JP2020171223A - Cryopreservation tool - Google Patents
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- JP2020171223A JP2020171223A JP2019074671A JP2019074671A JP2020171223A JP 2020171223 A JP2020171223 A JP 2020171223A JP 2019074671 A JP2019074671 A JP 2019074671A JP 2019074671 A JP2019074671 A JP 2019074671A JP 2020171223 A JP2020171223 A JP 2020171223A
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Abstract
Description
本発明は、細胞または組織を凍結保存する凍結保存用治具に関する。 The present invention relates to a cryopreservation jig for cryopreserving cells or tissues.
細胞または組織の優れた保存技術は、様々な産業分野で求められている。例えば、牛の胚移植技術においては、胚を凍結保存し、受胚牛の発情周期に合わせて胚を融解し、移植することが行われている。また、ヒトの不妊治療においては、母体から卵子または卵巣を採取後、移植に適したタイミングに合わせるために凍結保存しておき、移植時に融解して用いる手技がなされている。 Excellent cell or tissue preservation techniques are sought after in various industrial fields. For example, in the embryo transfer technique for cattle, embryos are cryopreserved, thawed and transplanted in accordance with the estrous cycle of the recipient cattle. Further, in human infertility treatment, after collecting an egg or an ovary from a mother, it is cryopreserved in order to adjust to a timing suitable for transplantation, and thawed at the time of transplantation for use.
一般に、生体内から採取された細胞または組織は、たとえ培養液の中であっても、次第に活性が失われていくことから、生体外での長期間の培養は好ましくない。そのため、生体活性を失わせずに長期間保存するための技術が重要になる。優れた保存技術により、採取された細胞または組織を、より正確に分析することが可能になる。また、優れた保存技術により、より高い生体活性を保ったまま、細胞または組織を移植に用いることが可能となり、移植後の生着率の向上が望める。さらには、生体外で培養した培養皮膚、生体外で構築した、いわゆる細胞シートのような移植のための人工の組織を、順次生産、保存しておき、必要な時に使用することも可能となり、医療、産業の両面において大きなメリットが期待できる。 In general, cells or tissues collected from a living body gradually lose their activity even in a culture solution, and thus long-term culture outside the living body is not preferable. Therefore, a technique for long-term storage without losing biological activity is important. Superior preservation techniques allow for more accurate analysis of harvested cells or tissues. In addition, excellent preservation technology makes it possible to use cells or tissues for transplantation while maintaining higher biological activity, and it is expected that the survival rate after transplantation will be improved. Furthermore, it is also possible to sequentially produce and store cultured skin cultured in vitro and artificial tissues for transplantation such as so-called cell sheets constructed in vitro and use them when necessary. Great benefits can be expected in both medical and industrial fields.
細胞または組織の保存方法として、例えば緩慢凍結法が知られている。この方法では、例えば、リン酸緩衝生理食塩水などの生理的溶液に耐凍剤を含有させることで得られた保存液に、細胞または組織を浸漬する。該耐凍剤としては、グリセロール、エチレングリコールなどの化合物が用いられる。該保存液に、細胞または組織を浸漬後、比較的遅い冷却速度（例えば０．３〜０．５℃／分の速度）で、−３０〜−３５℃まで冷却すると、細胞内外または組織内外の保存液が十分に冷却され、粘性が高くなる。このような状態で、該保存液中の細胞または組織を、さらに液体窒素の温度（−１９６℃）まで冷却すると、細胞内または組織内とその外の周囲の微少溶液が、いずれも非結晶のまま固体となるガラス化が起こる。ガラス化により、細胞内外または組織内外が固化すると、実質的に分子の動きがなくなるので、ガラス化された細胞または組織を液体窒素中に保存することで、半永久的に保存できると考えられる。 As a method for preserving cells or tissues, for example, a slow freezing method is known. In this method, cells or tissues are immersed in a preservation solution obtained by containing a freezing agent in a physiological solution such as, for example, phosphate buffered saline. As the antifreeze agent, compounds such as glycerol and ethylene glycol are used. After immersing the cells or tissues in the preservation solution, when the cells or tissues are cooled to -30 to -35 ° C. at a relatively slow cooling rate (for example, 0.3 to 0.5 ° C./min), the cells are inside or outside the cells or inside and outside the tissues. The storage solution is sufficiently cooled and becomes highly viscous. In such a state, when the cells or tissues in the storage solution are further cooled to the temperature of liquid nitrogen (-196 ° C.), the minute solutions inside and around the cells or tissues are all non-crystalline. Vitrification that becomes solid as it is occurs. When the inside and outside of cells or the inside and outside of tissues are solidified by vitrification, the movement of molecules is substantially stopped. Therefore, it is considered that the vitrified cells or tissues can be stored semi-permanently by storing them in liquid nitrogen.
例えば、特許文献１には緩慢凍結法によってブタ胚を凍結保存することが記載され、特許文献２にはウシ胚を緩慢凍結法によって保存することが記載されている。 For example, Patent Document 1 describes that pig embryos are cryopreserved by a slow freezing method, and Patent Document 2 describes that bovine embryos are stored by a slow freezing method.
しかしながら緩慢凍結法は、比較的遅い冷却速度で細胞または組織を冷却することから、凍結保存のための操作に時間を要する。また冷却速度を制御するための装置、または治具を必要とする問題がある。加えて細胞外または組織外の保存液中に氷晶が形成されるので、細胞または組織が該氷晶により物理的に損害を受けるおそれがある。 However, the slow freezing method cools cells or tissues at a relatively slow cooling rate, so that the operation for cryopreservation takes time. In addition, there is a problem that a device or a jig for controlling the cooling rate is required. In addition, since ice crystals are formed in the extracellular or extracellular storage solution, the cells or tissues may be physically damaged by the ice crystals.
一方、上記した緩慢凍結法における問題点を解消する保存方法として、ガラス化凍結法が知られている。ガラス化凍結法とは、グリセロール、エチレングリコール、ＤＭＳＯ（ジメチルスルホキシド）などの耐凍剤を多量に含む保存液の凝固点降下により、氷点下であっても氷晶ができにくくなる原理を用いたものである。この保存液を、急速に液体窒素中で冷却させると、氷晶を生じさせないまま固体化させることができる。このように固体化することをガラス化凍結という。ガラス化凍結に用いられる耐凍剤を多量に含む保存液は、ガラス化液と呼称される。 On the other hand, the vitrification freezing method is known as a storage method for solving the above-mentioned problems in the slow freezing method. The vitrification freezing method uses the principle that ice crystals are less likely to form even below freezing point due to the freezing point depression of a storage solution containing a large amount of freezing agent such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide). .. When this preservation solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. Solidification in this way is called vitrification freezing. A preservative solution containing a large amount of antifreeze used for vitrification freezing is called a vitrification solution.
ガラス化凍結法では、ガラス化液に細胞または組織を浸漬させ、その後、液体窒素の温度（−１９６℃）で急速に冷却する。ガラス化凍結法は、このような簡便かつ迅速な工程であるために、凍結保存のための操作に長い時間を必要としないほか、温度制御をするための装置または治具を必要としないという利点がある。 In the vitrification freezing method, cells or tissues are immersed in a vitrified solution and then rapidly cooled at the temperature of liquid nitrogen (-196 ° C.). Since the vitrification freezing method is such a simple and quick process, it does not require a long time for the operation for cryopreservation, and has the advantage that it does not require a device or jig for temperature control. There is.
ガラス化凍結法を用いた細胞または組織の凍結保存については、様々な方法で、様々な種類の細胞または組織を用いた例が示されている。例えば、特許文献３では、動物、ヒトの生殖細胞または体細胞へのガラス化凍結法の適用が、極めて有用であることが示されている。非特許文献１では、ショウジョウバエの胚の保存に、ガラス化凍結法が有効であることが示されている。さらに特許文献４では、植物培養細胞や組織の保存において、ガラス化凍結法が有効であることが示されている。 Regarding the cryopreservation of cells or tissues using the vitrification freezing method, examples using various types of cells or tissues are shown by various methods. For example, Patent Document 3 shows that the application of the vitrification freezing method to animal, human germ cells or somatic cells is extremely useful. Non-Patent Document 1 shows that the vitrification freezing method is effective for the preservation of Drosophila embryos. Further, Patent Document 4 shows that the vitrification freezing method is effective in the preservation of cultured plant cells and tissues.
しかしながら、ガラス化液に含まれる高濃度の耐凍剤には化学的毒性がある。このため細胞または組織の凍結保存時には、細胞または組織の周囲に存在するガラス化液は少ない方が望ましく、細胞が保存液に暴露される時間、つまり凍結までの時間が短時間であることが望ましい。さらには、解凍後ただちに保存液を希釈する必要がある。 However, the high concentration of antifreeze contained in the vitrified solution is chemically toxic. Therefore, during cryopreservation of cells or tissues, it is desirable that the amount of vitrification solution existing around the cells or tissues is small, and it is desirable that the time when the cells are exposed to the preservation solution, that is, the time until freezing is short. .. Furthermore, it is necessary to dilute the preservation solution immediately after thawing.
特許文献５、特許文献６には、短冊状の可撓性かつ無色透明なフィルムを卵付着保持用ストリップとして有する凍結保存用治具が記載されている。かかる文献では該ストリップの幅を制限することにより、細胞または組織の周囲に存在するガラス化液量を低減している。 Patent Documents 5 and 6 describe a cryopreservation jig having a strip-shaped flexible and colorless transparent film as an egg attachment holding strip. In such literature, limiting the width of the strip reduces the amount of vitrification liquid present around the cell or tissue.
特許文献７には、金網、紙などの天然物や、合成樹脂からなるフィルム状物で貫通孔を有した保存液除去材を有する凍結保存用治具が記載されている。かかる文献において卵子または胚は、ガラス化液と共に保存液除去材上に載置され、卵子または胚の周囲に付着した余分なガラス化液は、保存液除去材の下部から吸引することで取り除かれる。 Patent Document 7 describes a cryopreservation jig having a preservative solution removing material having through holes in a film-like material made of synthetic resin and a natural product such as wire mesh or paper. In such literature, the egg or embryo is placed on the preservation solution remover together with the vitrifying solution, and the excess vitrification solution adhering to the periphery of the egg or embryo is removed by suction from the lower part of the preservation solution remover. ..
余分なガラス化液を下部から吸引せずとも取り除くことが可能で、細胞を載置する際の作業性が改善された凍結保存用治具として、特許文献８には特定のヘーズ値を有する保存液吸収体を利用した凍結保存用治具が、特許文献９には、特定の屈折率を有する保存液吸収体を利用した凍結保存用治具が、特許文献１０には、特定の平均繊維径を有する素材で形成された多孔質構造体を保存液吸収体として有する凍結保存用治具がそれぞれ記載されている。 Patent Document 8 describes storage having a specific haze value as a cryopreservation jig that can remove excess vitrified liquid without sucking from the bottom and has improved workability when placing cells. A jig for cryopreservation using a liquid absorber is described in Patent Document 9, a jig for cryopreservation using a storage liquid absorber having a specific refractive index is described in Patent Document 9, and a specific average fiber diameter is described in Patent Document 10. Each of the cryopreservation jigs having a porous structure formed of the material having the above as a preservation solution absorber is described.
また、上記したような保存液吸収体を有する凍結保存用治具について、特許文献１１には、保存液吸収体と支持体が、部分的に接着層が存在しない形で接着されている例が記載されている。さらに、特許文献１２には、短冊状の可撓性かつ無色透明なフィルムを卵付着保持用ストリップとして有し、さらに該フィルム上の一部に吸水部（保存液吸収体）に取り囲まれた欠損部が設けられることにより、過剰なガラス化液の除去操作が不要な凍結保存用治具の例が記載されている。 Further, with respect to the cryopreservation jig having the preservation solution absorber as described above, Patent Document 11 provides an example in which the preservation solution absorber and the support are partially bonded in a form in which an adhesive layer does not exist. Are listed. Further, Patent Document 12 has a strip-shaped flexible and colorless transparent film as a strip for holding egg adhesion, and a defect in which a part of the film is surrounded by a water absorbing portion (preservative liquid absorber). An example of a cryopreservation jig that does not require an operation of removing excess vitrification liquid by providing a portion is described.
特許文献５、６に記載されている凍結保存用治具では、卵子または胚を載置するフィルムの幅を制限することにより、少ない量のガラス化液とともに卵子又は胚を凍結保存する方法が記載されている。該方法では、作業者の操作によって、極少量のガラス化液とともに、卵子又は胚をフィルム上に載置するが、フィルムが顕微鏡観察下で視認しづらく、載置箇所の幅が狭いため、操作の難度が高いといった問題があった。また該方法をもとにしたクライオトップ（登録商標）法では、より少ない量のガラス化液と共に卵子又は胚を凍結保存するために、一度ガラス化液とともに卵子又は胚をフィルム上に載せた後に、余分なガラス化液を吸引してフィルム上から除去するといった煩雑な操作がなされることもあった。 The cryopreservation jigs described in Patent Documents 5 and 6 describe a method of cryopreserving an egg or embryo together with a small amount of vitrifying solution by limiting the width of a film on which the egg or embryo is placed. Has been done. In this method, an egg or embryo is placed on a film together with a very small amount of vitrified solution by an operator's operation, but the film is difficult to see under a microscope and the width of the placement location is narrow. There was a problem that the difficulty level was high. In the cryotop (registered trademark) method based on this method, the egg or embryo is once placed on a film together with the vitrification solution in order to cryopreserve the egg or embryo with a smaller amount of the vitrification solution. In some cases, complicated operations such as sucking excess vitrifying liquid and removing it from the film may be performed.
特許文献７に記載されている凍結保存用治具では、ガラス化液除去材によって卵子又は胚の周囲に付着した余分なガラス化液を除くことにより、優れた生存率でこれらの生殖細胞を凍結保存させる方法が提案されている。該方法では、余分なガラス化液を除く際に、下部からの吸引操作を必要とするため、煩雑な操作となり、短時間でガラス化凍結保存操作を行うことは困難であった。 The cryopreservation jig described in Patent Document 7 freezes these germ cells with an excellent viability by removing excess vitrification solution adhering to the periphery of the egg or embryo with a vitrification solution removing material. A method of saving has been proposed. In this method, a suction operation from the lower part is required to remove the excess vitrification liquid, which is a complicated operation, and it is difficult to perform the vitrification cryopreservation operation in a short time.
特許文献８〜１１に記載されている凍結保存用治具では、上記いずれの課題も解決しており、細胞または組織の凍結保存作業を容易にかつ確実に行う上で有効である。これらの凍結保存用治具では、余分な保存液を、凍結保存用治具が有する保存液吸収体が吸収するため、上記した他の凍結保存用治具のように煩雑な操作を必要としない。また、保存液吸収体が元来有するヘーズにより、透過型顕微鏡観察下において、細胞または組織を載置する載置箇所の視認性に優れ、さらに特許文献９に記載される凍結保存用治具では、保存液吸収体の全光線透過率が保存液の吸収に伴い高まることで、載置された細胞または組織の視認性も良好となる。しかしながら、これらの凍結保存用治具に用いられている保存液吸収体は、細胞または組織を載置する際に、細胞または組織を保存液とともに載置箇所に滴下するために用いるピペット先端の像が保存液吸収体の像に隠れてしまい、ピペット先端の状況が把握しづらいという問題があった。 The cryopreservation jigs described in Patent Documents 8 to 11 solve any of the above problems and are effective in easily and surely performing the cryopreservation work of cells or tissues. In these cryopreservation jigs, the excess preservation solution is absorbed by the preservation solution absorber of the cryopreservation jig, so that a complicated operation is not required unlike the other cryopreservation jigs described above. .. Further, due to the haze originally possessed by the preservation solution absorber, the visibility of the placement location on which the cells or tissues are placed is excellent under observation with a transmission microscope, and the cryopreservation jig described in Patent Document 9 has excellent visibility. As the total light transmittance of the preservation solution absorber increases with the absorption of the preservation solution, the visibility of the placed cells or tissues is also improved. However, the preservation solution absorber used in these cryopreservation jigs is an image of the tip of a pipette used for dropping cells or tissues together with the preservation solution onto the placement site when placing cells or tissues. Was hidden in the image of the preservation solution absorber, and there was a problem that it was difficult to grasp the condition of the tip of the pipette.
特許文献１２に記載されている凍結保存用治具では、細胞または組織の載置箇所に保存液吸収体は存在せず、ピペット先端の状況は確認しやすいものの、載置された細胞または組織の下部領域が保存液の吸収性能を有さないことから、余分なガラス化液が残存しやすいという問題があった。 In the cryopreservation jig described in Patent Document 12, there is no preservative solution absorber at the place where the cells or tissues are placed, and although the state of the tip of the pipette can be easily confirmed, the cells or tissues placed on the jigs Since the lower region does not have the absorption performance of the storage liquid, there is a problem that excess vitrification liquid tends to remain.
しかるに本発明は、顕微鏡観察下において、細胞または組織を保存液とともに凍結保存用治具の載置部上に滴下する際、載置部とピペット先端の視認性に優れ、かつ載置部の細胞または組織周辺の余分な保存液を除く作業が不要な凍結保存用治具を提供することを課題とする。 However, the present invention has excellent visibility between the mounting portion and the tip of the pipette when the cells or tissues are dropped on the mounting portion of the cryopreservation jig together with the preservation solution under a microscope observation, and the cells in the mounting portion. Another object of the present invention is to provide a cryopreservation jig that does not require the work of removing excess preservative solution around the tissue.
本発明者らは、上記課題を解決するために鋭意検討した結果、以下の構成を有する凍結保存用治具によって、上記課題を解決できることを見出した。 As a result of diligent studies to solve the above problems, the present inventors have found that the above problems can be solved by a cryopreservation jig having the following configuration.
多孔性の保存液吸収体を有する凍結保存用治具であって、該保存液吸収体は、細胞または組織を載置する載置部と、該載置部よりも高吸収性の隣接部を有し、該載置部の全光線透過率が４０％以上であることを特徴とする凍結保存用治具。 A cryopreservation jig having a porous preservative solution absorber, the preservative solution absorber has a mounting portion on which cells or tissues are placed and an adjacent portion having a higher absorbency than the mounting portion. A jig for cryopreservation, which has a total light transmittance of 40% or more of the mounting portion.
上記の発明によれば、顕微鏡観察下において、細胞または組織を保存液とともに凍結保存用治具の載置部上に滴下する際、載置部とピペット先端の視認性に優れ、かつ載置部の細胞または組織周辺の余分な保存液を除く作業が不要な凍結保存用治具を提供することができる。 According to the above invention, when cells or tissues are dropped on a mounting portion of a cryopreservation jig together with a preservative solution under a microscope observation, the mounting portion and the tip of the pipette are excellent in visibility and the mounting portion. It is possible to provide a cryopreservation jig that does not require the work of removing excess preservative solution around the cells or tissues.
以下に、本発明の凍結保存用治具を詳細に説明する。 The cryopreservation jig of the present invention will be described in detail below.
本発明の凍結保存用治具は、細胞または組織を凍結保存する際に用いられるものである。かかる凍結保存のプロトコルとしては、前記した緩慢凍結法や、ガラス化凍結法を挙げることができるが、本発明は特にガラス化凍結法において好適である。 The cryopreservation jig of the present invention is used for cryopreserving cells or tissues. Examples of the cryopreservation protocol include the slow freezing method and the vitrification freezing method described above, but the present invention is particularly suitable for the vitrification freezing method.
本願明細書中で、細胞とは、単一の細胞のみならず、複数の細胞からなる細胞集団を含むものである。複数の細胞からなる細胞集団とは、単一の種類の細胞から構成される細胞集団でも良いし、複数の種類の細胞から構成される細胞集団でも良い。また、本願明細書中で、組織とは、単一の種類の細胞から構成される組織でも良いし、複数の種類の細胞から構成される組織でも良く、細胞以外に細胞外マトリックスのような非細胞性の物質を含むものでも良い。 In the specification of the present application, a cell includes not only a single cell but also a cell population composed of a plurality of cells. The cell population composed of a plurality of cells may be a cell population composed of a single type of cell or a cell population composed of a plurality of types of cells. Further, in the specification of the present application, the tissue may be a tissue composed of a single type of cells or a tissue composed of a plurality of types of cells, and may be a tissue other than cells, such as an extracellular matrix. It may contain a cellular substance.
本発明の凍結保存用治具を用いて凍結保存することができる細胞として、例えば、哺乳類（例えば、人（ヒト）、牛、豚、馬、ウサギ、ラット、マウスなど）の卵子、胚、精子などの生殖細胞；人工多能性幹細胞（ｉＰＳ細胞）、胚性幹細胞（ＥＳ細胞）などの多能性幹細胞が挙げられる。また、初代培養細胞、継代培養細胞、及び細胞株細胞などの培養細胞が挙げられる。また細胞は、一または複数の実施形態において、線維芽細胞、膵ガン・肝ガン細胞などのガン由来細胞、上皮細胞、血管内皮細胞、リンパ管内皮細胞、神経細胞、軟骨細胞、組織幹細胞、及び免疫細胞などの接着性細胞が挙げられる。さらに、凍結保存することができる組織として、同種または異種の細胞からなる組織、例えば、卵巣、皮膚、角膜上皮、歯根膜、心筋などの組織が挙げられる。本発明の凍結保存用治具は、特にシート状構造を有する組織（例えば、細胞シート、皮膚組織など）の凍結保存に好適である。本発明の凍結保存用治具は、直接生体から採取した組織だけでなく、例えば、生体外で培養し増殖させた培養皮膚、生体外で構築したいわゆる細胞シート、特開２０１２−２０５５１６号公報で提案されている三次元構造を有する組織モデルのような、人工の組織の凍結保存についても好適に用いることができる。本発明は、上記のような細胞または組織の凍結保存方法において好適に用いられる。 Examples of cells that can be cryopreserved using the cryopreservation jig of the present invention include eggs, embryos, and sperms of mammals (for example, humans, cows, pigs, horses, rabbits, rats, mice, etc.). Germ cells such as: induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) and other pluripotent stem cells. In addition, cultured cells such as primary cultured cells, subcultured cells, and cell line cells can be mentioned. In addition, the cells, in one or more embodiments, include fibroblasts, cancer-derived cells such as pancreatic and hepatic cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, cartilage cells, tissue stem cells, and Adhesive cells such as immune cells can be mentioned. Furthermore, tissues that can be cryopreserved include tissues composed of allogeneic or heterologous cells, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium. The cryopreservation jig of the present invention is particularly suitable for cryopreservation of tissues having a sheet-like structure (for example, cell sheets, skin tissues, etc.). The jig for cryopreservation of the present invention is not limited to tissues directly collected from a living body, for example, cultured skin cultured and grown in vitro, a so-called cell sheet constructed in vitro, JP-A-2012-205516. It can also be suitably used for cryopreservation of artificial tissues such as the proposed tissue model having a three-dimensional structure. The present invention is suitably used in the above-mentioned method for cryopreserving cells or tissues.
本発明の凍結保存用治具は、多孔性の保存液吸収体を有する。本発明における「多孔性」とは、気孔（細孔）を有する構造体であることを意味し、保存液吸収体表面及び内部に連続的な気孔を有することがより好ましい。該保存液吸収体としては、繊維からなるシート、多孔性樹脂シートなどの各種シートが例示される。 The cryopreservation jig of the present invention has a porous storage liquid absorber. "Porosity" in the present invention means a structure having pores (pores), and it is more preferable to have continuous pores on the surface and inside of the storage solution absorber. Examples of the storage liquid absorber include various sheets such as a sheet made of fibers and a porous resin sheet.
本発明において、保存液吸収体として用いる繊維からなるシートとしては、不織布が例示される。該不織布が含有する繊維としては、セルロース繊維、セルロース繊維からなる再生繊維であるレーヨン繊維やキュプラ繊維、さらにはセルロース繊維からの半合成繊維であるアセテート繊維、ポリエステル繊維、ナイロン繊維、アクリル繊維、ポリプロピレン繊維、ポリエチレン繊維、ポリ塩化ビニル繊維、ビニリデン繊維、ポリウレタン繊維、ビニロン（ポリビニルアルコール）繊維、ガラス繊維、絹繊維等が挙げられ、これら繊維を各種混合した不織布も用いることができる。中でもセルロース繊維、セルロース繊維由来の繊維でセルロース再生繊維であるレーヨン繊維やキュプラ繊維、更にはセルロース繊維からの半合成繊維であるアセテート繊維が好ましい。 In the present invention, a non-woven fabric is exemplified as a sheet made of fibers used as a storage solution absorber. The fibers contained in the non-woven fabric include cellulose fibers, rayon fibers and cupra fibers which are regenerated fibers composed of cellulose fibers, acetate fibers which are semi-synthetic fibers from cellulose fibers, polyester fibers, nylon fibers, acrylic fibers and polypropylene. Examples thereof include fibers, polyethylene fibers, polyvinyl chloride fibers, vinylidene fibers, polyurethane fibers, vinylon (polyvinyl alcohol) fibers, glass fibers, silk fibers and the like, and non-woven fabrics obtained by mixing various of these fibers can also be used. Among them, cellulosic fibers, fibers derived from cellulose fibers, rayon fibers and cupra fibers which are regenerated cellulose fibers, and acetate fibers which are semi-synthetic fibers from cellulose fibers are preferable.
前記した不織布は、密度が０．１〜０．４ｇ／ｃｍ３であり、坪量が１０〜１３０ｇ／ｍ２の不織布であることが好ましい。特に密度が０．１２〜０．３ｇ／ｃｍ３であり、坪量が１０〜１００ｇ／ｍ２である不織布は、保存液の吸収性に優れ、さらには細胞又は組織の視認性に優れた凍結保存用治具を提供することが可能となるため好ましい。 The non-woven fabric described above preferably has a density of 0.1 to 0.4 g / cm 3 and a basis weight of 10 to 130 g / m 2 . In particular, a non-woven fabric having a density of 0.12 to 0.3 g / cm 3 and a basis weight of 10 to 100 g / m 2 is excellent in absorption of a preservation solution and further excellent in visibility of cells or tissues. It is preferable because it is possible to provide a storage jig.
不織布は、バインダー等の結着剤成分の不織布に占める割合が１０質量％以下である不織布が好ましく、より好ましくは５質量％以下であり、更に３質量％以下である不織布が好ましい。特に結着剤を含有しない不織布が好ましい。 The non-woven fabric preferably has a binder component such as a binder in an amount of 10% by mass or less, more preferably 5% by mass or less, and further preferably 3% by mass or less. In particular, a non-woven fabric containing no binder is preferable.
不織布は紙と異なり様々な製造方法があるが、上記した結着剤成分が低減された不織布としては、スパンボンド法、メルトブロー法で製造された不織布、更には湿式法又は乾式法で繊維を並べた後、水流交絡法又はニードルパンチ法で製造された不織布が好適に使用できる。また上記した通り、本発明において不織布が含有する好ましい繊維としては、セルロース繊維、セルロース繊維由来の繊維でセルロース再生繊維であるレーヨン繊維やキュプラ繊維、更にはセルロース繊維からの半合成繊維であるアセテート繊維が挙げられるが、これら繊維を用いて製造する場合は、湿式法、乾式法関わらず水流交絡法又はニードルパンチ法での製造方法が好適である。 Unlike paper, there are various manufacturing methods for non-woven fabrics, but as the non-woven fabrics with reduced binder components described above, non-woven fabrics manufactured by the spunbond method and the melt blow method, and further, fibers are arranged by a wet method or a dry method. After that, a non-woven fabric produced by the water flow entanglement method or the needle punch method can be preferably used. Further, as described above, the preferable fibers contained in the non-woven fabric in the present invention are cellulose fibers, fibers derived from cellulose fibers such as rayon fibers and cupra fibers which are regenerated cellulose fibers, and acetate fibers which are semi-synthetic fibers from cellulose fibers. However, in the case of manufacturing using these fibers, the manufacturing method by the water flow entanglement method or the needle punching method is preferable regardless of the wet method or the dry method.
本発明において保存液吸収体として用いる多孔性樹脂シートとしては、例えば特公昭４２−１３５６０号公報や、特開平０８−２８３４４７号公報に記載される、少なくとも一軸方向に延伸し、樹脂の融点以上に加熱し焼結することで得た微細繊維状構造により多孔質構造を形成した樹脂シート、特開２００９−２３５４１７号公報に記載される、乳化重合又は粉砕等の方法によって得られた熱可塑性樹脂の固体粉末を金型に充填し、加熱、焼結して粉末粒子表面を融着させて冷却することにより、多孔質構造を形成した樹脂シート等が挙げられる。多孔性樹脂シートを保存液吸収体として用いた場合、保存液の吸収性に優れ、さらには細胞又は組織の視認性に優れた凍結保存用治具を提供することが可能となるため、より好ましい。 The porous resin sheet used as the preservation solution absorber in the present invention is, for example, described in Japanese Patent Application Laid-Open No. 42-13560 and Japanese Patent Application Laid-Open No. 08-283447, which is stretched at least in the uniaxial direction to exceed the melting point of the resin. A resin sheet having a porous structure formed by a fine fibrous structure obtained by heating and sintering, and a thermoplastic resin obtained by a method such as emulsion polymerization or pulverization described in JP-A-2009-235417. Examples thereof include a resin sheet in which a porous structure is formed by filling a mold with solid powder, heating and sintering, and fusing and cooling the surface of powder particles. When the porous resin sheet is used as a preservation solution absorber, it is more preferable because it is possible to provide a cryopreservation jig having excellent absorption of the preservation solution and further excellent visibility of cells or tissues. ..
上記した多孔性樹脂シートを形成する樹脂としては、低密度ポリエチレン、高密度ポリエチレン、超高分子ポリエチレン等の各種ポリエチレンやポリプロピレン、ポリメチルメタクリレート、ポリスチレン、ポリテトラフルオロエチレンやポリビニルジフロライド等のフッ素樹脂、エチレン−酢酸ビニル共重合体、ポリアミド、スチレン−アクリロニトリル共重合体、スチレン−ブタジエン−アクリロニトリル三元共重合体、ポリカーボネート、ポリ塩化ビニル等が挙げられる。中でもポリテトラフルオロエチレンやポリビニリデンジフロライド等のフッ素樹脂の多孔性樹脂シートは、細胞又は組織を保存液とともに該樹脂シート上に載置した場合、透過顕微鏡観察下での細胞又は組織の視認性が飛躍的に高まり、細胞または組織の視認性にとりわけ優れた凍結保存用治具を提供することが可能となるため好適である。また多孔性樹脂シートとしては、理化学実験用途や研究用途として市販されている、濾過用のメンブレンフィルターも使用できる。 Examples of the resin forming the above-mentioned porous resin sheet include various polyethylenes such as low-density polyethylene, high-density polyethylene, and ultra-high-density polyethylene, and fluorine such as polypropylene, polymethylmethacrylate, polystyrene, polytetrafluoroethylene, and polyvinyldifluorolide. Examples thereof include resins, ethylene-vinyl acetate copolymers, polyamides, styrene-acrylonitrile copolymers, styrene-butadiene-acrylonitrile ternary copolymers, polycarbonates and polyvinyl chlorides. Among them, the porous resin sheet of fluororesin such as polytetrafluoroethylene or polyvinylidene difluoride allows the cells or tissues to be visually recognized under a transmission microscope when the cells or tissues are placed on the resin sheet together with the preservation solution. It is suitable because the properties are dramatically improved and it becomes possible to provide a cryopreservation jig having particularly excellent visibility of cells or tissues. Further, as the porous resin sheet, a membrane filter for filtration, which is commercially available for physics and chemistry experiment use and research use, can also be used.
本発明の凍結保存用治具は、前述した多孔性の保存液吸収体を有し、該保存液吸収体は、細胞または組織を載置する載置部と、該載置部よりも高吸収性の隣接部を有する。本発明における細胞または組織を載置する載置部とは、細胞または組織が保存液とともに載置される部分に相当する箇所であり、本発明では該載置部の隣接箇所に、該載置部よりも高吸収性の隣接部を配置したことを特徴とする。該隣接部は、載置部を取り囲むように配置されていてもよいし、載置部の一部に隣接するよう配置されていてもよい。隣接部が載置部の一部に隣接する具体的な例としては、載置部を細胞または組織を載置する載置面から見た際の形状が多角形の場合、該多角形の一辺に隣接部が接している状態、載置部が円形の場合、該円周の一部に隣接部が接している状態が例示されるが、少なくとも載置部を中心として対向する二方向に隣接部が接していることが好ましい。 The cryopreservation jig of the present invention has the above-mentioned porous preservation solution absorber, and the preservation solution absorber has a mounting portion on which cells or tissues are placed and a higher absorption than the mounting portion. Has sex adjacencies. The placement portion on which the cells or tissues are placed in the present invention corresponds to a portion on which the cells or tissues are placed together with the preservation solution, and in the present invention, the placement portion is adjacent to the placement portion. It is characterized in that an adjacent portion having higher absorption than the portion is arranged. The adjacent portion may be arranged so as to surround the mounting portion, or may be arranged so as to be adjacent to a part of the mounting portion. As a specific example in which the adjacent portion is adjacent to a part of the mounting portion, when the shape of the mounting portion when viewed from the mounting surface on which the cells or tissues are placed is a polygon, one side of the polygon. In the case where the adjacent portion is in contact with the mounting portion and the mounting portion is circular, the state in which the adjacent portion is in contact with a part of the circumference is exemplified, but at least adjacent to the mounting portion in two opposite directions. It is preferable that the parts are in contact with each other.
載置部の空隙率は、保存液に対する吸収性の観点から、１容量％以上であることが好ましく、より好ましくは５容量％以上である。上限は３５容量％未満であることが好ましい。また、保存液吸収体内部の気孔は、厚み方向のみならず、厚み方向に対して垂直な方向に対しても連続的な構造であることが好ましい。このような構造を有すると、多孔体内部の気孔を有効に用いることができるために、保存液の高い吸収性能が得られる。載置部の保存液吸収体の厚みや多孔体の空隙容量は、用いる細胞または組織の種類や細胞または組織とともに滴下される保存液の滴下量などに応じて、適宜選択することができる。 The porosity of the mounting portion is preferably 1% by volume or more, more preferably 5% by volume or more, from the viewpoint of absorbability to the storage solution. The upper limit is preferably less than 35% by volume. Further, it is preferable that the pores inside the storage liquid absorber have a continuous structure not only in the thickness direction but also in the direction perpendicular to the thickness direction. With such a structure, the pores inside the porous body can be effectively used, so that high absorption performance of the preservation solution can be obtained. The thickness of the storage solution absorber in the placement portion and the void volume of the porous body can be appropriately selected depending on the type of cells or tissues used, the amount of the preservation solution dropped together with the cells or tissues, and the like.
上記した空隙率は、以下の式で定義される。ここで空隙容量Ｖは水銀ポロシメーター（測定器名称 Ａｕｔｏｐｏｒｅ ＩＩ ９２２０ 製造者 ｍｉｃｒｏｍｅｒｉｔｉｃｓ ｉｎｓｔｒｕｍｅｎｔ ｃｏｒｐｏｒａｔｉｏｎ）を用い測定・処理された、保存液吸収体における細孔半径３ｎｍから４００ｎｍまでの累積細孔容積（ｍｌ／ｇ）に、保存液吸収体の乾燥固形分量（ｇ／平方メートル）を乗ずることで、単位面積（平方メートル）当たりの数値として求めることができる。
Ｐ＝（Ｖ／Ｔ）×１００（％）
Ｐ：空隙率（％）
Ｖ：空隙容量（ｍｌ／ｍ２）
Ｔ：厚み（μｍ）
The porosity described above is defined by the following equation. Here, the void volume V is the cumulative pore volume (ml / g) from a pore radius of 3 nm to 400 nm in the storage solution absorber, which was measured and processed using a mercury porosimeter (measuring instrument name Autopore II 9220 manufacturer micromerits instrument corporation). ) Is multiplied by the dry solid content (g / square meter) of the preservative solution absorber, and can be obtained as a numerical value per unit area (square meter).
P = (V / T) x 100 (%)
P: Porosity (%)
V: Void volume (ml / m 2 )
T: Thickness (μm)
載置部の細孔径は、０．０１〜１０μｍであることが好ましく、より好ましくは０．０１〜２μｍである。なお、該細孔径は、多孔性樹脂シートの場合には、バブルポイント試験により測定される最も大きい細孔の直径である。 The pore size of the mounting portion is preferably 0.01 to 10 μm, more preferably 0.01 to 2 μm. In the case of a porous resin sheet, the pore diameter is the diameter of the largest pore measured by the bubble point test.
載置部は、全光線透過率が４０％以上である。載置部の全光線透過率が４０％未満の場合、細胞または組織を保存液とともに凍結保存用治具の載置部上に滴下する際、透過型顕微鏡観察下において、ピペット先端の良好な視認性が得られない場合がある。載置部の全光線透過率は、ＪＩＳ−Ｋ−７３６１−１：１９９７に準拠して、例えば、スガ試験機（株）のヘーズメーターＨＺ−Ｖ３により測定することができる。 The mounting portion has a total light transmittance of 40% or more. When the total light transmittance of the mounting part is less than 40%, when cells or tissues are dropped on the mounting part of the cryopreservation jig together with the preservation solution, the tip of the pipette is clearly visible under a transmission microscope observation. Sex may not be obtained. The total light transmittance of the mounting portion can be measured by, for example, a haze meter HZ-V3 manufactured by Suga Test Instruments Co., Ltd., in accordance with JIS-K-7361-1: 1997.
本発明の凍結保存用治具は、上記した載置部周辺に、保存液に対してより高吸収性の隣接部を有する。 The cryopreservation jig of the present invention has an adjacent portion having higher absorption to the storage liquid around the above-mentioned mounting portion.
隣接部の空隙率は、保存液に対する吸収性の観点から、３５容量％以上であることが好ましく、より好ましくは５０容量％以上である。上限は、９０容量％未満であることが好ましい。隣接部に用いる保存液吸収体内部の気孔も、厚み方向のみならず、厚み方向に対して垂直な方向に対して連続的な構造であることが好ましい。隣接部の保存液吸収体の厚みや多孔体の空隙率も、用いる細胞または組織の種類や細胞または組織とともに滴下される保存液の滴下量などに応じて、適宜選択することができる。隣接部の空隙率も、前述した載置部の空隙率と同様の方法で求めることができる。 The porosity of the adjacent portion is preferably 35% by volume or more, more preferably 50% by volume or more, from the viewpoint of absorbability to the storage solution. The upper limit is preferably less than 90% by volume. It is preferable that the pores inside the storage liquid absorber used in the adjacent portion have a structure that is continuous not only in the thickness direction but also in the direction perpendicular to the thickness direction. The thickness of the storage solution absorber in the adjacent portion and the porosity of the porous body can also be appropriately selected depending on the type of cells or tissues used, the amount of the preservation solution dropped together with the cells or tissues, and the like. The porosity of the adjacent portion can also be obtained by the same method as the porosity of the mounting portion described above.
隣接部の細孔径は、０．０２〜２０μｍであることが好ましく、より好ましくは０．０２〜５μｍである。 The pore size of the adjacent portion is preferably 0.02 to 20 μm, more preferably 0.02 to 5 μm.
載置部、隣接部各部の面積は、細胞または組織と共に滴下される保存液の滴下量などに応じて適宜設定すればよく、特に限定されないが、例えば、滴下する保存液１μｌにつき１ｍｍ２以上とすることが好ましく、２〜４００ｍｍ２とすることがより好ましい。載置部は、保存液吸収体上のいずれかの場所にあればよく、その面積は保存液吸収体の面積に準じる。 The area of each part of the placement portion and the adjacent portion may be appropriately set according to the amount of the preservation solution dropped together with the cells or tissues, and is not particularly limited. For example, 1 mm 2 or more per 1 μl of the preservation solution to be dropped. It is preferably 2 to 400 mm 2 . The mounting portion may be located anywhere on the storage liquid absorber, and its area conforms to the area of the storage liquid absorber.
載置部、隣接部を構成する保存液吸収体の厚みは、１０μｍ〜５ｍｍであることが好ましく、より好ましくは２０μｍ〜２．５ｍｍである。該保存液吸収体の厚みは、載置部または隣接部のいずれかが有する保存液吸収体の最大厚みである。 The thickness of the storage liquid absorber constituting the mounting portion and the adjacent portion is preferably 10 μm to 5 mm, more preferably 20 μm to 2.5 mm. The thickness of the preservation solution absorber is the maximum thickness of the preservation solution absorber that either the mounting portion or the adjacent portion has.
本発明の凍結保存用治具において、保存液吸収体を有する部位は、保存液吸収体のみで構成することも可能であるが、保存液吸収体を支持する支持体として、光透過性支持体を有することが好ましい。かかる光透過性支持体としては保存液吸収体と積層した際に載置部の全光線透過率を低下させないものが好ましく、全光線透過率が８０％以上である光透過性支持体が好ましい。このような光透過性支持体としては、各種樹脂フィルム、ガラス、ゴム等が例示され、本発明の効果を損なわない限り、２種以上の光透過性支持体を組み合わせて用いてもよい。上記した中でも、樹脂フィルムは、取扱いがしやすく、好適に用いられる。樹脂フィルムの具体的な例としては、ポリエチレンテレフタレートやポリエチレンナフタレートなどのポリエステル樹脂、ポリカーボネート樹脂、ポリメチルメタクリレート、メチルメタクリレート−スチレン共重合体、メチルメタクリレート−ポリカーボネート共重合体などのアクリル樹脂、低密度ポリエチレン、高密度ポリエチレン、超高分子量ポリエチレンなどの各種ポリエチレン、ポリプロピレン、ポリスチレン、ポリテトラフルオロエチレンやポリビニルジフロライドなどのフッ素樹脂、ポリ塩化ビニル、エポキシ樹脂、シリコーン樹脂、ジアセテート樹脂、トリアセテート樹脂、ポリアリレート樹脂、ポリスルフォン樹脂、ポリエーテルスルフォン樹脂、ポリイミド樹脂、ポリアミド樹脂、ポリオレフィン樹脂などが例示される。上記した全光線透過率は、ＪＩＳ−Ｋ−７３６１−１：１９９７に準拠して、例えば、前述したスガ試験機（株）のヘーズメーターＨＺ−Ｖ３により測定することができる。 In the cryopreservation jig of the present invention, the portion having the preservation solution absorber may be composed of only the preservation solution absorber, but as a support for supporting the preservation solution absorber, a light-transmitting support It is preferable to have. As such a light-transmitting support, one that does not reduce the total light transmittance of the mounting portion when laminated with the storage liquid absorber is preferable, and a light-transmitting support having a total light transmittance of 80% or more is preferable. Examples of such a light-transmitting support include various resin films, glass, rubber, and the like, and two or more kinds of light-transmitting supports may be used in combination as long as the effects of the present invention are not impaired. Among the above, the resin film is easy to handle and is preferably used. Specific examples of the resin film include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, polycarbonate resins, polymethyl methacrylate, methyl methacrylate-styrene copolymers, acrylic resins such as methyl methacrylate-polycarbonate copolymers, and low densities. Various polyethylenes such as polyethylene, high-density polyethylene and ultra-high molecular weight polyethylene, polypropylene, polystyrene, fluororesins such as polytetrafluoroethylene and polyvinyl difluororide, polyvinyl chloride, epoxy resin, silicone resin, diacetate resin, triacetate resin, Examples thereof include polyallylate resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, and polyolefin resin. The above-mentioned total light transmittance can be measured in accordance with JIS-K-7361-1: 1997, for example, by the above-mentioned haze meter HZ-V3 of Suga Test Instruments Co., Ltd.
上記した保存液吸収体と支持体との間に、接着層を設けることは好ましい。接着層は後述する水溶性高分子化合物や非水溶性樹脂を含有することが好ましく、あるいは湿気硬化性の接着物質に代表されるような瞬間接着組成物、ホットメルト接着組成物、光硬化性接着組成物などを含有することも可能である。例えば、水溶性高分子化合物としては、ポリビニルアルコール、ヒドロキシセルロース、ポリビニルピロリドン、澱粉糊等が例示され、非水溶性樹脂としては、酢酸ビニル系樹脂、アクリル系樹脂、エポキシ系樹脂、ウレタン系樹脂、エラストマー系樹脂、シアノアクリレート系樹脂、フッ素系樹脂、シリコン系樹脂、ニトロセルロース系樹脂、ニトリルゴム系樹脂、スチレン―ブタジエン系樹脂、ユリア系樹脂、スチレン系樹脂、フェノール系樹脂、ポリイミド系樹脂、ポリアミド系樹脂、ポリエステル系樹脂、ビスマレイミド系樹脂、オレフィン系樹脂、ＥＶＡ系樹脂などが例示される。接着層は、一種類の樹脂を含有してもよいし、複数種類の樹脂を含有してもよい。接着層の固形分量は、０．０１〜１００ｇ／ｍ２の範囲が好ましく、更に０．１〜５０ｇ／ｍ２の範囲がより好ましい。 It is preferable to provide an adhesive layer between the above-mentioned storage solution absorber and the support. The adhesive layer preferably contains a water-soluble polymer compound or a water-insoluble resin, which will be described later, or an instant adhesive composition, a hot melt adhesive composition, or a photocurable adhesive such as a moisture-curable adhesive substance. It is also possible to contain a composition or the like. For example, examples of the water-soluble polymer compound include polyvinyl alcohol, hydroxycellulose, polyvinylpyrrolidone, and starch paste, and examples of the water-insoluble resin include vinyl acetate resin, acrylic resin, epoxy resin, and urethane resin. Elastomer-based resin, cyanoacrylate-based resin, fluorine-based resin, silicon-based resin, nitrocellulose-based resin, nitrile rubber-based resin, styrene-butadiene-based resin, urea-based resin, styrene-based resin, phenol-based resin, polyimide-based resin, polyamide Examples thereof include based resins, polyester resins, bismaleimide resins, olefin resins, and EVA resins. The adhesive layer may contain one kind of resin or may contain a plurality of kinds of resins. Solid content of the adhesive layer is preferably in the range of 0.01 to 100 g / m 2, further range of 0.1 to 50 g / m 2 is more preferable.
上記した接着層は、載置部外の箇所（隣接部）に設けることが好ましい。接着層が載置部と支持体の間に存在する場合、顕微鏡観察下で細胞または組織の視認性に悪影響を与える場合がある。載置部外に接着層を設けるための具体的な一例としては、支持体上にマスキングテープによるマスキングを施し、接着層敷設後にマスキングテープを剥離するなどして、支持体上の隣接部にあたる位置に接着層を設け、支持体上の載置部にあたる箇所に接着層が存在しない部分を設け、該接着層上に保存液吸収体を貼り合わせる方法が例示される。 The above-mentioned adhesive layer is preferably provided at a location (adjacent portion) outside the mounting portion. The presence of an adhesive layer between the rest and the support can adversely affect the visibility of cells or tissues under microscopic observation. As a specific example for providing an adhesive layer outside the mounting portion, a position corresponding to an adjacent portion on the support is provided by masking the support with masking tape and peeling off the masking tape after laying the adhesive layer. An example is a method in which an adhesive layer is provided on the support, a portion where the adhesive layer does not exist is provided at a portion corresponding to a mounting portion on the support, and a storage liquid absorber is bonded onto the adhesive layer.
保存液吸収体に載置部と隣接部を形成する方法を例示する。例えば、保存液吸収体の片面に、保存液吸収体よりも長辺、短辺ともにサイズが小さい正方形の金属板をホットプレスし、該保存液吸収体が有する細孔の一部を一軸、加圧方向に圧縮することにより、全光線透過率が４０％以上で、保存液に対して低吸収性の載置部と、該載置部よりも高吸収性の隣接部を形成する方法（方法１）が例示される。また、保存液吸収性が異なる２種類以上の多孔性を有する保存液吸収体を、支持体の片面に隣接して配置し、全光線透過率が４０％以上で吸収性が低い保存液吸収体を載置部として、吸収性が高い方の保存液吸収体を隣接部として用いる方法（方法２）が例示される。他方法としては、保存液吸収体の細孔の一部に、浸透性や流動性に優れる樹脂や水溶性高分子化合物を浸透させ、一部の細孔を閉塞した状態で樹脂や水溶性高分子化合物を固定化することで、全光線透過率が４０％以上の載置部と、該載置部よりも高吸収性の隣接部を形成する方法（方法３）が例示される。また別方法としては、部分的にエンボス加工が施された不織布をトリミングして、該不織布のエンボス加工部を全光線透過率が４０％以上の載置部として、未加工部を高吸収性の隣接部として用いる方法（方法４）が例示される。 A method of forming a mounting portion and an adjacent portion on the storage liquid absorber will be illustrated. For example, a square metal plate whose long side and short side are smaller than those of the preservation solution absorber is hot-pressed on one side of the preservation solution absorber, and a part of the pores of the preservation solution absorber is uniaxially added. A method (method) of forming a mounting portion having a total light transmittance of 40% or more and having low absorption with respect to a preservative solution and an adjacent portion having higher absorption than the mounting portion by compressing in the pressure direction. 1) is illustrated. Further, two or more types of preservative liquid absorbers having different porosity are arranged adjacent to one side of the support, and the preservative liquid absorber having a total light transmittance of 40% or more and low absorbency. An example is a method (method 2) in which the storage solution absorber having higher absorbency is used as an adjacent portion. As another method, a resin having excellent permeability and fluidity or a water-soluble polymer compound is impregnated into a part of the pores of the preservation solution absorber, and the resin or the water-soluble is highly soluble in a state where some of the pores are closed. An example is a method (method 3) of forming a mounting portion having a total light transmittance of 40% or more and an adjacent portion having higher absorption than the mounting portion by immobilizing a molecular compound. Alternatively, the partially embossed non-woven fabric is trimmed, the embossed portion of the non-woven fabric is used as a mounting portion having a total light transmittance of 40% or more, and the unprocessed portion is highly absorbent. An example is a method (method 4) used as an adjacent portion.
本発明の凍結保存用治具は、把持部を有していても良い。把持部を有すると、凍結保存作業時および融解作業時の作業性が良好になるため好ましい。把持部は、耐液体窒素素材であることが好ましい。このような素材としては、例えばアルミ、鉄、銅、ステンレス合金などの各種金属、ＡＢＳ樹脂、ポリプロピレン樹脂、ポリエチレン樹脂、フッ素系樹脂や各種エンジニアリングプラスチック、更にはガラスなどを好適に用いることができる。 The cryopreservation jig of the present invention may have a grip portion. Having a grip portion is preferable because it improves workability during cryopreservation work and thawing work. The grip portion is preferably made of a liquid nitrogen resistant material. As such a material, for example, various metals such as aluminum, iron, copper, and stainless alloy, ABS resin, polypropylene resin, polyethylene resin, fluororesin, various engineering plastics, and glass can be preferably used.
本発明の凍結保存用治具を用いて細胞または組織を長期凍結保存する場合、細胞または組織を外界と遮断するために凍結保存用治具にキャップを被せる、または、該凍結保存用治具を任意の形状の容器に入れて密閉することが可能である。液体窒素が滅菌されておらず、細胞または組織を直接液体窒素に接触させて凍結させる場合は、凍結保存用治具が滅菌されていても滅菌状態を保証できない場合がある。よって、凍結前に細胞または組織を付着させた保存液吸収体にキャップをする、または凍結保存用治具を容器中に密閉することにより、細胞または組織を直接液体窒素に接触させずに凍結させることがある。上記理由から、キャップおよび容器は耐液体窒素性のある素材である各種金属、各種樹脂、ガラス、セラミックなどで作製することが好ましい。形状としては特に限定されず、例えば、キャップは、鉛筆用のキャップのような半紡錘状またはドーム状などのキャップ、円柱状のストローキャップなど、凍結保存用治具の保存液吸収体と接触せず、細胞または組織を外界と遮断できるような形状ならどのような形状でもよい。容器は、載置された細胞または組織に接触せずに、凍結保存用治具を被包または収納して密閉できるものであればよく、その形状は特に限定されない。 When cells or tissues are cryopreserved for a long period of time using the cryopreservation jig of the present invention, the cryopreservation jig is capped in order to block the cells or tissues from the outside world, or the cryopreservation jig is used. It can be placed in a container of any shape and sealed. If liquid nitrogen is not sterilized and cells or tissues are frozen in direct contact with liquid nitrogen, the sterilized state may not be guaranteed even if the cryopreservation jig is sterilized. Therefore, the cells or tissues are frozen without being in direct contact with liquid nitrogen by capping the storage solution absorber to which the cells or tissues are attached before freezing, or by sealing the cryopreservation jig in the container. Sometimes. For the above reasons, it is preferable that the cap and the container are made of various metals, various resins, glass, ceramics, etc., which are materials having liquid nitrogen resistance. The shape is not particularly limited, and for example, the cap is brought into contact with a storage solution absorber of a cryopreservation jig such as a semi-spindle-shaped or dome-shaped cap such as a pencil cap, or a columnar straw cap. However, any shape may be used as long as it can block cells or tissues from the outside world. The shape of the container is not particularly limited as long as it can be sealed by encapsulating or storing the cryopreservation jig without contacting the placed cells or tissues.
本発明の凍結保存用治具は、本発明の効果を損なわない限り、凍結保存用治具を上記したようなキャップ、容器と組み合わせて使用することができる。また、このような、キャップまたは容器と組み合わせて使用される形態の凍結保存用治具も、本発明に包含される。 The cryopreservation jig of the present invention can be used in combination with the above-mentioned cap and container as long as the effect of the present invention is not impaired. In addition, such a cryopreservation jig in a form used in combination with a cap or a container is also included in the present invention.
本発明の凍結保存用治具は、例えば、クライオトップ法において好適に用いられるものである。また、従来のクライオトップ法は、通常、単一の細胞または１０個未満の少数の細胞の保存に用いられるが、本発明は、より多くの細胞の保存（例えば、１０〜１００００００個の細胞の保存）においても好適に用いることができる。本発明を用いると、細胞または組織周辺に存在する余分な保存液を、保存液吸収体ですばやく除去することが可能である。 The cryopreservation jig of the present invention is preferably used in, for example, the cryotop method. Also, while conventional cryotop methods are typically used to conserve a single cell or a small number of cells less than 10, the present invention conserves more cells (eg, of 10 to 1,000,000 cells). It can also be suitably used in storage). Using the present invention, excess preservation solution existing around cells or tissues can be quickly removed by a preservation solution absorber.
本発明の凍結保存用治具を用いて細胞または組織を凍結保存する手順は特に限定されない。例を以下に示す。まず、保存液に浸漬した細胞または組織を、保存液とともに載置部上に滴下する。該細胞または該組織の周囲には余分な保存液が付着しているが、載置部および隣接部の保存液吸収体により（特に高吸収性の隣接部により）、余分な保存液が自動的かつ速やかに除かれる。次いで、前記細胞または組織を載置部に保持させたまま液体窒素などの中に浸漬することにより、細胞または組織を凍結する。この時、前記した載置部上の細胞または組織を外界と遮断することができるキャップを保存液吸収体に装着、または凍結保存用治具を前記した容器に密閉して、液体窒素などの中に浸漬することもできる。保存液は、通常卵子、胚などの細胞の凍結のために使用されるものを使用することができ、例えば、リン酸緩衝生理食塩水などの生理的溶液に耐凍剤（グリセロール、エチレングリコールなど）を含有させることで得られた保存液や、グリセロールやエチレングリコール、ＤＭＳＯ（ジメチルスルホキシド）などの各種耐凍剤を多量に（少なくとも保存液の全質量に対して１０質量％以上、より好ましくは２０質量％以上）含む保存液を使用できる。融解作業の際は、液体窒素などの冷却溶媒中から、該凍結保存用治具を取り出し、凍結された細胞または組織を載せた載置部近傍を融解液中に浸漬させ、必要に応じて保存液吸収体を融解液中で揺するなどして細胞または組織を回収する。 The procedure for cryopreserving cells or tissues using the cryopreservation jig of the present invention is not particularly limited. An example is shown below. First, the cells or tissues immersed in the preservation solution are dropped onto the placement portion together with the preservation solution. Extra preservatives adhere to the cells or around the tissue, but the preservative absorbers in the placement and adjoining areas (especially due to the highly absorptive adjacencies) automatically remove the excess preservatives. And it is removed promptly. Then, the cells or tissues are frozen by immersing the cells or tissues in liquid nitrogen or the like while holding them in the placement portion. At this time, a cap capable of blocking cells or tissues on the mounting portion from the outside world is attached to the preservative solution absorber, or a cryopreservation jig is sealed in the above-mentioned container in liquid nitrogen or the like. It can also be immersed in. As the storage solution, those usually used for freezing cells such as eggs and embryos can be used. For example, a freezing agent (glycerol, ethylene glycol, etc.) is used in a physiological solution such as phosphate buffered saline. A large amount of various antifreeze agents such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide) (at least 10% by mass or more, more preferably 20% by mass, based on the total mass of the preservation solution). % Or more) can be used. During the thawing operation, the cryopreservation jig is taken out from a cooling solvent such as liquid nitrogen, and the vicinity of the place where the frozen cells or tissues are placed is immersed in the thawing solution and stored as necessary. Collect cells or tissues by shaking the liquid absorber in the melt.
以下に、本発明の凍結保存用治具の構造を、図面を用いてより詳細に説明する。 Hereinafter, the structure of the cryopreservation jig of the present invention will be described in more detail with reference to the drawings.
図１は、本発明の凍結保存用治具の一例を示す全体図である。図１において凍結保存用治具６は把持部１を有し、該把持部１には支持体５が接続されており、該支持体５上、把持部１との接続箇所と反対側の端部近傍にあたる箇所に隣接部３に取り囲まれた載置部２を有する。 FIG. 1 is an overall view showing an example of the cryopreservation jig of the present invention. In FIG. 1, the cryopreservation jig 6 has a grip portion 1, and a support 5 is connected to the grip portion 1, and an end on the support 5 opposite to the connection portion with the grip portion 1. A mounting portion 2 surrounded by an adjacent portion 3 is provided at a portion near the portion.
図１中、把持部１は円柱形であるが、その形状は任意である。また、前述したように、細胞または組織を直接液体窒素に接触させないことを目的に、凍結前に細胞または組織を付着させた載置部２にキャップを被せることがあるが、この場合、把持部１を、載置部２を有さない側から、載置部２を有する側に向かって、円柱の径が連続的に小さくなる形状（テーパー状）とすることで、キャップを被せる際の作業性を向上させることも可能である。載置部２の形状は、ハンドリング上、短冊状またはシート状であることが好ましい。 In FIG. 1, the grip portion 1 has a cylindrical shape, but the shape is arbitrary. Further, as described above, for the purpose of preventing the cells or tissues from coming into direct contact with liquid nitrogen, the mounting portion 2 to which the cells or tissues are attached may be covered with a cap before freezing. Work for covering the cap by forming 1 into a shape (tapered shape) in which the diameter of the cylinder is continuously reduced from the side having no mounting portion 2 toward the side having the mounting portion 2. It is also possible to improve the sex. The shape of the mounting portion 2 is preferably strip-shaped or sheet-shaped in terms of handling.
図１の把持部１と支持体５の接続方法について説明する。把持部１が樹脂の場合、例えば、成形加工する時にインサート成形により支持体５を把持部１に接続することができる。さらに、把持部１に図示しない構造体挿入部を作製し、接着剤にて支持体５を接続することもできる。接着剤としては様々なものが使用できるが、低温に強いシリコン系やフッ素系の接着剤を好適に用いることができる。 A method of connecting the grip portion 1 and the support 5 of FIG. 1 will be described. When the grip portion 1 is made of resin, for example, the support 5 can be connected to the grip portion 1 by insert molding during molding. Further, a structure insertion portion (not shown) may be formed in the grip portion 1, and the support 5 may be connected with an adhesive. Although various adhesives can be used, silicon-based or fluorine-based adhesives that are resistant to low temperatures can be preferably used.
図２は、図１の凍結保存用治具における載置部を、細胞または組織を載置する載置面側から見た概略図であり、隣接部３ａに囲まれた正方形状の単一の載置部２ａが、図示しない支持体５と貼り合わせられた保存液吸収体中に設けられている。また図３は、図１の凍結保存用時治具における載置部の断面構造概略図であり、図１のＡ−Ａ′間における断面構造の概略図である。かかる構造の載置部２ａを有する凍結保存用治具を得るための方法としては、前述した方法１により保存液吸収体に載置部２ａと隣接部３ａを形成し、該保存液吸収体を短冊状にトリミングした後、該保存液吸収体と短冊状の支持体５を、支持体５上に形成した接着層４を介して貼り合わせる方法が例示される。 FIG. 2 is a schematic view of the mounting portion of the cryopreservation jig of FIG. 1 as viewed from the mounting surface side on which cells or tissues are placed, and is a single square shape surrounded by the adjacent portion 3a. The mounting portion 2a is provided in a storage liquid absorber bonded to a support 5 (not shown). Further, FIG. 3 is a schematic cross-sectional structure of the mounting portion of the jig for cryopreservation of FIG. 1, and is a schematic view of the cross-sectional structure between A and A'of FIG. As a method for obtaining a cryopreservation jig having a mounting portion 2a having such a structure, a mounting portion 2a and an adjacent portion 3a are formed on the storage liquid absorber by the above-mentioned method 1, and the storage liquid absorber is used. An example is a method in which the storage liquid absorber and the strip-shaped support 5 are bonded to each other via an adhesive layer 4 formed on the support 5 after being trimmed into strips.
図４は、本発明の凍結保存用治具における載置部の別の一例であり、載置部２ｂを中心として対向する二方向に隣接部３ｂを設けた概略図である。かかる構造の載置部２ｂは、例えば、前述した方法１において、ホットプレスに用いる金属板を、一辺が保存液吸収体の短辺よりも長く、もう一辺が保存液吸収体の長辺よりも短いものとすることにより得られる。 FIG. 4 is another example of the mounting portion in the cryopreservation jig of the present invention, and is a schematic view in which adjacent portions 3b are provided in two directions facing each other with the mounting portion 2b as the center. In the mounting portion 2b having such a structure, for example, in the method 1 described above, one side of the metal plate used for the hot press is longer than the short side of the storage liquid absorber, and the other side is longer than the long side of the storage liquid absorber. Obtained by making it shorter.
図５は、本発明の凍結保存用治具における載置部のまた別の一例であり、隣接部３ｃに囲まれた正方形状の複数の載置部２ｃが、図示しない支持体５と貼り合わせられた保存液吸収体中に設けられている。かかる構造の載置部２ｃは、例えば、前述した方法１において、金属板を保存液吸収体の長辺方向に複数回位置をずらしながらホットプレスすることにより得られる。 FIG. 5 is another example of the mounting portion in the cryopreservation jig of the present invention, in which a plurality of square mounting portions 2c surrounded by adjacent portions 3c are bonded to a support 5 (not shown). It is provided in the storage solution absorber provided. The mounting portion 2c having such a structure can be obtained, for example, by hot-pressing the metal plate in the above-mentioned method 1 while shifting the position a plurality of times in the long side direction of the storage liquid absorber.
図６は、本発明の凍結保存用治具における載置部のまた別の一例であり、隣接部３ｄに囲まれた正円の単一の載置部２ｄを設けた概略図である。かかる構造の載置部２ｄは、例えば、前述した方法１において、ホットプレスに用いる金属板を、保存液吸収体の短辺よりも直径が小さい正円のものとすることにより得られる。 FIG. 6 is another example of the mounting portion in the cryopreservation jig of the present invention, and is a schematic view in which a single mounting portion 2d of a perfect circle surrounded by the adjacent portion 3d is provided. The mounting portion 2d having such a structure can be obtained, for example, by forming the metal plate used for the hot press into a perfect circle having a diameter smaller than the short side of the storage liquid absorber in the above-mentioned method 1.
図７は、本発明の凍結保存用治具における載置部の断面構造概略図の一例であり、吸収性の異なる保存液吸収体（２ｅ、３ｅ）が、支持体５上に接着層４を介して貼り合わせられている凍結保存用治具の載置部についての、図１のＢ−Ｂ′間における断面構造の概略図である。かかる構造の載置部２ｅは、例えば、前述した方法２により得られる。 FIG. 7 is an example of a schematic cross-sectional structure of a mounting portion in the cryopreservation jig of the present invention, in which storage liquid absorbers (2e, 3e) having different absorbency form an adhesive layer 4 on the support 5. It is the schematic of the cross-sectional structure between BB'in FIG. 1 about the mounting part of the cryopreservation jig which is bonded through. The mounting portion 2e having such a structure can be obtained, for example, by the method 2 described above.
以下に、本発明を実施例によりさらに詳細に示し、本発明を具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, and the present invention will be specifically described, but the present invention is not limited to the following Examples.
（実施例１）
保存液吸収体として、アドバンテック東洋（株）製のポリテトラフルオロエチレン多孔体（細孔径０．２μｍ、空隙率７１％、厚み３５μｍ、全光線透過率２８．４％）の片面の一部に、平坦なニッケル板（１ｍｍ×１ｍｍ）を重ね合わせ、サイトウエンヂニアーズ（株）製のロール式エンボス加工機ＥＭＢＯＳＴＡＲ（登録商標）を用いて、２５０℃の温度条件でホットプレスを施した。ホットプレス後にニッケル板を取り除き、その後１．５ｍｍ×１０ｍｍにカットし、前述した図２の形態で載置部２ａおよび隣接部３ａが形成された保存液吸収体を得た。他方、支持体として、東レ（株）製のポリエチレンテレフタレートフィルムである、ルミラー（登録商標）Ｔ６０（短辺が１．５ｍｍ、長辺が２５ｍｍで厚み１８８μｍ、全光線透過率８８．９％）を準備し、該ポリエチレンテレフタレートフィルムに、（株）寺岡製作所製のマスキングテープ（厚み５５μｍ）を用いて、図３に示したような形態の接着層４（支持体５上の隣接部３ａとの貼り合わせ箇所のみ接着層４が存在）が形成できるようにマスキングを施し、該マスキング後のポリエチレンテレフタレートフィルム上に、接着層４として、ヘンケルジャパン（株）製のホットメルトウレタン樹脂Ｐｕｒｍｅｌｔ（登録商標）ＱＲ １７０−７１４１Ｐを、乾燥時の固形分量が３０ｇ／ｍ２となるように塗布した。接着層４塗布後、マスキングテープを剥離し、接着剤が流動性を有するうちに、上記したホットプレス後の保存液吸収体を、接着層４非塗布部と載置部２ａが重なり合うように貼り合わせた。また、保存液吸収体と支持体５の密着性を高めるため、載置部２ａにニッケル板を再度軽く押しつけた後、室温で２４時間エイジングを行い、接着層４の硬化を促した。その後、保存液吸収体を有していない側の支持体５の一端を、ＡＢＳ樹脂製の把持部１と接合させ、実施例１の凍結保存用治具を図１に示すような形態で得た。なお、実施例１の載置部２ａの全光線透過率を、ＪＩＳ−Ｋ−７３６１−１：１９９７に準拠して、スガ試験機（株）のヘーズメーターＨＺ−Ｖ３により測定したところ、６２．９％であった。また、実施例１の載置部２ａについて、前述した方法で空隙率を測定したところ、６％であり、隣接部３ａ（空隙率７１％）よりも低吸収性であった。
(Example 1)
As a storage solution absorber, a part of one side of a polytetrafluoroethylene porous body (pore diameter 0.2 μm, porosity 71%, thickness 35 μm, total light transmittance 28.4%) manufactured by Advantech Toyo Co., Ltd. Flat nickel plates (1 mm × 1 mm) were superposed and hot-pressed under a temperature condition of 250 ° C. using a roll-type embossing machine EMBOSTAR (registered trademark) manufactured by Cytowengenies Co., Ltd. After hot pressing, the nickel plate was removed and then cut into 1.5 mm × 10 mm to obtain a storage liquid absorber in which the mounting portion 2a and the adjacent portion 3a were formed in the form of FIG. 2 described above. On the other hand, as a support, Lumirer (registered trademark) T60 (short side 1.5 mm, long side 25 mm, thickness 188 μm, total light transmittance 88.9%), which is a polyethylene terephthalate film manufactured by Toray Co., Ltd., is used. Prepare and attach the polyethylene terephthalate film to the adhesive layer 4 (adjacent portion 3a on the support 5) in the form shown in FIG. 3 using masking tape (thickness 55 μm) manufactured by Teraoka Seisakusho Co., Ltd. Masking is applied so that the adhesive layer 4 exists only at the mating portion), and the hot melt urethane resin Purmelt (registered trademark) QR manufactured by Henkel Japan Ltd. is used as the adhesive layer 4 on the masked polyethylene terephthalate film. 170-7141P was applied so that the solid content at the time of drying was 30 g / m 2 . After applying the adhesive layer 4, the masking tape is peeled off, and while the adhesive has fluidity, the above-mentioned preservative liquid absorber after hot pressing is attached so that the non-applied portion of the adhesive layer 4 and the mounting portion 2a overlap. I matched it. Further, in order to improve the adhesion between the storage solution absorber and the support 5, the nickel plate was lightly pressed against the mounting portion 2a and then aged at room temperature for 24 hours to promote the curing of the adhesive layer 4. After that, one end of the support 5 on the side that does not have the storage liquid absorber is joined to the grip portion 1 made of ABS resin, and the cryopreservation jig of Example 1 is obtained in the form as shown in FIG. It was. The total light transmittance of the mounting portion 2a of Example 1 was measured by a haze meter HZ-V3 of Suga Test Instruments Co., Ltd. in accordance with JIS-K-7361-1: 1997. It was 9%. Further, when the porosity of the mounting portion 2a of Example 1 was measured by the method described above, it was 6%, which was lower absorption than the adjacent portion 3a (porosity 71%).
（実施例２）
実施例１において、ホットプレス時の温度条件を１５０℃とする以外は実施例１と同様にして、実施例２の凍結保存用治具を得た。なお、実施例２の凍結保存用治具における載置部２ａの全光線透過率は５３．９％、空隙率は１８％であった。
(Example 2)
In Example 1, a jig for cryopreservation of Example 2 was obtained in the same manner as in Example 1 except that the temperature condition at the time of hot pressing was 150 ° C. The total light transmittance of the mounting portion 2a in the cryopreservation jig of Example 2 was 53.9%, and the porosity was 18%.
（実施例３）
実施例１において、ホットプレス時の温度条件を４０℃とする以外は実施例１と同様にして、実施例３の凍結保存用治具を得た。なお、実施例３の凍結保存用治具における載置部２ａの全光線透過率は４３．１％、空隙率は３０％であった。
(Example 3)
In Example 1, a jig for cryopreservation of Example 3 was obtained in the same manner as in Example 1 except that the temperature condition during hot pressing was 40 ° C. The total light transmittance of the mounting portion 2a in the cryopreservation jig of Example 3 was 43.1%, and the porosity was 30%.
（比較例１）
実施例１において、ホットプレスを施していないアドバンテック東洋（株）製のポリテトラフルオロエチレン多孔体（細孔径０．２μｍ、空隙率７１％、厚み３５μｍ、全光線透過率２７．３％）を保存液吸収体として用いる以外は、実施例１と同様にして、比較例１の凍結保存用治具を得た。比較例１の凍結保存用治具における載置部の全光線透過率は２７．３％、空隙率は７１％であった。
(Comparative Example 1)
In Example 1, a polytetrafluoroethylene porous body (pore diameter 0.2 μm, porosity 71%, thickness 35 μm, total light transmittance 27.3%) manufactured by Advantech Toyo Co., Ltd., which was not hot-pressed, was stored. A jig for cryopreservation of Comparative Example 1 was obtained in the same manner as in Example 1 except that it was used as a liquid absorber. The total light transmittance of the mounting portion of the cryopreservation jig of Comparative Example 1 was 27.3%, and the porosity was 71%.
（比較例２）
実施例１において、載置部２ａの保存液吸収体をカッターナイフにより除去し、支持体のポリエチレンテレフタレートフィルムを露出させることにより、比較例２の凍結保存用治具を得た。比較例２の載置部の全光線透過率は８８．９％、空隙率は０％であった。
(Comparative Example 2)
In Example 1, the storage liquid absorber of the mounting portion 2a was removed with a cutter knife to expose the polyethylene terephthalate film of the support, thereby obtaining a cryopreservation jig of Comparative Example 2. The total light transmittance of the mounting portion of Comparative Example 2 was 88.9%, and the porosity was 0%.
＜マウス卵子の凍結操作＞
実施例１〜３および比較例１〜２の凍結保存用治具の各々について、保存液として、細胞または組織のガラス化保存のために一般的に用いられている、Ｉｒｖｉｎｅ Ｓｃｉｅｎｔｉｆｉｃ社製Ｖｉｔ Ｋｉｔガラス化液を使用し、実体顕微鏡観察下で、載置部上にガラス化液０．２μＬと、直径約１００μｍのマウス卵子をマイクロピペットにより滴下する操作を行った。該操作において、以下の基準により、顕微鏡観察下における載置部およびピペット先端の視認性と、マウス卵子周辺の余分なガラス化液に対する保存液吸収体の吸収性を評価した。
<Freezing operation of mouse egg>
For each of the cryopreservation jigs of Examples 1 to 3 and Comparative Examples 1 and 2, a Vit Kit glass manufactured by Irvine Scenicific Co., Ltd., which is generally used for vitrification and preservation of cells or tissues as a preservation solution. Using the chemical solution, 0.2 μL of the vitrified solution and a mouse egg having a diameter of about 100 μm were dropped onto the placement portion by a micropipette under a stereomicroscopic observation. In this operation, the visibility of the placement portion and the tip of the pipette under microscopic observation and the absorbency of the storage solution absorber to the excess vitrified solution around the mouse egg were evaluated according to the following criteria.
＜載置部およびピペットの視認性の評価基準＞
○：顕微鏡観察下において、載置部とピペット先端を明瞭に観察することができた。
×：顕微鏡観察下において、載置部は明瞭に観察できたが、ピペット先端はよく見えなかった。
<Evaluation criteria for visibility of mounting part and pipette>
◯: The placement portion and the tip of the pipette could be clearly observed under a microscope.
X: Under microscopic observation, the placement part could be clearly observed, but the tip of the pipette could not be seen well.
＜保存液吸収体の吸収性の評価基準＞
○：マウス卵子周辺の余分なガラス化液が、保存液吸収体に１０秒未満で吸収された。
×：マウス卵子周辺の余分なガラス化液が、保存液吸収体に１０秒以上経っても吸収されず、作業者が余分なガラス化液を吸引除去する必要性を感じた。
<Evaluation criteria for absorbency of preservative solution absorber>
◯: Excess vitrified solution around the mouse egg was absorbed by the preservation solution absorber in less than 10 seconds.
X: The excess vitrified solution around the mouse egg was not absorbed by the preservation solution absorber even after 10 seconds or more, and the operator felt the need to suck and remove the excess vitrified solution.
表１の結果から、本発明の凍結保存用治具は、顕微鏡観察下における載置部とピペット先端の視認性が良好で、かつ載置部の細胞または組織周辺の余分な保存液を除く作業が不要な凍結保存用治具であることがわかる。 From the results in Table 1, the cryopreservation jig of the present invention has good visibility of the placement portion and the tip of the pipette under microscopic observation, and removes excess storage solution around the cells or tissues of the placement portion. It can be seen that this is an unnecessary cryopreservation jig.
本発明は、牛などの家畜や動物の胚移植や人工授精、人への人工授精などの他、ｉＰＳ細胞、ＥＳ細胞、一般に用いられている培養細胞、生体から採取した検査用または移植用の細胞または組織、生体外で培養した細胞または組織などの凍結保存に用いることができる。 The present invention relates to embryo transfer and artificial insemination of domestic animals such as cattle and animals, artificial insemination to humans, iPS cells, ES cells, commonly used cultured cells, and for examination or transplantation collected from a living body. It can be used for cryopreservation of cells or tissues, cells or tissues cultured in vitro.
１ 把持部
２、２ａ〜２ｅ 載置部
３、３ａ〜３ｅ 隣接部
４ 接着層
５ 支持体
６ 凍結保存用治具
1 Grips 2, 2a to 2e Mounting parts 3, 3a to 3e Adjacent parts 4 Adhesive layer 5 Supports 6 Jig for cryopreservation
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014183757A (en) * | 2013-03-22 | 2014-10-02 | Kitasato Institute | Jig for vitrification cryopreservation of ovum or embryo |
| JP2017060457A (en) * | 2014-10-23 | 2017-03-30 | 三菱製紙株式会社 | Tool for cryopreservation of cell or tissue and cryopreservation method |
| WO2018230477A1 (en) * | 2017-06-12 | 2018-12-20 | 三菱製紙株式会社 | Cryopreservation jig for cryopreserving cells or tissues |
| WO2019004300A1 (en) * | 2017-06-30 | 2019-01-03 | 株式会社北里コーポレーション | Biological cell cryopreservation tool and tool for biological cell cryopreservation |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014183757A (en) * | 2013-03-22 | 2014-10-02 | Kitasato Institute | Jig for vitrification cryopreservation of ovum or embryo |
| JP2017060457A (en) * | 2014-10-23 | 2017-03-30 | 三菱製紙株式会社 | Tool for cryopreservation of cell or tissue and cryopreservation method |
| WO2018230477A1 (en) * | 2017-06-12 | 2018-12-20 | 三菱製紙株式会社 | Cryopreservation jig for cryopreserving cells or tissues |
| WO2019004300A1 (en) * | 2017-06-30 | 2019-01-03 | 株式会社北里コーポレーション | Biological cell cryopreservation tool and tool for biological cell cryopreservation |
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