JP2020039326A - Frost damage protection liquid of mesenchymal stem cells, and use of the same - Google Patents
Frost damage protection liquid of mesenchymal stem cells, and use of the same Download PDFInfo
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本発明は、間葉系幹細胞を、−８０℃よりも高い温度条件下でも、長時間安定的に保存することを可能とする細胞凍害保護液及びその利用に関するものである。 TECHNICAL FIELD The present invention relates to a cell cryoprotection solution that enables mesenchymal stem cells to be stably stored for a long period of time even at a temperature higher than -80 ° C, and to use thereof.
近年、様々な間葉系幹細胞を用いた再生医療等製品が承認を受けているが、使用時の輸送においては、常温での短時間の輸送の形態か、冷却手段として液体窒素を利用した極低温輸送の形態が採用されている。 In recent years, various products such as regenerative medicine using mesenchymal stem cells have been approved.However, in transport during use, the form of short-time transport at room temperature or extreme use of liquid nitrogen as cooling means A low-temperature transport mode is employed.
しかしながら、常温輸送品では輸送に長時間かかる海外への展開が難しく、一方、極低温輸送では、特別な輸送機器や液体窒素などを要するため、輸送や保管管理のコストが嵩むという問題がある。 However, it is difficult to develop overseas products that require a long time to transport, while cryogenic transport requires special transport equipment, liquid nitrogen, and the like.
そこで、低コストで再生医療等製品の長時間輸送が可能な、再生医療等製品の保存方法の開発が切望されている。 Therefore, development of a method for storing regenerative medicine products, which enables long-term transportation of regenerative medicine products at low cost, has been desired.
なお、造血幹細胞については、ヒドロキシルエチルデンプン及びジメチルスルホキシドを有効成分とする細胞凍害保護液であるＣＰ−１（極東製薬工業株式会社製）を用いると、−８０℃で長期間の凍結保存ができることが報告されている（非特許文献１〜４）。 The hematopoietic stem cells can be cryopreserved at -80 ° C for a long period of time using CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.), which is a cell cryoprotectant containing hydroxylethyl starch and dimethyl sulfoxide as active ingredients. Have been reported (Non-Patent Documents 1 to 4).
本発明は、このような状況に鑑みてなされたものであり、その目的は、細胞の保存方法であって、安価で細胞の長時間輸送を可能とする方法を提供することにある。さらなる本発明の目的は、このような保存方法に用いられる、細胞の保護液を提供することにある。 The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method for preserving cells, which is inexpensive and enables long-term transport of cells. A further object of the present invention is to provide a cell protection solution used in such a storage method.
本発明者らは、従来の液体窒素による極低温輸送に代わる、安価な凍結細胞の輸送方法として、ドライアイスによる輸送を構想した。ドライアイスそのものの温度は−７９℃であるが、輸送中の周囲の環境あるいは輸送の条件によっては、凍結細胞の温度が上昇することが考えられる。このため、本発明者らは、ドライアイスによる凍結細胞の輸送において、より高温な条件（例えば、−７８℃〜−６０℃）でも凍結細胞の生存や増殖能を維持することが可能な細胞凍害保護液につき、鋭意検討を行った。 The present inventors have conceived of dry ice transport as an inexpensive method for transporting frozen cells instead of conventional cryogenic transport using liquid nitrogen. The temperature of dry ice itself is -79C, but the temperature of the frozen cells may increase depending on the surrounding environment during transportation or the conditions of transportation. For this reason, the present inventors have found that, in transporting frozen cells by dry ice, cell cryotoxicity capable of maintaining the survival and proliferation ability of frozen cells even under higher temperature conditions (for example, -78 ° C to -60 ° C). The protective liquid was studied diligently.
上記の通り、造血幹細胞では、ヒドロキシルエチルデンプン及びジメチルスルホキシドを有効成分とする細胞凍害保護液であるＣＰ−１を用いると、−８０℃の冷凍温度で長期間の凍結保存ができることが報告されているが、ＣＰ−１がより高温での凍結保存に適しているか否かは不明であり、また、このような高温での凍結保存に適した細胞種についても不明である。そこで、本発明者らは、より高温でのＣＰ−１による凍結保存に適した細胞の探索を行った。 As described above, in hematopoietic stem cells, it has been reported that long-term cryopreservation can be performed at a freezing temperature of −80 ° C. by using CP-1 which is a cell cryoprotective solution containing hydroxylethyl starch and dimethyl sulfoxide as active ingredients. However, it is unclear whether CP-1 is suitable for cryopreservation at higher temperatures, and it is also unknown about cell types suitable for cryopreservation at such high temperatures. Thus, the present inventors searched for cells suitable for cryopreservation with CP-1 at higher temperatures.
その結果、間葉系幹細胞については、より高温条件で長時間、ＣＰ−１により凍結保存した場合あっても、その解凍後の生存及び増殖能が維持できることが判明した。しかしながら、ＣＰ−１を利用しない常法で同様に凍結保存した場合には、増殖能が顕著に低下した。一方、ヒト新生児皮膚繊維芽細胞については、間葉系幹細胞とは対照的に、ＣＰ−１を用いた場合であっても、解凍後の増殖能が顕著に低下した。 As a result, it was found that the mesenchymal stem cells can maintain their survival and proliferative ability after thawing even when they are cryopreserved with CP-1 for a long time under higher temperature conditions. However, when the cells were similarly cryopreserved by a conventional method not using CP-1, the proliferation ability was remarkably reduced. On the other hand, in human neonatal skin fibroblasts, in contrast to mesenchymal stem cells, even when CP-1 was used, the proliferation ability after thawing was significantly reduced.
以上から、本発明者らは、より高温でのＣＰ−１による凍結細胞の保存においては、ＣＰ−１への適性が細胞種によって異なり、間葉系幹細胞が優れた適性を有することを見出し、本発明を完成するに至った。 From the above, the present inventors have found that in storing frozen cells with CP-1 at higher temperatures, the suitability for CP-1 differs depending on the cell type, and that mesenchymal stem cells have excellent suitability, The present invention has been completed.
本発明は、より詳しくは、以下を提供するものである。 The present invention more specifically provides:
［１］ヒドロキシルエチルデンプン及びジメチルスルホキシドを有効成分とする、間葉系幹細胞を凍害から保護するための溶液。 [1] A solution for protecting mesenchymal stem cells from freezing damage, comprising hydroxylethyl starch and dimethyl sulfoxide as active ingredients.
［２］間葉系幹細胞を凍結保存する方法であって、間葉系幹細胞を、ヒドロキシルエチルデンプン及びジメチルスルホキシドを含む溶液に懸濁して凍結させることを特徴とする方法。 [2] A method for cryopreserving mesenchymal stem cells, wherein the mesenchymal stem cells are suspended in a solution containing hydroxylethyl starch and dimethyl sulfoxide and frozen.
［３］溶液が、さらに、血清アルブミンまたは血清を含む、［２］に記載の方法。 [3] The method according to [2], wherein the solution further contains serum albumin or serum.
［４］間葉系幹細胞の凍結細胞を輸送する方法であって、当該凍結細胞がヒドロキシルエチルデンプン及びジメチルスルホキシドを含む溶液中で凍結されたものであり、当該輸送における当該凍結細胞の温度を−８０℃よりも高い温度とする方法。 [4] A method for transporting frozen mesenchymal stem cells, wherein the frozen cells are frozen in a solution containing hydroxylethyl starch and dimethyl sulfoxide, and the temperature of the frozen cells in the transport is- A method in which the temperature is higher than 80C.
［５］溶液が、さらに、血清アルブミンまたは血清を含む、［４］に記載の方法。 [5] The method according to [4], wherein the solution further contains serum albumin or serum.
［６］輸送における当該凍結細胞の冷却手段としてドライアイスを利用する、［４］に記載の方法。 [6] The method according to [4], wherein dry ice is used as a cooling means for the frozen cells in transportation.
本発明により、凍結された間葉系幹細胞が−８０℃よりも高温に曝されても、長時間安定的に細胞の機能を維持させることが可能となった。これにより、国内外を問わず、ドライアイスによる凍結細胞の輸送が可能となり、輸送中の凍結細胞の温度上昇による機能低下の問題を回避することができる。また、ドライアイス輸送では、高価な液体窒素や特別な輸送機器は不要であるため、従来の極低温輸送と比較して、輸送コストを大幅に削減することができ、かつ、簡便である。また、間葉系幹細胞の輸送時のみならず、保管においても、液体窒素や特別な機器を使用する必要はないことから、保管コストも削減することができる。このため、幅広い医療機関において間葉系幹細胞の冷凍保存が可能となる。 According to the present invention, even if frozen mesenchymal stem cells are exposed to a temperature higher than -80 ° C, it has become possible to stably maintain cell functions for a long time. This enables the transport of frozen cells by dry ice both in Japan and abroad, thereby avoiding the problem of functional deterioration due to a rise in the temperature of the frozen cells during transportation. Moreover, since dry ice transport does not require expensive liquid nitrogen or special transport equipment, transport costs can be significantly reduced and simpler than conventional cryogenic transport. In addition, not only during transportation of mesenchymal stem cells but also during storage, there is no need to use liquid nitrogen or special equipment, so that storage costs can be reduced. This allows mesenchymal stem cells to be cryopreserved in a wide range of medical institutions.
本発明は、ヒドロキシルエチルデンプン及びジメチルスルホキシドを有効成分とする、間葉系幹細胞を凍害から保護するための溶液（以下、「本発明の凍害保護液」と称する）を提供する。 The present invention provides a solution for protecting mesenchymal stem cells from frost damage (hereinafter, referred to as “the cryoprotectant solution of the present invention”), which contains hydroxylethyl starch and dimethyl sulfoxide as active ingredients.
本発明において「凍害」とは、細胞の凍結や融解によって引き起こされる、細胞の損傷、死滅、機能低下などの負の影響を意味する。また、「間葉系幹細胞」は、骨芽細胞、脂肪細胞、筋細胞、軟骨細胞など、間葉系に属する細胞への分化能をもつ幹細胞である。間葉系幹細胞には、採取する組織に応じて、骨髄由来間葉系幹細胞や脂肪組織由来間葉系幹細胞などが含まれるが、本発明の凍害保護液を適用する間葉系幹細胞は、その由来は問わない。実際、本発明の凍結保護液は、骨髄由来間葉系幹細胞及び脂肪組織由来間葉系幹細胞のいずれに対しても、凍害からの顕著な保護作用を示すことができる（図１、２、４）。 In the present invention, the term "freezing damage" means a negative effect such as cell damage, death, and functional deterioration caused by freezing or thawing of cells. “Mesenchymal stem cells” are stem cells that have the ability to differentiate into cells belonging to the mesenchymal system, such as osteoblasts, adipocytes, muscle cells, and chondrocytes. The mesenchymal stem cells include bone marrow-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells depending on the tissue to be collected, and the mesenchymal stem cells to which the cryoprotectant solution of the present invention is applied are The origin does not matter. In fact, the cryoprotectant solution of the present invention can show a remarkable protective effect against frost damage on both bone marrow-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells (FIGS. 1, 2, and 4). ).
本発明の凍害保護液は、ヒドロキシルエチルデンプン及びジメチルスルホキシドを有効成分として含む。間葉系幹細胞の凍結保存時のヒドロキシルエチルデンプン及びジメチルスルホキシドの濃度（終濃度）は、間葉系幹細胞に対して凍害からの保護作用がある限り、特に制限はない。ヒドロキシルエチルデンプンの濃度は、通常、４〜８％（ｗ／ｖ）であり、好ましくは５〜７％（ｗ／ｖ）であり、特に好ましくは６％（ｗ／ｖ）である。また、ジメチルスルホキシドの濃度は、通常、３〜７％（ｖ／ｖ）であり、好ましくは、４〜６％（ｖ／ｖ）であり、特に好ましくは５％（ｖ／ｖ）である。従って、例えば、本発明の凍害保護液を間葉系幹細胞の懸濁液と等量で混和して凍結する場合には、本発明の凍害保護液におけるヒドロキシルエチルデンプン及びジメチルスルホキシドの濃度は、上記終濃度の２倍の濃度とすればよい。本発明の凍害保護液は、例えば、生理食塩水をベースに、上記有効成分を添加して調製することができる。 The cryoprotectant solution of the present invention contains hydroxyl ethyl starch and dimethyl sulfoxide as active ingredients. The concentration (final concentration) of hydroxylethyl starch and dimethyl sulfoxide during cryopreservation of the mesenchymal stem cells is not particularly limited as long as the mesenchymal stem cells have a protective effect against frost damage. The concentration of hydroxylethyl starch is usually 4 to 8% (w / v), preferably 5 to 7% (w / v), particularly preferably 6% (w / v). The concentration of dimethyl sulfoxide is usually 3 to 7% (v / v), preferably 4 to 6% (v / v), and particularly preferably 5% (v / v). Therefore, for example, when the cryoprotectant solution of the present invention is mixed with an equal amount of a suspension of mesenchymal stem cells and frozen, the concentrations of hydroxylethyl starch and dimethyl sulfoxide in the cryoprotectant solution of the present invention are as described above. The concentration may be twice the final concentration. The frost protection solution of the present invention can be prepared, for example, by adding the above-mentioned active ingredient to physiological saline.
また、本発明は、間葉系幹細胞を凍結保存する方法であって、間葉系幹細胞をヒドロキシルエチルデンプン及びジメチルスルホキシドを含む溶液に懸濁して凍結させることを特徴とする方法（以下、「本発明の凍結保存方法」と称する）を提供する。 Further, the present invention relates to a method for cryopreserving mesenchymal stem cells, wherein the mesenchymal stem cells are suspended in a solution containing hydroxylethyl starch and dimethyl sulfoxide and frozen (hereinafter referred to as “the present invention”). A method for cryopreservation of the invention ").
間葉系幹細胞のヒドロキシルエチルデンプン及びジメチルスルホキシドを含む溶液への懸濁は、例えば、間葉系幹細胞を含む細胞懸濁液に、上記凍害保護液を添加し、両溶液を混和することにより行うことができる。 Suspension of mesenchymal stem cells in a solution containing hydroxylethyl starch and dimethyl sulfoxide is performed, for example, by adding the cryoprotective solution to a cell suspension containing mesenchymal stem cells and mixing both solutions. be able to.
凍結保存時の間葉系幹細胞の濃度（終濃度）は、特に制限はないが、例えば、１ｘ１０４ｃｅｌｌｓ／ｍＬ〜１ｘ１０８ｃｅｌｌｓ／ｍＬ（例えば、１ｘ１０５ｃｅｌｌｓ／ｍＬ〜１ｘ１０７ｃｅｌｌｓ／ｍＬ）の濃度を採用することができる。従って、例えば、本発明の凍害保護液を間葉系幹細胞の懸濁液と等量で混和して凍結する場合には、細胞懸濁液における間葉系幹細胞の濃度は、上記終濃度の２倍の濃度とすればよい。細胞懸濁液の調製においては、例えば、生理食塩水や培地を用いることができる。培地としては、例えば、ＲＰＭＩ１６４０培地が好適である。細胞懸濁液には、適宜、凝固防止剤（例えば、ヘパリンやＡＣＤ液など）を添加してもよい。 The concentration (final concentration) of the mesenchymal stem cells during cryopreservation is not particularly limited, but is, for example, 1 × 10 4 cells / mL to 1 × 10 8 cells / mL (eg, 1 × 10 5 cells / mL to 1 × 10 7 cells / mL). Can be adopted. Therefore, for example, when the cryoprotectant solution of the present invention is mixed with an equal amount of the mesenchymal stem cell suspension and frozen, the concentration of the mesenchymal stem cells in the cell suspension is 2% of the final concentration. The concentration may be doubled. In preparing a cell suspension, for example, physiological saline or a medium can be used. As the medium, for example, RPMI1640 medium is suitable. An anticoagulant (for example, heparin or ACD solution) may be appropriately added to the cell suspension.
本発明の凍結保存方法においては、間葉系幹細胞を含む細胞懸濁液に、さらに、血清アルブミン（例えば、ヒト血清アルブミン）または血清が添加されることが好ましい。血清アルブミンまたは血清の凍結保存時の濃度（終濃度）は、通常、３〜５％（ｗ／ｖ）であり、好ましくは４％（ｗ／ｖ）である。血清アルブミンまたは血清は、直接的に、または、本発明の凍害保護液への添加を通じて間接的に、細胞懸濁液に添加することができる。 In the cryopreservation method of the present invention, it is preferable that serum albumin (for example, human serum albumin) or serum is further added to the cell suspension containing mesenchymal stem cells. The concentration (final concentration) of serum albumin or serum during cryopreservation is usually 3 to 5% (w / v), preferably 4% (w / v). Serum albumin or serum can be added to the cell suspension directly or indirectly through addition to the cryoprotectant solution of the invention.
本発明の凍結保存方法において、凍結工程は、例えば、プログラムフリーザーによる緩速凍結や、超低温フリ−ザ−による簡易式凍結により行うことができる。プログラムフリーザーによる緩速凍結においては、例えば、毎分−２℃〜−３℃に計画された速さで冷却することができる。一方、簡易凍結法においては、例えば、間葉系幹細胞を含む凍結チューブを凍結処理容器や発砲スチロール箱に入れ、フリーザー内に入れて、上記と同様の速度で緩慢凍結させればよい。凍結チューブや凍結処理容器は、例えば、ＢＩＣＥＬＬ（日本フリーザー株式会社）などの市販品を利用することができる。本発明の凍結保存方法においては、簡易凍結法により簡便で効率良く作業を行うことができる。凍結工程における、フリーザーの設定温度は、−８０℃とすることができるが、より高温（例えば、−７５℃、−７０℃、−６５℃、−６０℃）であってもよい。また、当該緩慢凍結後に、液体窒素中で保存してもよい。 In the cryopreservation method of the present invention, the freezing step can be performed, for example, by slow freezing using a program freezer or simple freezing using an ultra-low temperature freezer. In slow freezing by a program freezer, for example, cooling can be performed at a planned rate of −2 ° C. to −3 ° C. per minute. On the other hand, in the simple freezing method, for example, a freezing tube containing mesenchymal stem cells may be placed in a freezing treatment container or a foamed styrene box, placed in a freezer, and slowly frozen at the same speed as described above. Commercial products such as BICELL (Japan Freezer Co., Ltd.) can be used for the freezing tube and the freezing treatment container, for example. In the cryopreservation method of the present invention, simple and efficient work can be performed by the simple freezing method. The set temperature of the freezer in the freezing step may be -80 ° C, but may be higher (eg, -75 ° C, -70 ° C, -65 ° C, -60 ° C). After the slow freezing, it may be stored in liquid nitrogen.
また、本発明は、間葉系幹細胞の凍結細胞を輸送する方法であって、当該凍結細胞がヒドロキシルエチルデンプン及びジメチルスルホキシドを含む溶液中で凍結されたものであり、当該輸送における当該凍結細胞の温度を−８０℃よりも高い温度とする方法（以下、「本発明の輸送方法」と称する）を提供する。 Further, the present invention is a method for transporting frozen cells of mesenchymal stem cells, wherein the frozen cells are frozen in a solution containing hydroxylethyl starch and dimethyl sulfoxide, and the frozen cells in the transport are transported. A method for raising the temperature to a temperature higher than -80 ° C (hereinafter, referred to as “the transportation method of the present invention”) is provided.
本発明の輸送方法における凍結細胞の冷却手段としては、上記温度で凍結細胞を輸送しうる限り、特に制限はないが、例えば、ドライアイスや各種冷却機器を利用することができる。簡便かつ安価な手段である点で、特に、ドライアイスが好ましい。 The means for cooling the frozen cells in the transport method of the present invention is not particularly limited, as long as the frozen cells can be transported at the above-mentioned temperature. For example, dry ice or various cooling devices can be used. Dry ice is particularly preferred because it is a simple and inexpensive means.
本発明の輸送方法においては、例えば、凍結された間葉系幹細胞を含むチューブを適当な輸送用の容器（例えば、発砲スチロール箱）に入れ、当該チューブの周囲にドライアイスを詰めて、凍結状態を維持しながら輸送することができる。ドライアイスそのものの温度は−７９℃であるが、輸送中の周囲の環境により、凍結細胞の温度が上昇しうる。しかしながら、本発明によれば、凍結細胞の温度が、さらに高温（例えば、−７８℃〜−６０℃）となった場合でも、間葉系幹細胞の生存や増殖能を長時間維持することが可能であり、幅広く海外への輸送も可能である。ドライアイスを利用した輸送時間は、通常、４週間以内、好ましくは２週間以内であり、より好ましくは１週間以内である。 In the transportation method of the present invention, for example, a tube containing frozen mesenchymal stem cells is placed in a suitable transportation container (for example, a foamed styrene box), and dry ice is packed around the tube to form a frozen state. Can be transported while maintaining. The temperature of dry ice itself is -79C, but the temperature of the frozen cells may increase due to the surrounding environment during transportation. However, according to the present invention, even when the temperature of the frozen cells is further increased (for example, -78 ° C to -60 ° C), the survival and proliferation ability of the mesenchymal stem cells can be maintained for a long time. It is possible to transport a wide range of overseas. The transport time using dry ice is usually within 4 weeks, preferably within 2 weeks, and more preferably within 1 week.
なお、間葉系幹細胞の使用の段階においては、例えば、チューブ中の凍結細胞を３７〜４０℃の恒温槽での急速融解を行うことが好ましい。急速融解の間は、チューブを振盪することが好ましい。また、解凍時間は、５分以内で終了することが好ましく、３分以内で終了することがより好ましい。解凍後の間葉系幹細胞は、適切な密度でフラスコに播種し、通常の条件（例えば、３７℃、５％ＣＯ２）で、インキュベーターにて培養することができる。 In the stage of using the mesenchymal stem cells, for example, it is preferable that the frozen cells in the tube be rapidly thawed in a thermostat at 37 to 40 ° C. Preferably, the tube is shaken during rapid thawing. Further, the thawing time is preferably completed within 5 minutes, and more preferably completed within 3 minutes. The mesenchymal stem cells after thawing can be seeded in a flask at an appropriate density and cultured in an incubator under ordinary conditions (for example, 37 ° C., 5% CO 2 ).
以下、実施例に基づいて本発明をより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 more specifically with reference to Examples, but the present invention is not limited to the following Examples.
Ａ．実験材料
（１）細胞
本実施例に用いた細胞は、次の通りである。ヒト骨髄由来間葉系幹細胞（ｈＭＳＣ−ＢＭ）（Ｌｏｎｚａ、製品コード：ＰＴ−２５０１）、ヒト脂肪組織由来間葉系幹細胞（ｈＭＳＣ−ＡＴ）（ＰｒｏｍｏＣｅｌｌ、製品コードＣ−１２９７７）、ヒト新生児皮膚繊維芽細胞（ＮＨＤＦ−Ｎｅｏ）。
A. Experimental Materials (1) Cells The cells used in this example are as follows. Human bone marrow-derived mesenchymal stem cells (hMSC-BM) (Lonza, product code: PT-2501), human adipose tissue-derived mesenchymal stem cells (hMSC-AT) (PromoCell, product code C-12977), human neonatal skin fiber Blast cells (NHDF-Neo).
（２）培地
ＭＳＣＧＭ ＢｕｌｌｅｔＫｉｔ（Ｌｏｎｚａ、ｒｅｆ：ＰＴ−３００１）をヒト骨髄由来間葉系幹細胞の培養に使用した。間葉系幹細胞増殖培地（ＰｒｏｍｏＣｅｌｌ、製品コード：Ｃ−２８００９）をヒト脂肪組織由来間葉系幹細胞の培養に使用した。ＤＭＥＭ（Ｌ）（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ、ｒｅｆ：１１８８５０８４）をＮＨＤＦ−Ｎｅｏの培養に使用した。
(2) Medium MSCGM BulletKit (Lonza, ref: PT-3001) was used for culturing human bone marrow-derived mesenchymal stem cells. Mesenchymal stem cell growth medium (PromoCell, product code: C-28009) was used for culturing mesenchymal stem cells derived from human adipose tissue. DMEM (L) (Thermo Fisher Scientific, ref: 11885084) was used for culture of NHDF-Neo.
（３）凍害保護液
用いた凍害保護液は、以下の通りである（表１）。
(3) Freezing protection liquid The following freezing protection liquid was used (Table 1).
Ｂ．機材
（１）フリーザー（日本フリーザー ＶＴ−７８ＨＣ）
−８０℃または−６０℃に設定して使用した（実際の温度は、設定温度±２℃程度）。
B. Equipment (1) Freezer (Japan Freezer VT-78HC)
The temperature was set at −80 ° C. or −60 ° C. (the actual temperature was about ± 2 ° C. set temperature).
（２）緩慢凍結用容器（日本フリーザー ＢＩＣＥＬＬ）
予め４℃に冷却した状態で使用した。使用前に重量がカタログ値である１９０ｇ〜２００ｇに収まっている事を確認した。
(2) Slow freezing container (Japan Freezer BICELL)
It was used after being cooled to 4 ° C in advance. Before use, it was confirmed that the weight was within the catalog value of 190 g to 200 g.
（３）セルカウンター（ベックマンコールター Ｖｉ−ＣＥＬＬ ＸＲ）
トリパンブルー染色法で５００μＬのサンプルを染色した後、サンプルを５０回に分けて測定し、その生細胞密度及び生存率の平均値を算出した。
(3) Cell counter (Beckman Coulter Vi-CELL XR)
After staining 500 μL of the sample by the trypan blue staining method, the sample was measured in 50 times, and the average value of the viable cell density and the viability was calculated.
（４）実験方法
各種細胞を解凍して培養を開始し数継代を実施した後に、対数増殖期の細胞を回収し、１ｘ１０６ｃｅｌｌｓ／ｍＬの密度でＣＰ−１若しくは対照（１０％ＤＭＳＯ、４％ ＨＳＡ）で凍結した。凍結には日本フリーザー社のＢＩＣＥＬＬを使用し、−８０℃フリーザーにて緩慢凍結を実施した。緩慢凍結２４時間後に細胞をＬＮ２タンクに移動させ、そのままＬＮ２タンクで１週間以上保存した後、細胞を各温度に設定したフリーザー（−８０、−６０℃）へ移動させた。フリーザーで一定期間（例えば、１週間）保存した後に各細胞を解凍し、解凍直後生存率、２４時間後の生存率及び培養器への接着率等を確認した。
(4) Experimental method After thawing various cells to start culturing and performing several passages, cells in the logarithmic growth phase were collected and collected at a density of 1 × 10 6 cells / mL in CP-1 or control (10% DMSO, 4% HSA). BICELL of Japan Freezer Co., Ltd. was used for freezing, and slow freezing was performed in a -80 ° C freezer. After 24 hours of slow freezing, the cells were transferred to the LN2 tank, and stored in the LN2 tank for at least one week, and then the cells were transferred to a freezer (-80, -60 ° C) set at each temperature. After being stored in a freezer for a certain period (for example, one week), each cell was thawed, and the survival rate immediately after thawing, the survival rate after 24 hours, the adhesion rate to the incubator, and the like were confirmed.
（ａ）解凍
１５ｍＬ遠心管に、９ｍＬの培地（ｗ／１０％ＦＢＳ）を分注した（洗浄用培地）。ＬＮ２タンクで保管していた細胞を３７℃水浴にて振盪解凍した。解凍した細胞全量を洗浄用培地へ加え、遠心した（５００ｇ、５分間、室温）。上清をアスピレートし、ペレットをタッピングした。ペレットに培地（ｗ／１０％ＦＢＳ）を５ｍＬ加え、Ｖｉ−ＣＥＬＬ ＸＲでセルカウントした（５５０μＬ使用）。細胞を適切な密度でフラスコに播種し、３７℃、５％ＣＯ２にて、インキュベーターで培養した。
(A) Thawing 9 mL of medium (w / 10% FBS) was dispensed into a 15 mL centrifuge tube (washing medium). The cells stored in the LN2 tank were thawed by shaking in a 37 ° C water bath. The whole amount of the thawed cells was added to the washing medium and centrifuged (500 g, 5 minutes, room temperature). The supernatant was aspirated and the pellet was tapped. 5 mL of medium (w / 10% FBS) was added to the pellet, and the cells were counted using Vi-CELL XR (550 μL was used). Cells were seeded in flasks at appropriate density and cultured in an incubator at 37 ° C., 5% CO 2 .
（ｂ）継代（接着細胞）
フラスコから培地をアスピレートした。細胞にＤ−ＰＢＳ（−）を適量加え、洗浄した。Ｄ−ＰＢＳ（−）をアスピレートし、０．２５％トリプシンを適量加えた。ここで、顕微鏡下で細胞の剥離を確認しつつトリプシン反応時間を調整した。フラスコを軽く叩き、細胞をフラスコから剥離させ、培地（ｗ／１０％ＦＢＳ）を適量加えた。細胞全量を１５ｍＬ遠心管に回収し、遠心（５００ｇ、５分、室温）を行った。上清をアスピレートし、ペレットをタッピングした。ペレットに培地（ｗ／１０％ＦＢＳ）を適量加え、Ｖｉ−ＣＥＬＬ ＸＲでセルカウントした（５５０μＬ使用）。適切な密度で細胞をフラスコに播種し、３７℃、５％ＣＯ２にて、インキュベーターで培養した。
(B) Passage (adherent cells)
The medium was aspirated from the flask. An appropriate amount of D-PBS (-) was added to the cells, and the cells were washed. D-PBS (-) was aspirated, and an appropriate amount of 0.25% trypsin was added. Here, the trypsin reaction time was adjusted while confirming the detachment of the cells under a microscope. The flask was gently tapped to detach the cells from the flask, and an appropriate amount of medium (w / 10% FBS) was added. The total amount of cells was collected in a 15 mL centrifuge tube, and centrifuged (500 g, 5 minutes, room temperature). The supernatant was aspirated and the pellet was tapped. An appropriate amount of medium (w / 10% FBS) was added to the pellet, and the cells were counted using Vi-CELL XR (550 μL was used). Cells were seeded in flasks at the appropriate density and cultured in an incubator at 37 ° C., 5% CO 2 .
（ｃ）凍結保存（接着細胞）
フラスコから培地をアスピレートした。細胞にＤ−ＰＢＳ（−）を適量加え、洗浄した。Ｄ−ＰＢＳ（−）をアスピレートし、０．２５％トリプシンを適量加えた。ここで、顕微鏡下で細胞の剥離を確認しつつトリプシン反応時間を調整した。フラスコを軽く叩き、細胞をフラスコから剥離させた。細胞に培地（ｗ／１０％ＦＢＳ）を適量を加えた。空になったフラスコに培地（ｗ／１０％ＦＢＳ）を適量加え、洗浄した。細胞全量を遠心管に回収し、セルカウントした（５５０μＬ使用）。凍結に必要な細胞量（１ｘ１０６ｃｅｌｌｓ／ｃｒｙｏｔｕｂｅ）を２本の遠心管に分注し、遠心（５００ｇ、５分、室温）を行った。上清をアスピレートし、ペレットをタッピングした。ペレットにＣＰ−１若しくは対照を加え、１ｘ１０６ｃｅｌｌｓ／ｍＬの密度にした。細胞を１ｍＬずつクライオチューブに分注した。予め４℃に冷やしておいたＢＩＣＥＬＬにクライオチューブを入れ、−８０℃フリーザーにて緩慢凍結した。２４時間後、凍結した細胞をＬＮ２タンクへ移動させた。
(C) Cryopreservation (adherent cells)
The medium was aspirated from the flask. An appropriate amount of D-PBS (-) was added to the cells, and the cells were washed. D-PBS (-) was aspirated, and an appropriate amount of 0.25% trypsin was added. Here, the trypsin reaction time was adjusted while confirming the detachment of the cells under a microscope. The flask was gently tapped to detach the cells from the flask. An appropriate amount of medium (w / 10% FBS) was added to the cells. An appropriate amount of a medium (w / 10% FBS) was added to the empty flask and washed. The total amount of cells was collected in a centrifuge tube, and the cells were counted (using 550 μL). The amount of cells required for freezing (1 × 10 6 cells / cryotube) was dispensed into two centrifuge tubes and centrifuged (500 g, 5 minutes, room temperature). The supernatant was aspirated and the pellet was tapped. CP-1 or control was added to the pellet to a density of 1 × 10 6 cells / mL. The cells were dispensed in 1 mL portions into a cryotube. The cryotube was placed in BICELL which had been cooled to 4 ° C. in advance, and slowly frozen in a −80 ° C. freezer. Twenty-four hours later, the frozen cells were transferred to the LN2 tank.
（ｄ）細胞保存試験（−６０℃）
ＬＮ２タンクで１週間以上保存した細胞を、−６０℃に設定したフリーザーに移動し、そのまま１週間静置した。
(D) Cell storage test (-60 ° C)
The cells stored in the LN2 tank for one week or longer were moved to a freezer set at -60 ° C, and allowed to stand for one week.
（ｅ）細胞解凍
１５ｍＬ遠心管に９ｍＬの培地（ｗ／１０％ＦＢＳ）を分注した（洗浄用培地）。−６０℃で一定期間保存した細胞を３７℃温水浴にて振盪解凍した。解凍した細胞全量を洗浄用培地へ加え、遠心（５００ｇ、５分、室温）を行った。上清をアスピレートし、ペレットをタッピングした。ペレットに培地（ｗ／１０％ＦＢＳ）を５ｍＬ加え、Ｖｉ−ＣＥＬＬ ＸＲでセルカウントした（５５０μＬ使用）。細胞を適切な密度でフラスコに播種し、３７℃、５％ＣＯ２にて、インキュベーターで培養した。
(E) Cell thawing 9 mL of medium (w / 10% FBS) was dispensed into a 15 mL centrifuge tube (washing medium). The cells stored at −60 ° C. for a certain period were thawed by shaking in a 37 ° C. warm water bath. The whole amount of the thawed cells was added to the washing medium, and centrifuged (500 g, 5 minutes, room temperature). The supernatant was aspirated and the pellet was tapped. 5 mL of medium (w / 10% FBS) was added to the pellet, and the cells were counted using Vi-CELL XR (550 μL was used). Cells were seeded in flasks at appropriate density and cultured in an incubator at 37 ° C., 5% CO 2 .
（ｆ）２４時間後生存率確認（接着細胞）
顕微鏡接続カメラで細胞像を記録した。フラスコ内の培地をアスピレートし、細胞にＤ−ＰＢＳ（−）を適量加え、洗浄した。Ｄ−ＰＢＳ（−）をアスピレートし、０．２５％トリプシンを適量加えた。ここで、顕微鏡下で細胞の剥離を確認しつつトリプシン反応時間を調整した。フラスコを軽く叩き、細胞をフラスコから剥離させ、細胞に培地（ｗ／１０％ＦＢＳ）を適量を加えた。空になったフラスコに培地（ｗ／１０％ＦＢＳ）を適量加え、洗浄した。細胞全量を遠心管に回収し、遠心（５００ｇ、５分、室温）を行った。上清をアスピレートし、ペレットをタッピングした。ペレットに培地（ｗ／１０％ＦＢＳ）を加え、Ｖｉ−ＣＥＬｌ ＸＲでセルカウントした。生存率及び生細胞数、接着率（接着細胞数／播種細胞数ｘ１００）を記録した。
(F) Confirmation of survival rate after 24 hours (adherent cells)
Cell images were recorded with a camera connected to a microscope. The medium in the flask was aspirated, D-PBS (-) was added to the cells in an appropriate amount, and the cells were washed. D-PBS (-) was aspirated, and an appropriate amount of 0.25% trypsin was added. Here, the trypsin reaction time was adjusted while confirming the detachment of the cells under a microscope. The flask was tapped gently to detach the cells from the flask, and an appropriate amount of medium (w / 10% FBS) was added to the cells. An appropriate amount of a medium (w / 10% FBS) was added to the empty flask and washed. The entire amount of cells was collected in a centrifuge tube, and centrifuged (500 g, 5 minutes, room temperature). The supernatant was aspirated and the pellet was tapped. A medium (w / 10% FBS) was added to the pellet, and the cells were counted using Vi-CELL XR. The viability, viable cell count, and adhesion rate (number of adherent cells / number of seeded cells x 100) were recorded.
Ｃ．結果
各細胞ともＣＰ−１若しくは対照で凍結したどちらの条件においても、ＬＮ２タンク内保存と比較して、−６０℃に設定したフリーザーで保存後に生存率が急激に減少することは無かった（表２）。
C. Results Under either condition where each cell was frozen with CP-1 or control, there was no sharp decrease in viability after storage in the freezer set at -60 ° C, compared to storage in the LN2 tank (Table 1). 2).
しかし、−６０℃フリーザーで１週間凍結保存後に細胞を培養したところ、ＣＰ−１で凍結した細胞は解凍翌日にほぼ全ての細胞がフラスコに接着しているのに対し、対照で凍結した細胞ではフラスコに接着していない細胞が多数認められた（図１〜４）。また、骨髄由来間葉系幹細胞及び脂肪組織由来間葉系幹細胞の方が、ヒト新生児皮膚繊維芽細胞よりも、解凍翌日の接着率が顕著に高かった。 However, when cells were cultured after cryopreservation for 1 week in a -60 ° C. freezer, almost all of the cells frozen with CP-1 adhered to the flask the day after thawing, whereas the cells frozen with the control did not. Many cells not adhering to the flask were observed (FIGS. 1-4). In addition, bone marrow-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells had significantly higher adhesion rates on the day after thawing than human neonatal dermal fibroblasts.
以上説明したように、本発明により、間葉系幹細胞の凍結細胞を−８０℃よりも高温で長時間保存した場合でも、凍害を抑制し、解凍後の生存及び増殖能が維持することが判明し、国内外を問わず、間葉系幹細胞のドライアイス輸送が可能となった。間葉系幹細胞は、再生医療への応用が行われていることから、本発明は、特に、再生医療分野に大きく貢献しうるものである。 As described above, according to the present invention, it has been found that even when frozen cells of mesenchymal stem cells are stored at a temperature higher than -80 ° C for a long time, frost damage is suppressed, and survival and proliferation ability after thawing are maintained. This has enabled mesenchymal stem cells to be transported on dry ice both in Japan and overseas. Since mesenchymal stem cells have been applied to regenerative medicine, the present invention can greatly contribute to the regenerative medicine field in particular.
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