm以下、好ましくは0.35mm以下、さらに好ましくは0.15mm以下である。   When the outer edge of the opening of the recess is circular, the pitch between adjacent recesses is X + m + n (where X represents the maximum diameter of the cell and m is the diameter of the circle formed by the outer edge of the opening of the recess. It is also possible to define a length obtained by subtracting the maximum diameter of n, and n represents the length of the partition between the recesses. The partition between the recesses refers to the shortest distance between the outer edge and the outer edge between the adjacent recesses. Here, m is usually 0.1 mm or less, preferably 0.07 mm or less, more preferably 0.05 mm or less, and n is usually 0.6 mm or less, preferably 0.35 mm or less, more preferably 0.15 mm or less. is there.

上記のようなピッチで凹部を密に配置することにより、細胞を個別に管理しつつ多くの細胞を同時に培養でき、さらに顕微鏡の一視野に多くの細胞が入るため、一度に多くの細胞の画像を取得することができる。   By densely arranging the recesses with the pitch as described above, many cells can be cultured simultaneously while managing the cells individually, and many cells enter a single field of view of the microscope, so many cells can be imaged at once. Can be obtained.

細胞収容部の凹部の開口部は、細胞を収容可能な開口幅を有する。ここで、凹部の開口部の開口幅は、凹部の開口部の外縁が形成する図形の最短径の長さをさす。従って、凹部の開口部の外縁が円形である場合、開口幅は円の直径に等しく、その直径は、培養する細胞の最大寸法より大きいものとなる。本発明の培養容器により受精卵を培養する場合、胚盤胞の段階まで培養することが望ましいため、円形の開口部の直径は、胚盤胞の段階の細胞の最大寸法より大きいものであることが望ましい。胚盤胞の段階の細胞の最大寸法は通常100μm〜280μmであることから、円形の開口部の直径は、通常100μm以上である。また、上述のように、凹部の開口部の外縁の寸法(開口部の外縁が形成する図形の最大径と最短径の平均値)は、凹部間のピッチより小さい。開口部の外縁が円形である場合、その直径は、凹部間のピッチ、すなわち円の中心間の距離より短く、通常1mm未満である。上記開口幅及び寸法は収容する細胞の種類に依存して異なるが、通常は、凹部の開口部の開口幅を、例えば100μm〜300μmとすることが好ましい。   The opening part of the recessed part of a cell accommodating part has the opening width which can accommodate a cell. Here, the opening width of the opening of the recess refers to the length of the shortest diameter of the figure formed by the outer edge of the opening of the recess. Therefore, when the outer edge of the opening of the recess is circular, the opening width is equal to the diameter of the circle, and the diameter is larger than the maximum dimension of the cells to be cultured. When fertilized eggs are cultured in the culture container of the present invention, it is desirable to culture to the blastocyst stage, so that the diameter of the circular opening is larger than the maximum cell size of the blastocyst stage Is desirable. Since the maximum size of cells in the blastocyst stage is usually 100 μm to 280 μm, the diameter of the circular opening is usually 100 μm or more. Further, as described above, the dimension of the outer edge of the opening of the recess (the average value of the maximum diameter and the shortest diameter of the figure formed by the outer edge of the opening) is smaller than the pitch between the recesses. When the outer edge of the opening is circular, its diameter is shorter than the pitch between the recesses, ie the distance between the centers of the circles, and is usually less than 1 mm. Although the said opening width and dimension differ depending on the kind of cell to accommodate, Usually, it is preferable that the opening width of the opening part of a recessed part shall be 100 micrometers-300 micrometers, for example.

例えば、ウシ受精卵の場合、凹部の開口部の開口幅(凹部が円形である場合は直径)は、通常0.25mm以上、好ましくは0.26mm以上、さらに好ましくは0.27mm以上であり、通常1mm未満、好ましくは0.7mm未満、さらに好ましくは0.45mm未満である。また、上記凹部の開口部の開口幅は、X+m(ここでXは細胞の最大径を表す)と規定することもできる。ここで、mは、好ましくは0.01mm以上、さらに好ましくは0.02mm以上である。   For example, in the case of a bovine fertilized egg, the opening width of the opening of the recess (the diameter when the recess is circular) is usually 0.25 mm or more, preferably 0.26 mm or more, more preferably 0.27 mm or more, Usually, it is less than 1 mm, preferably less than 0.7 mm, more preferably less than 0.45 mm. Moreover, the opening width of the opening of the recess can be defined as X + m (where X represents the maximum cell diameter). Here, m is preferably 0.01 mm or more, and more preferably 0.02 mm or more.

細胞収容部の凹部の壁面は、凹部の最も低い位置から凹部の外縁に進むに従って高くなるような傾斜面を有する。細胞収容部の凹部は、通常、培養容器の底壁側に底がくるように形成される。凹部の傾斜面の形状(プロファイル)は、凹部の最も低い位置から凹部の外縁へ向かって曲線状に高くなる場合、階段状に高くなる場合等、適宜採用することができるが、特に直線部分を含むこと、すなわち凹部の最も低い位置から凹部の外縁へ進むに従い、その経路の全区間もしくは一部の区間が直線状に高くなる傾斜面であることが好ましい。直線部分を含むことで、凹部内に配置した細胞の移動が抑制され、細胞を凹部の最深部に固定し易くなる。したがって、顕微鏡で観察した場合に鮮明な画像を得ることができる。   The wall surface of the concave portion of the cell accommodating portion has an inclined surface that becomes higher from the lowest position of the concave portion toward the outer edge of the concave portion. The concave portion of the cell accommodating portion is usually formed so that the bottom comes to the bottom wall side of the culture vessel. The shape (profile) of the inclined surface of the concave portion can be appropriately adopted when it becomes higher in a curved shape from the lowest position of the concave portion toward the outer edge of the concave portion, when it becomes higher in a stepped shape, etc. Including, that is, an inclined surface in which all or a part of the path linearly increases as it proceeds from the lowest position of the recess to the outer edge of the recess. By including the straight part, the movement of the cell arranged in the recess is suppressed, and the cell is easily fixed to the deepest part of the recess. Therefore, a clear image can be obtained when observed with a microscope.

傾斜面の表面粗さは、大きい値であると、顕微鏡で透過観察を行った画像を輪郭抽出処理に付す際に、傾斜面上の凹凸に起因して明瞭な輪郭が得られない恐れがあるため、可能な限り小さい値であることが好ましい。具体的には、最大高さRy(粗さ曲線からその平均線の方向に基準長さだけを抜き取り、この抜き取り部分における山頂線と谷底線との間隔をいう)が1.0μm未満、特に0.5μm未満であることが好ましい。なお、傾斜面の表面粗さは、培養容器の鋳型を作製する際に磨き処理を施す等して、鋳型の加工精度を高めることにより小さくすることができる。   When the surface roughness of the inclined surface is a large value, there is a possibility that a clear contour cannot be obtained due to unevenness on the inclined surface when an image subjected to transmission observation with a microscope is subjected to contour extraction processing. Therefore, it is preferable that the value is as small as possible. Specifically, the maximum height Ry (extracting only the reference length from the roughness curve in the direction of the average line, which means the interval between the peak line and the valley line in the extracted part) is less than 1.0 μm, particularly 0. It is preferably less than 5 μm. In addition, the surface roughness of the inclined surface can be reduced by increasing the processing accuracy of the mold, for example, by performing a polishing process when producing the mold for the culture vessel.

細胞収容部の凹部の壁面は、好ましくは円錐状又は円錐台状の部分を含む。円錐状又は円錐台状の部分は、培養容器の底壁側に、円錐の頂点又は円錐台の上面及び下面のうち面積の狭い方がくるように形成される。円錐状には、円錐及び楕円錐、これらに類似の形状、例えば、円錐又は楕円錐の頂点が丸みを帯びている形状、円錐面が外側に膨らんでいる形状、円錐面が内側に凹んでいる形状などが含まれる。円錐台状には、円錐台及び楕円錐台、これらに類似の形状、例えば、円錐台又は楕円錐台の上面又は下面と円錐面との接合部が丸みを帯びている形状、円錐面が外側に膨らんでいる形状、円錐面が内側に凹んでいる形状などが含まれる。細胞収容部の凹部の壁面が、円錐台状の部分を含む場合、円錐台の上面及び下面のうち培養容器の底壁側にくる面積の狭い方の直径は、細胞の最大径より小さいことが好ましい。具体的には、面積の狭い方の直径が、細胞の最大径の2分の1以下であることが好ましい。2分の1以下であると、細胞が凹部の中心に配置されやすくなり、移動も抑制され、細胞の撮影が容易になる。また、特に、面積の狭い方の直径が、10μm以下であることが好ましい。直径が10μm以下であると、撮影の際に、光の屈折、散乱などの影響を受けて観察しにくくなる中央の領域が狭くなり、鮮明な画像が得られるため有効である。なお、凹部の円錐状及び円錐台状の部分において円錐面は曲面であり、平面を含まないことが好ましい。細胞収容部の凹部の壁面は、通常、培養容器の底壁側に、上記のような傾斜面、好ましくは円錐状又は円錐台状の部分を有する。細胞収容部の凹部の壁面は、上記のような傾斜面より開口部側に、培養容器の底壁に垂直な壁面を有していてもよい。   The wall surface of the concave portion of the cell accommodating portion preferably includes a conical or truncated cone portion. The conical or frustoconical portion is formed on the bottom wall side of the culture vessel so that the apex of the cone or the upper surface and the lower surface of the truncated cone has a smaller area. Conical shapes include cones and elliptical cones, and similar shapes, for example, cones or elliptical cones with rounded vertices, conical surfaces bulging outward, and conical surfaces recessed inward Shape etc. are included. The truncated cone has a truncated cone and an elliptical truncated cone, and shapes similar to these, for example, a shape in which the joint between the upper or lower surface of the truncated cone or the truncated truncated cone and the truncated cone is rounded, and the truncated cone is outside. And a shape having a conical surface recessed inward. When the wall surface of the concave portion of the cell containing portion includes a truncated cone-shaped part, the diameter of the narrower area on the bottom wall side of the culture vessel of the upper and lower surfaces of the truncated cone should be smaller than the maximum diameter of the cell. preferable. Specifically, the diameter of the narrower area is preferably less than or equal to one half of the maximum cell diameter. When the ratio is less than or equal to one half, the cells are easily placed at the center of the recess, the movement is also suppressed, and the imaging of the cells is facilitated. In particular, the diameter of the narrower area is preferably 10 μm or less. When the diameter is 10 μm or less, the central region that is difficult to observe due to the influence of light refraction, scattering, etc. is narrowed during photographing, and this is effective because a clear image can be obtained. In addition, it is preferable that the conical surface is a curved surface in the conical part and the truncated cone part of the concave part, and does not include a flat surface. The wall surface of the concave portion of the cell accommodating portion usually has an inclined surface as described above, preferably a conical or frustoconical portion on the bottom wall side of the culture vessel. The wall surface of the concave portion of the cell storage unit may have a wall surface perpendicular to the bottom wall of the culture vessel on the opening side from the inclined surface as described above.

細胞収容部の凹部の壁面が円錐状又は円錐台状の部分を含む場合、円錐又は円錐台の中心線と母線とのなす角度は、通常89〜45°、好ましくは88〜65°、より好ましくは85〜80°である。一定の角度以上とすることにより、重力を駆動源として、細胞を配置したい場所(最深部)へ移動させやすく、一定の角度以下とすることにより、顕微鏡で透過観察する際の傾斜面での反射、散乱が起こりにくくなり、鮮明な観察像を得ることができる。   When the wall surface of the concave portion of the cell containing portion includes a conical or frustoconical portion, the angle formed between the center line of the cone or the truncated cone and the generatrix is usually 89 to 45 °, preferably 88 to 65 °, more preferably Is 85 to 80 °. By making the angle greater than a certain angle, it is easy to move to the place (deepest part) where cells are to be placed using gravity as the driving source, and by making the angle less than a certain angle, reflection on the inclined surface during transmission observation with a microscope Scattering is less likely to occur, and a clear observation image can be obtained.

細胞収容部の凹部が培養容器の底壁に平行な底面とそれに垂直な側面とからなる場合は、細胞が凹部内で移動して側面に接触する場合があり、その状態で細胞の撮像を行うと、撮影された画像において輪郭抽出処理により細胞の画像を抽出することが困難であるという問題があるが、細胞収容部の凹部の壁面が、凹部の最も低い位置から凹部の外縁に進むに従って高くなるような傾斜面を有する場合、好ましくは円錐状又は円錐台状の部分を含む場合は、培養される細胞は自動的に凹部の底の部分に存在することとなり、凹部が培養容器の底壁に垂直な側面を傾斜面より開口部側に有していたとしても、これに接触したままとなることはなく、撮像された細胞の画像の輪郭抽出処理を問題なく実施することができる。   In the case where the concave portion of the cell storage portion is composed of a bottom surface parallel to the bottom wall of the culture vessel and a side surface perpendicular thereto, the cell may move in the concave portion and come into contact with the side surface. However, it is difficult to extract an image of the cell by the contour extraction process in the photographed image, but the wall surface of the concave portion of the cell accommodating portion increases as it proceeds from the lowest position of the concave portion to the outer edge of the concave portion. If it has an inclined surface, and preferably includes a conical or frustoconical portion, the cells to be cultured will automatically exist in the bottom portion of the recess, and the recess is the bottom wall of the culture vessel. Even if it has a side surface perpendicular to the inclined surface closer to the opening than the inclined surface, it does not remain in contact therewith, and the contour extraction processing of the captured cell image can be carried out without any problem.

また、細胞収容部の凹部の深さは、特に限定されるものではないが、浅過ぎると、培養容器の輸送時や細胞の分裂時などに細胞が動き、細胞が凹部の範囲外に出てしまう恐れがあるため、確実に細胞を凹部内に保持できるように適宜設定される。例えば、細胞を凹部内に保持するには、深さが細胞の最大径の1/3以上であることが好ましく、1/2以上であることがさらに好ましい。一方、深過ぎると、凹部内に培養液や細胞を導入することが難しくなるため、細胞を凹部内に保持しつつ、深過ぎない値になるよう適宜設定される。例えば、深さの上限を凹部の開口部の直径に対して3倍以下とすることができる。さらに、培養液の導入を容易にするためには、深さは凹部直径の1倍以下であることが好ましく、1/2以下であることが特に好ましい。また、凹部の直径が小さく、深さが深いほど対流が起きにくくなるため、細胞の呼吸や代謝に伴って、周辺の培養液の組成変化が起きやすくなる可能性がある。細胞は、周辺の培養液の組成の影響を受けて成長しやすさが変化するため、細胞の成長を促すように生物学的な影響を考慮して直径と深さを設定することが好ましい。一般的には、細胞の種類によっても異なるが、凹部の深さを50μm〜200μm程度とすることが好ましい。例えば、ウシ受精卵の場合、最大径が250μm程度であるため、深さは80μm以上、さらに好ましくは125μm以上とすることが好ましい。なお、この深さは、傾斜面と、さらに存在する場合は培養容器の底壁に垂直な側面とを有する凹部において、開口部から最深部までを垂直に測った深さをいう。   In addition, the depth of the concave portion of the cell accommodating portion is not particularly limited, but if it is too shallow, the cell moves when the culture vessel is transported or the cell is divided, and the cell comes out of the concave portion. Therefore, it is set appropriately so that the cells can be reliably held in the recesses. For example, in order to hold the cell in the recess, the depth is preferably 1/3 or more of the maximum cell diameter, and more preferably 1/2 or more. On the other hand, if the depth is too deep, it becomes difficult to introduce the culture medium or cells into the recess, so that the value is appropriately set so that the value is not too deep while the cells are held in the recess. For example, the upper limit of the depth can be 3 times or less with respect to the diameter of the opening of the recess. Furthermore, in order to facilitate the introduction of the culture solution, the depth is preferably 1 time or less of the diameter of the recess, and particularly preferably 1/2 or less. Further, since the convection is less likely to occur as the diameter of the recess is smaller and the depth is deeper, there is a possibility that the composition change of the surrounding culture medium is likely to occur with the respiration and metabolism of the cells. Since the ease of growth of cells changes under the influence of the composition of the surrounding culture medium, it is preferable to set the diameter and depth in consideration of biological effects so as to promote cell growth. In general, the depth of the recess is preferably about 50 μm to 200 μm, although it varies depending on the cell type. For example, in the case of a bovine fertilized egg, since the maximum diameter is about 250 μm, the depth is preferably 80 μm or more, more preferably 125 μm or more. This depth refers to the depth measured vertically from the opening to the deepest part in a recess having an inclined surface and, if present, a side surface perpendicular to the bottom wall of the culture vessel.

4個以上近接して形成された凹部は、それらを囲む内壁により、培養容器内のその他の部分と隔てられていてもよい。当該実施形態では、近接した凹部(細胞収容部)の群ごとに内壁で囲まれており、複数の凹部の群が培養容器の底壁に存在する場合は、各群ごとに内壁で囲まれることになる。通常、受精卵等の培養においては、培養容器に受精卵を含む培地の液滴を形成し、液滴をオイルで覆うことにより培地の乾燥が防止されている。4個以上近接して形成された凹部の群をさらに内壁で囲むことにより、その内部(中心側)に培地を収容して培地の分散を防ぐことができる。培地をミネラルオイル等のオイルで覆う場合も同様である。   Four or more recesses formed close to each other may be separated from other parts in the culture vessel by an inner wall surrounding them. In this embodiment, each group of adjacent recesses (cell storage units) is surrounded by an inner wall, and when a plurality of groups of recesses are present on the bottom wall of the culture vessel, each group is surrounded by an inner wall. become. Usually, in culturing a fertilized egg or the like, the medium is prevented from drying by forming droplets of a medium containing the fertilized egg in a culture container and covering the droplets with oil. By enclosing a group of four or more recesses formed close to each other with an inner wall, the medium can be accommodated inside (center side) to prevent the medium from being dispersed. The same applies when the medium is covered with oil such as mineral oil.

さらに、本発明の培養容器は、培養容器の外周部であって細胞収容部を有しない部分に液体収容部を有していてもよい。換言すれば、細胞収容部が存在する部分より外周側に、さらなる内壁と培養容器の側壁によって形成される、液体を収容可能な外堀があるような形状である。近接した凹部を囲む内壁の内部(中心側)には上記と同様に培地を収容し、外堀より内側(中心側)にミネラルオイル等のオイルを収容し、外堀に培地や水などを収容することができる。外堀の容量は、通常1ml以上である。この外堀に培地や水を収容することにより、凹部に細胞を入れる際にピペットやガラスキャピラリーを洗うのに用いることができたり、培養器内の湿度を高められる。従って、培地の乾燥を防止するためのミネラルオイルを使わなくてもすむ場合がある。   Furthermore, the culture container of the present invention may have a liquid storage part in a portion that is an outer peripheral part of the culture container and does not have a cell storage part. In other words, there is an outer moat that is formed by a further inner wall and a side wall of the culture vessel on the outer peripheral side of the portion where the cell storage portion exists, and that can store the liquid. The inside of the inner wall that surrounds the adjacent recess (center side) contains the medium in the same manner as above, the inside of the outer moat (center side) contains mineral oil and the like, and the outer moat contains medium and water. Can do. The capacity of the outer moat is usually 1 ml or more. By storing the culture medium and water in this outer moat, it can be used to wash pipettes and glass capillaries when cells are put into the recess, and the humidity in the incubator can be increased. Therefore, it may not be necessary to use mineral oil for preventing the culture medium from drying.

また、4個以上近接して形成された凹部は、その内部の培地が互いに連通しうるような溝を有していてもよい。溝が存在することにより、細胞培養中に培地が凹部間を循環することができ、オートクライン・パラクライン効果により凹部にそれぞれ収容される細胞の成長を増進させることができる。   Moreover, the four or more recessed parts formed close may have a groove | channel which the culture medium inside can communicate with each other. Due to the presence of the groove, the medium can circulate between the recesses during cell culture, and the growth of the cells respectively accommodated in the recesses can be promoted by the autocrine / paracrine effect.

本発明の培養容器の材質は、特に制限されない。具体的には、金属、ガラス、及びシリコン等の無機材料、プラスチック(例えば、ポリスチレン樹脂、ポリエステル樹脂、ポリエチレン樹脂、ポリプロピレン樹脂、ABS樹脂、ナイロン、アクリル樹脂、フッ素樹脂、ポリカーボネート樹脂、ポリウレタン樹脂、メチルペンテン樹脂、フェノール樹脂、メラミン樹脂、エポキシ樹脂、塩化ビニル樹脂)で代表される有機材料を挙げることができる。本発明の培養容器は、当業者に公知の方法で製造することができる。例えば、プラスチック材料からなる培養容器を製造する場合には、慣用の成形法、例えば射出成形により製造することができる。   The material of the culture container of the present invention is not particularly limited. Specifically, inorganic materials such as metal, glass and silicon, plastics (for example, polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methyl resin) Organic materials represented by pentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). The culture container of the present invention can be produced by a method known to those skilled in the art. For example, when a culture container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding.

本発明の培養容器は、受精卵の発育を促進するような表面処理又は表面コートがなされていてもよい。特に、受精卵の発育を促進するために、他の器官の細胞(例えば、子宮内膜細胞や卵管上皮細胞)と共培養をする場合、これらの細胞をあらかじめ培養容器に接着させる必要がある。このような場合に、培養容器の表面に細胞接着性の材料をコートすると有利である。   The culture container of the present invention may be surface-treated or surface-coated so as to promote the development of fertilized eggs. In particular, in order to promote the development of a fertilized egg, when co-culturing with cells of other organs (for example, endometrial cells or fallopian tube epithelial cells), it is necessary to adhere these cells to a culture vessel in advance. . In such a case, it is advantageous to coat the surface of the culture vessel with a cell adhesive material.

図2aに本発明の培養容器の一実施形態を示す。図2aは、細胞収容部の凹部の壁面が、培養容器の底壁側に円錐状(頂点が丸みを帯びている形状)の部分を有し、それより開口部側に、培養容器の底壁に垂直な壁面を有する実施形態である。図2aに示すとおり、培養容器は、側壁1と底壁2を有し、底壁2には細胞収容部3の凹部4が9個近接して形成され、凹部4の9個の群が4群形成されている。凹部4には細胞6が収容されており、凹部4は円錐状の部分を有し、その壁面(円錐面)7は、凹部の外縁13に進むに従って高くなるように傾斜している。円錐の中心線と母線とのなす角度はαで表される。図2aの実施形態では、9個の近接する凹部の群が、それらを囲む内壁5により、培養容器内のその他の部分と隔てられている。内壁5の内部には培地8が収容され、さらに培養容器全体にミネラルオイル9が収容されている。   FIG. 2a shows an embodiment of the culture vessel of the present invention. FIG. 2a shows that the wall surface of the concave portion of the cell accommodating portion has a conical portion (a shape whose apex is rounded) on the bottom wall side of the culture vessel, and the bottom wall of the culture vessel on the opening side from there. It is embodiment which has a wall surface perpendicular | vertical to. As shown in FIG. 2 a, the culture vessel has a side wall 1 and a bottom wall 2, and nine concave portions 4 of the cell accommodating portion 3 are formed in the bottom wall 2, and nine groups of the concave portions 4 are divided into four groups. A group is formed. Cells 6 are accommodated in the recess 4, and the recess 4 has a conical portion, and the wall surface (conical surface) 7 is inclined so as to increase toward the outer edge 13 of the recess. The angle between the center line of the cone and the generatrix is represented by α. In the embodiment of FIG. 2a, a group of nine adjacent recesses is separated from other parts in the culture vessel by an inner wall 5 surrounding them. Inside the inner wall 5, a medium 8 is accommodated, and further, mineral oil 9 is accommodated in the entire culture container.

図2bに本発明の培養容器の別の実施形態を示す。図2bは、細胞収容部の凹部の壁面が、培養容器の底壁側に円錐状(頂点が丸みを帯びている形状)の部分を有し、それより開口部側に、培養容器の底壁に垂直な壁面を有する実施形態である。図2bに示すとおり、培養容器は、側壁1と底壁2を有し、底壁2には細胞収容部3の凹部4が9個近接して形成され、凹部4の9個の群が4群形成されている。凹部4には細胞6が収容されており、凹部4は円錐状の部分を有し、その壁面(円錐面)7は、凹部の外縁13に進むに従って高くなるように傾斜している。図2bの実施形態では、9個の近接する凹部の群が、それらを囲む内壁5により、培養容器内のその他の部分と隔てられている。さらに、培養容器の外周部であって細胞収容部を有しない部分に液体収容部が存在する。すなわち、培養容器の外周部に、さらなる内壁10と培養容器の側壁とにより形成される外堀11があるような形状である。近接した凹部を囲む内壁の内部(中心側)には培地8が収容され、外堀の内側(中心側)にミネラルオイル9が収容され、外堀11に培地や水12が収容される。ミネラルオイル9は省略することができる。なお、図2a及び図2bでは、近接した凹部が正方格子状に配置されている。   FIG. 2b shows another embodiment of the culture vessel of the present invention. FIG. 2b shows that the wall surface of the concave portion of the cell accommodating portion has a conical portion (a shape in which the apex is rounded) on the bottom wall side of the culture vessel, and the bottom wall of the culture vessel on the opening side from there. It is embodiment which has a wall surface perpendicular | vertical to. As shown in FIG. 2 b, the culture container has a side wall 1 and a bottom wall 2, and nine recesses 4 of the cell accommodating part 3 are formed in the bottom wall 2, and nine groups of the recesses 4 are divided into four groups. A group is formed. Cells 6 are accommodated in the recess 4, and the recess 4 has a conical portion, and the wall surface (conical surface) 7 is inclined so as to increase toward the outer edge 13 of the recess. In the embodiment of FIG. 2b, a group of nine adjacent recesses is separated from other parts in the culture vessel by an inner wall 5 surrounding them. Furthermore, a liquid storage part exists in the outer peripheral part of a culture container and does not have a cell storage part. That is, the outer moat 11 formed by the further inner wall 10 and the side wall of the culture vessel is formed on the outer periphery of the culture vessel. Medium 8 is accommodated inside (center side) the inner wall surrounding the adjacent recess, mineral oil 9 is accommodated inside (center side) of the outer moat, and medium and water 12 are accommodated in outer moat 11. Mineral oil 9 can be omitted. In FIG. 2a and FIG. 2b, the adjacent recesses are arranged in a square lattice pattern.

本発明の培養容器は、培養細胞を自動判別する場合に特に好適に用いられる。培養細胞の判別には、培養細胞の質の判別が含まれる。培養細胞の質の判別には、例えば受精卵を培養する場合には、子宮への移植に適した良質な受精卵であるか否かの判別が含まれる。   The culture container of the present invention is particularly preferably used when automatically distinguishing cultured cells. The discrimination of the cultured cells includes discrimination of the quality of the cultured cells. For example, in the case of culturing a fertilized egg, determination of the quality of the cultured cell includes determination of whether or not it is a high-quality fertilized egg suitable for transplantation into the uterus.

培養細胞の自動判別においては、顕微鏡により取得された培養容器内の細胞の画像をCCDカメラ等の検出装置によって撮像し、得られた像を輪郭抽出処理に付し、画像中の細胞に該当する部分を抽出し、抽出された細胞の画像を画像解析装置で解析することによりその質を判別することができる。画像の輪郭抽出処理については、例えば、特開2006−337110号公報に記載された処理を利用できる。   In automatic discrimination of cultured cells, an image of the cells in the culture vessel obtained by a microscope is picked up by a detection device such as a CCD camera, the obtained image is subjected to contour extraction processing, and corresponds to the cells in the image The quality can be determined by extracting the portion and analyzing the extracted cell image with an image analysis apparatus. As the image contour extraction processing, for example, the processing described in Japanese Patent Laid-Open No. 2006-337110 can be used.

従って一実施形態において本発明は、培養細胞の判別方法に関する。本発明の判別方法は、本発明の培養容器の細胞収容部の凹部に、受精卵、卵細胞、ES細胞及びiPS細胞からなる群から選択される細胞をそれぞれ導入して培養し、顕微鏡により取得された培養細胞の画像をCCDカメラ等の検出装置で撮像し、得られた像を輪郭抽出処理に付すことを含む。   Therefore, in one embodiment, the present invention relates to a method for distinguishing cultured cells. The discrimination method of the present invention is obtained by introducing a cell selected from the group consisting of a fertilized egg, an egg cell, an ES cell, and an iPS cell into the recess of the cell container of the culture container of the present invention and culturing the cells. And taking an image of the cultured cells with a detection device such as a CCD camera, and subjecting the obtained image to contour extraction processing.

培養は、通常、培養容器を培養細胞の発育及び維持に必要なガスを含む環境雰囲気及び一定の環境温度をもたらすインキュベータに入れることにより実施される。必要なガスには、水蒸気、遊離酸素(O)及び二酸化炭素(CO)が含まれる。環境温度とCO含有量を調節することにより、培地のpHを一定時間内に安定させることができる。安定なCO含有量と安定な温度により安定なpHが得られる。培養のための培地は、細胞を培養する能力を有するものであれば特に制限はないが、受精卵培養用の培養液としては、例えば、M16が挙げられる。画像比較プログラムにより、培養中の細胞の画像を予め保存された画像と比較することにより、培養の際の温度、ガス及び培地などの培養条件を調節することもできる。 Cultivation is usually carried out by placing the culture vessel in an incubator that provides an environmental atmosphere containing gas necessary for the growth and maintenance of cultured cells and a constant environmental temperature. Necessary gases include water vapor, free oxygen (O 2 ) and carbon dioxide (CO 2 ). By adjusting the environmental temperature and the CO 2 content, the pH of the medium can be stabilized within a certain time. A stable pH is obtained by a stable CO 2 content and a stable temperature. The medium for culturing is not particularly limited as long as it has the ability to cultivate cells. Examples of the culture medium for fertilized egg culture include M16. By comparing an image of cells in culture with an image stored in advance by an image comparison program, culture conditions such as temperature, gas, and culture medium can be adjusted.

培養細胞の判別は、例えば、特許第3693907号公報に記載の方法を採用できる。
以下、本発明を実施例により説明するが、本発明は実施例の範囲に限定されるものではない。
For example, the method described in Japanese Patent No. 3693907 can be used for discrimination of cultured cells.
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to the range of an Example.

製造例1 培養容器Aの製造
図2aに示す培養容器を、一般的な射出成形加工により製造した。まず、図2aの逆パターンの鋳型を加工し、該鋳型上へ加熱溶融したポリスチレン材料を流し込み、冷却、離型して傾斜付き培養容器Aを得た。該傾斜付き培養容器Aは、バイオクリーンベンチ内のUVライト下に40分程度置いて滅菌してから培養に用いた。
Production Example 1 Production of Culture Container A The culture container shown in FIG. 2a was produced by a general injection molding process. First, the mold having the reverse pattern shown in FIG. 2a was processed, and the polystyrene material heated and melted was poured onto the mold, cooled, and released from the mold to obtain a culture vessel A with an inclination. The inclined culture vessel A was placed in a bioclean bench under UV light for about 40 minutes and sterilized before being used for culture.

製造例2 培養容器Bの製造
直径約35mmのポリスチレン製ディッシュを用意し、その底面へ直径0.4mmの貫通口を1mm間隔で加工して貫通口付きディッシュを得た。次に、貫通口付きディッシュの底面へ、ディッシュの外側からカバーガラスを接着して培養容器Bを得た(図4)。貫通孔の側面はカバーガラス接着には、接着剤を用いた。該培養容器Bは、バイオクリーンベンチ内のUVライト下に40分程度置いて滅菌してから培養に用いた。
Production Example 2 Production of Culture Container B A polystyrene dish having a diameter of about 35 mm was prepared, and through-holes with a diameter of 0.4 mm were processed on the bottom surface at intervals of 1 mm to obtain a dish with a through-hole. Next, a cover glass was adhered from the outside of the dish to the bottom surface of the dish with a through-hole to obtain a culture vessel B (FIG. 4). The adhesive was used for the cover glass adhesion on the side surface of the through hole. The culture vessel B was placed in a bioclean bench under UV light for about 40 minutes and sterilized before use for culture.

製造例3 培養容器Cの製造
図11〜16に示す培養容器Cを、一般的な射出成形加工により製造した。まず、図11の逆パターンの鋳型を加工し、磨き加工により平滑性を高めた。該鋳型上へ加熱溶融したポリスチレン材料を流し込み、冷却、離型して傾斜付き培養容器Cを得た。この培養容器Cの細胞収容部3には、5×5の正方格子状に配置された25個の凹部4が形成されている。製造した培養容器Cの外観写真を図17に示す。該傾斜付き培養容器Cは、エタノール中に1時間浸漬して洗浄した後、バイオクリーンベンチ内のUVライト下に40分程度置いて滅菌してから培養に用いた。なお、図12において、内壁5で囲まれた領域の直径dは7mmであり、図13及び14で示す凹部4のピッチaは420μmである。また、図15及び16において、凹部の直径rは270μm、深さLは150μmに設定した。さらに、円錐状に形成した壁面7について、円錐の中心線と母線とのなす角度αは83°に設定した。
Production Example 3 Production of Culture Container C Culture container C shown in FIGS. 11 to 16 was produced by a general injection molding process. First, the reverse pattern mold of FIG. 11 was processed, and the smoothness was improved by polishing. A polystyrene material heated and melted was poured onto the mold, cooled, and released from the mold to obtain an inclined culture vessel C. In the cell container 3 of the culture vessel C, 25 concave portions 4 arranged in a 5 × 5 square lattice are formed. An appearance photograph of the produced culture vessel C is shown in FIG. The inclined culture vessel C was immersed in ethanol for 1 hour, washed, placed under UV light in a bioclean bench for about 40 minutes, and then used for culture. In FIG. 12, the diameter d of the region surrounded by the inner wall 5 is 7 mm, and the pitch a of the recesses 4 shown in FIGS. 13 and 14 is 420 μm. 15 and 16, the diameter r of the recess is set to 270 μm and the depth L is set to 150 μm. Further, with respect to the wall surface 7 formed in a conical shape, the angle α formed between the center line of the cone and the generatrix was set to 83 °.

図18に、製造した培養容器Cの細胞収容部3の断面形状計測結果(計測装置:キーエンス社製ダブルスキャン高精度レーザ測定器LT−9010M及び高精度形状測定システムKS−1100、測定間隔5μm)、図19に凹部4の断面形状計測結果(計測装置及び測定条件は上記と同様)をそれぞれ示す。また、図20には、図19の右側傾斜面に対して、線形フィッティングを行い、さらに傾きを補正した図を示す。図20から、最大高さRyは0.47μmと求められた。   FIG. 18 shows a cross-sectional shape measurement result of the cell container 3 of the manufactured culture vessel C (measuring device: double-scan high-precision laser measuring instrument LT-9010M and high-precision shape measuring system KS-1100 manufactured by Keyence Corporation, measurement interval 5 μm). FIG. 19 shows a cross-sectional shape measurement result of the recess 4 (measurement apparatus and measurement conditions are the same as above). FIG. 20 shows a diagram in which linear fitting is performed on the right inclined surface of FIG. 19 and the inclination is corrected. From FIG. 20, the maximum height Ry was determined to be 0.47 μm.

製造例4 培養容器Dの製造
鋳型の製造時に、磨き処理を施さない以外は上記製造例3と同様の手順で培養容器Dを製造した。図21に培養容器Dの凹部4の断面形状計測結果を示す。また、図22には、図21の右側傾斜面に対して、線形フィッティングを行い、さらに傾きを補正した図を示す。図22から、最大高さRyは4.9μmと求められた。
Production Example 4 Production of Culture Container D Culture container D was produced in the same manner as in Production Example 3 except that no polishing treatment was performed during the production of the casting mold. FIG. 21 shows a cross-sectional shape measurement result of the recess 4 of the culture vessel D. FIG. 22 shows a diagram in which linear fitting is performed on the right inclined surface of FIG. 21 and the inclination is further corrected. From FIG. 22, the maximum height Ry was determined to be 4.9 μm.

製造例5 培養容器Eの製造
図23〜図28に示す培養容器Eを、上記製造例3と同様の手順で製造した。なお、培養容器Eでは、凹部4の壁面7を、凹部の最深部から凹部の外縁13へ向かって曲線状に高くなるような傾斜面としている。培養容器Eのその他の寸法は図11〜16に示す培養容器Cと同一である。
Production Example 5 Production of Culture Container E Culture vessel E shown in FIGS. 23 to 28 was produced in the same procedure as in Production Example 3 above. In the culture vessel E, the wall surface 7 of the recess 4 is an inclined surface that increases in a curved shape from the deepest portion of the recess toward the outer edge 13 of the recess. Other dimensions of the culture vessel E are the same as those of the culture vessel C shown in FIGS.

図29に培養容器Eの凹部4の断面形状計測結果を示す。また、図30には、図29の右側傾斜面に対して、線形フィッティングを行い、さらに傾きを補正した図を示す。図30から、最大高さRyは2.9μmと求められた。   FIG. 29 shows the cross-sectional shape measurement result of the recess 4 of the culture vessel E. FIG. 30 shows a diagram in which linear fitting is performed on the right inclined surface of FIG. 29 and the inclination is corrected. From FIG. 30, the maximum height Ry was determined to be 2.9 μm.

(実施例1)
製造例1で製造した培養容器Aを用いて、ラット受精卵を培養し、観察した画像を用いて、受精卵の輪郭抽出処理を行った。
Example 1
A rat fertilized egg was cultured using the culture container A manufactured in Production Example 1, and the contour extraction process of the fertilized egg was performed using the observed image.

ラット受精卵を製造例1の培養容器Aの細胞収容部の凹部にガラスキャピラリーを用いて導入した。さらに、培地の蒸発を防ぐために、培地を覆うようにミネラルオイルを入れた。培養は、COインキュベータ(5%CO、5%O及び90%空気、37℃、湿度飽和)にて行った。 The rat fertilized egg was introduced into the recess of the cell housing part of the culture container A of Production Example 1 using a glass capillary. In addition, mineral oil was added to cover the medium in order to prevent the medium from evaporating. The culture was performed in a CO 2 incubator (5% CO 2 , 5% O 2 and 90% air, 37 ° C., humidity saturation).

受精卵が初期の卵割をした後に、倍率20倍の観察装置(高倍率のレンズと撮像用カメラを備えている)で撮影した写真を図5に示す。
受精卵の輪郭を抽出するため、以下のような閾値処理を施した。
FIG. 5 shows a photograph taken with an observation device (equipped with a high-magnification lens and an imaging camera) after the fertilized egg had an initial cleavage.
In order to extract the outline of a fertilized egg, the following threshold processing was performed.

図6は図5に対して閾値処理を行った画像である。製造例1の培養容器Aを用いた場合、閾値処理を施すことによって、図6に示すように、凹部の壁面の影響無く受精卵の輪郭を識別することが可能であった。   FIG. 6 is an image obtained by performing threshold processing on FIG. When the culture container A of Production Example 1 was used, it was possible to identify the outline of a fertilized egg without being affected by the wall surface of the recess, by performing threshold processing, as shown in FIG.

閾値処理は、例えばカメラで撮影した受精卵画像の各画素値(=輝度値)を予め設定された第1の閾値と第2の閾値(第1の閾値<第2の閾値)に基づいて、第1の閾値以上かつ第2の閾値以下の場合には1(白)0(黒)を設定し、第1の閾値より小さい又は2の閾値より大きい場合には0(黒)1(白)を設定するといった処理である(=2値化処理)。   The threshold processing is based on, for example, a first threshold value and a second threshold value (first threshold value <second threshold value) set in advance for each pixel value (= luminance value) of a fertilized egg image captured by a camera. 1 (white) 0 (black) is set if it is greater than or equal to the first threshold and less than or equal to the second threshold, and 0 (black) or 1 (white) if it is less than the first threshold or greater than the second threshold. Is set (= binarization process).

(実施例2)
製造例3で製造した培養容器Cを用いて、マウス受精卵を培養し、観察した画像を用いて、受精卵の輪郭抽出処理を行った。
(Example 2)
A mouse fertilized egg was cultured using the culture container C manufactured in Production Example 3, and the contour extraction process of the fertilized egg was performed using the observed image.

マウス受精卵を製造例3の培養容器Cの細胞収容部の凹部にガラスキャピラリーを用いて導入した。さらに、培地の蒸発を防ぐために、培地を覆うようにミネラルオイルを入れた。培養は、COインキュベータ(5%CO、5%O及び90%空気、37℃、湿度飽和)にて行った。 A mouse fertilized egg was introduced into the concave portion of the cell housing part of the culture container C of Production Example 3 using a glass capillary. In addition, mineral oil was added to cover the medium in order to prevent the medium from evaporating. The culture was performed in a CO 2 incubator (5% CO 2 , 5% O 2 and 90% air, 37 ° C., humidity saturation).

受精卵が初期の卵割をした後に、倍率10倍の観察装置(高倍率のレンズと撮像用カメラを備えている)で撮影した写真を図31に示す。実施例1と同様の閾値処理により、受精卵の輪郭を抽出した。図32は図31に対して閾値処理を行った画像である。製造例3の培養容器Cを用いた場合、閾値処理を施すことによって、図32に示すように、凹部の壁面の影響無くマウス受精卵の輪郭を識別することが可能であった。   FIG. 31 shows a photograph taken by an observation apparatus (equipped with a high-magnification lens and an imaging camera) after the fertilized egg has split the initial stage. The outline of the fertilized egg was extracted by the threshold processing similar to that in Example 1. FIG. 32 is an image obtained by performing threshold processing on FIG. When the culture container C of Production Example 3 was used, it was possible to identify the outline of a mouse fertilized egg without being affected by the wall surface of the recess, as shown in FIG. 32, by performing threshold processing.

(実施例3)
実施例2と同様に培養容器Cにウシ受精卵を導入した直後に、倍率4倍の観察装置(高倍率のレンズと撮像用カメラを備えている)で撮影し、一部を切り出した写真を図33に示す。実施例1と同様の閾値処理により、受精卵の輪郭を抽出した。図34は図33に対して閾値処理を行った画像である。製造例3の培養容器Cを用いた場合、閾値処理を施すことによって、図34に示すように、凹部の壁面の影響無くウシ受精卵の輪郭を識別することが可能であった。
(Example 3)
Immediately after introducing the fertilized bovine egg into the culture vessel C as in Example 2, a photograph taken with an observation device (equipped with a high-magnification lens and an imaging camera) was taken at a magnification of 4 times, and a photograph of a part cut out was taken. As shown in FIG. The outline of the fertilized egg was extracted by the threshold processing similar to that in Example 1. FIG. 34 is an image obtained by performing threshold processing on FIG. When the culture container C of Production Example 3 was used, by performing threshold processing, it was possible to identify the contour of the bovine fertilized egg without being affected by the wall surface of the recess as shown in FIG.

(実施例4)
実施例2と同様に培養容器Dにマウス受精卵を導入し初期の卵割をした後に、倍率10倍の観察装置(高倍率のレンズと撮像用カメラを備えている)で撮影した。実施例1と同様の閾値処理により、受精卵の輪郭を抽出したところ、凹部の壁面の影響無くマウス受精卵の輪郭を識別することが可能であった。しかしながら、細胞収容部の凹部の傾斜面が平滑化されていないため、透過率が低く、ノイズの影響があり、実施例2よりも識別が困難であった。
Example 4
In the same manner as in Example 2, a mouse fertilized egg was introduced into the culture vessel D, and the initial cleavage was performed. Then, the image was taken with an observation device having a magnification of 10 (including a high-magnification lens and an imaging camera). When the contour of the fertilized egg was extracted by the threshold processing similar to that in Example 1, it was possible to identify the contour of the mouse fertilized egg without the influence of the wall surface of the recess. However, since the inclined surface of the concave portion of the cell accommodating portion is not smoothed, the transmittance is low, there is an influence of noise, and identification is more difficult than in Example 2.

(実施例5)
実施例2と同様に培養容器Eにマウス受精卵を導入し初期の卵割をした後に、倍率10倍の観察装置(高倍率のレンズと撮像用カメラを備えている)で撮影した。実施例1と同様の閾値処理により、受精卵の輪郭を抽出したところ、凹部の壁面の影響無くマウス受精卵の輪郭を識別することが可能であった。しかしながら、細胞収容部の凹部における傾斜面が培養容器Cのように直線状ではなく、曲率を有するため、受精卵が凹部の壁面に接触している確率が実施例2よりも高かった。以上の実施例2、4及び5の結果から、凹部の傾斜面は直線部分を含み且つRy値が小さいほど、細胞を凹部の中央に集めることができ、細胞の判別に有利であることが分かった。
(Example 5)
In the same manner as in Example 2, a mouse fertilized egg was introduced into the culture vessel E and the initial cleavage was performed, and then the image was taken with an observation device having a magnification of 10 times (equipped with a high magnification lens and an imaging camera). When the contour of the fertilized egg was extracted by the threshold processing similar to that in Example 1, it was possible to identify the contour of the mouse fertilized egg without the influence of the wall surface of the recess. However, since the inclined surface in the concave portion of the cell accommodating portion is not linear as in the culture vessel C and has a curvature, the probability that the fertilized egg is in contact with the wall surface of the concave portion is higher than in Example 2. From the results of Examples 2, 4 and 5 described above, it can be seen that the inclined surface of the concave portion includes a straight portion and the smaller the Ry value, the more cells can be collected at the center of the concave portion, which is advantageous for cell discrimination. It was.

(比較例1)
比較例1として、製造例2で製造した培養容器Bを用いて、マウス受精卵を培養し、観察した画像を用いて、受精卵の輪郭抽出処理を行った。
(Comparative Example 1)
As Comparative Example 1, mouse fertilized eggs were cultured using the culture container B manufactured in Production Example 2, and the contour extraction process of the fertilized eggs was performed using the observed images.

マウス受精卵を製造例2の培養容器Bの細胞収容部の凹部にガラスキャピラリーを用いて導入した。さらに、培地の蒸発を防ぐために、培地を覆うようにミネラルオイルを入れた。培養は、COインキュベータ(5%CO、5%O及び90%空気、37℃、湿度飽和)にて行った。 A mouse fertilized egg was introduced into the recess of the cell housing part of the culture vessel B of Production Example 2 using a glass capillary. In addition, mineral oil was added to cover the medium in order to prevent the medium from evaporating. The culture was performed in a CO 2 incubator (5% CO 2 , 5% O 2 and 90% air, 37 ° C., humidity saturation).

製造例2で製造した培養容器Bに受精卵を導入した直後に、倍率10の観察装置(高倍率のレンズと撮像用カメラを備えている)で撮影した写真を図7及び9に示す。   Immediately after introducing a fertilized egg into the culture container B produced in Production Example 2, photographs taken with an observation device having a magnification of 10 (equipped with a high-magnification lens and an imaging camera) are shown in FIGS.

実施例1と同様の閾値処理により、受精卵の輪郭を抽出した。図8は図7に対して閾値処理をしたもの、図10は図9に対して閾値処理をしたものである。   The outline of the fertilized egg was extracted by the threshold processing similar to that in Example 1. FIG. 8 shows the result of performing threshold processing on FIG. 7, and FIG. 10 shows the result of performing threshold processing on FIG.

図8のように、偶然に細胞収容部の凹部の壁面から離れている受精卵については、受精卵の輪郭を抽出できたが、図10のように、細胞収容部の凹部の壁面に接触している受精卵については、受精卵の輪郭を抽出することができなかった。   As shown in FIG. 8, the outline of the fertilized egg could be extracted from a fertilized egg that was accidentally separated from the wall surface of the concave portion of the cell accommodating portion. However, as shown in FIG. As for the fertilized egg, the outline of the fertilized egg could not be extracted.

1:側壁、2:底壁、3:細胞収容部、4:細胞収容部の凹部、5:内壁、6:細胞、7:凹部の壁面(円錐面)、8:培地、9:ミネラルオイル、10:内壁、11:外堀、12:培地又は水、13:外縁 1: side wall, 2: bottom wall, 3: cell accommodating part, 4: concave part of cell accommodating part, 5: inner wall, 6: cell, 7: wall surface (conical surface) of concave part, 8: medium, 9: mineral oil, 10: inner wall, 11: outer moat, 12: medium or water, 13: outer edge

Claims (14)

個別管理が必要とされる細胞を培養するための、底壁と側壁とを有する培養容器であって、
底壁に、凹部を有する細胞収容部が配置されており、
凹部が4個以上近接しており、
凹部の壁面が、凹部の最も低い位置から凹部の外縁に進むに従って高くなるような傾斜面と、前記傾斜面よりも前記凹部の開口部側に位置し、前記傾斜面から起ち上がる壁面とを有する、
前記培養容器。
A culture vessel having a bottom wall and a side wall for culturing cells that require individual management,
A cell housing portion having a recess is disposed on the bottom wall,
4 or more concave parts are close to each other,
The wall surface of the recess has an inclined surface that becomes higher from the lowest position of the recess toward the outer edge of the recess, and a wall surface that is positioned closer to the opening of the recess than the inclined surface and rises from the inclined surface ,
The culture container.
近接する凹部間のピッチが1mm以下である、請求項1に記載の培養容器。   The culture container according to claim 1, wherein a pitch between adjacent concave portions is 1 mm or less. 凹部の壁面が、直線部分を含む傾斜面を有する、請求項1又は2に記載の培養容器。   The culture vessel according to claim 1 or 2, wherein the wall surface of the recess has an inclined surface including a straight portion. 傾斜面の表面粗さについて、最大高さRyが1.0μm未満である、請求項1〜3のいずれか1項に記載の培養容器。   The culture container according to any one of claims 1 to 3, wherein the maximum height Ry is less than 1.0 µm with respect to the surface roughness of the inclined surface. 凹部の開口部の開口幅が100μm以上である、請求項1〜のいずれか1項に記載の培養容器。 The culture container according to any one of claims 1 to 4 , wherein the opening width of the opening of the recess is 100 µm or more. 凹部の深さが50μm〜200μmである、請求項1〜のいずれか1項に記載の培養容器。 The culture container according to any one of claims 1 to 5 , wherein the recess has a depth of 50 µm to 200 µm. 凹部の壁面が、円錐状又は円錐台状の部分を含み、前記円錐状又は円錐台状の部分が、培養容器の底壁側に、円錐の頂点又は円錐台の上面及び下面のうち面積の狭い方が位置するように形成され、前記面積の狭い方の直径は、100μm〜280μmである細胞の最大径の2分の1以下である、請求項1〜6のいずれか1項に記載の培養容器。The wall surface of the recess includes a conical or frustoconical portion, and the conical or frustoconical portion has a narrow area on the bottom wall side of the culture vessel, of the apex of the cone or the upper and lower surfaces of the frustoconical The culture according to any one of claims 1 to 6, wherein a diameter of the narrower area is not more than one half of a maximum diameter of a cell having a size of 100 µm to 280 µm. container. 面積の狭い方の直径が、10μm以下である、請求項7に記載の培養容器。The culture container according to claim 7, wherein the diameter of the narrower area is 10 µm or less. 円錐又は円錐台の中心線と母線とのなす角度が、89°〜45°である、請求項7又は8に記載の培養容器。The culture vessel according to claim 7 or 8, wherein an angle formed between the center line of the cone or the truncated cone and the generatrix is 89 ° to 45 °. 近接する4個以上の凹部が、それらを囲む内壁により、培養容器内のその他の部分と隔てられている、請求項1〜9のいずれか1項に記載の培養容器。   The culture container according to claim 1, wherein four or more adjacent recesses are separated from other parts in the culture container by an inner wall surrounding them. 内壁の平面視形状が円形である、請求項10に記載の培養容器。   The culture vessel according to claim 10, wherein the shape of the inner wall in plan view is circular. 培養容器の外周部であって細胞収容部を有しない部分に液体収容部を有する、請求項10又は11に記載の培養容器。   The culture container according to claim 10 or 11, wherein the culture container has a liquid container in a portion that is an outer peripheral part of the culture container and does not have a cell container. 近接する凹部を連通する溝を有する、請求項1〜12のいずれか1項に記載の培養容器。   The culture container according to any one of claims 1 to 12, which has a groove communicating with the adjacent recess. 細胞が、受精卵、卵細胞、ES細胞及びiPS細胞からなる群から選択される、請求項1〜13のいずれか1項に記載の培養容器。   The culture container according to any one of claims 1 to 13, wherein the cell is selected from the group consisting of a fertilized egg, an egg cell, an ES cell, and an iPS cell.
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