JP7072147B2 - Mesenchymal stem cell frost protection solution and its use - Google Patents
Mesenchymal stem cell frost protection solution and its use 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。 The present invention relates to a cell frost damage protective solution capable of stably storing mesenchymal stem cells for a long period of time even under temperature conditions higher than −80 ° C. and its utilization.
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, products such as regenerative medicine using various mesenchymal stem cells have been approved, but for transportation during use, either a short-time transportation form at room temperature or a pole using liquid nitrogen as a cooling means. A form of low temperature transportation is adopted.
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 expand overseas, which takes a long time to transport products at room temperature, while ultra-low temperature transportation requires special transportation equipment, liquid nitrogen, etc., so there is a problem that the cost of transportation and storage management increases.
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, there is an urgent need to develop a storage method for products such as regenerative medicine, which enables long-term transportation of products such as regenerative medicine at low cost.
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）。
For hematopoietic stem cells, CP-1 (manufactured by Far East Pharmaceutical Co., Ltd.), which is a cell frost damage protective solution containing hydroxylethyl starch and dimethyl sulfoxide as active ingredients, can be used for long-term cryopreservation at -80 ° C. Has been reported (Non-Patent
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 a situation, 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 protective solution for cells used in such a preservation 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 transport by dry ice as an inexpensive method for transporting frozen cells in place of the conventional ultra-low temperature transport by liquid nitrogen. The temperature of the dry ice itself is -79 ° C, but it is possible that the temperature of the frozen cells will rise depending on the surrounding environment during transportation or the conditions of transportation. Therefore, the present inventors can maintain the survival and proliferation ability of frozen cells even under higher temperature conditions (for example, −78 ° C. to −60 ° C.) in the transport of frozen cells by dry ice. A diligent study was conducted on the protective solution.
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, it has been reported that hematopoietic stem cells can be cryopreserved for a long period of time at a freezing temperature of -80 ° C by using CP-1, which is a cell frost damage protective solution containing hydroxylethyl starch and dimethylsulfoxide as active ingredients. However, it is unclear whether CP-1 is suitable for cryopreservation at higher temperatures, and it is also unclear as to which cell type is suitable for such cryopreservation at high temperatures. Therefore, the present inventors searched for cells suitable for cryopreservation by CP-1 at a higher temperature.
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 cryopreserved by CP-1 for a long time under higher temperature conditions. However, when the cells were cryopreserved in the same manner by a conventional method without using CP-1, the proliferation ability was significantly reduced. On the other hand, in human neonatal skin fibroblasts, in contrast to mesenchymal stem cells, the proliferative ability after thawing was significantly reduced even when CP-1 was used.
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 the storage of frozen cells by CP-1 at a higher temperature, the suitability for CP-1 differs depending on the cell type, and the 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, in more detail, 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 containing hydroxylethyl starch and dimethyl sulfoxide as active ingredients to protect mesenchymal stem cells from frost damage.
［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, which comprises suspending the mesenchymal stem cells in a solution containing hydroxylethyl starch and dimethyl sulfoxide and freezing them.
［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 comprises 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 cells of mesenchymal stem cells, in which the frozen cells are frozen in a solution containing hydroxylethyl starch and dimethylsulfoxide, and the temperature of the frozen cells in the transport is set to-. A method of setting the temperature higher than 80 ° C.
［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 comprises 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 means for cooling 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 is possible to stably maintain the cell function for a long period of time. This makes it possible to transport frozen cells by dry ice both in Japan and overseas, and it is possible to avoid the problem of functional deterioration due to the temperature rise of the frozen cells during transportation. Further, since dry ice transportation does not require expensive liquid nitrogen or special transportation equipment, the transportation cost can be significantly reduced and is simple as compared with the conventional ultra-low temperature transportation. In addition, since it is not necessary to use liquid nitrogen or special equipment not only for transporting mesenchymal stem cells but also for storage, storage costs can be reduced. Therefore, mesenchymal stem cells can 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 containing hydroxylethyl starch and dimethyl sulfoxide as active ingredients for protecting mesenchymal stem cells from frost damage (hereinafter referred to as "the frost damage protective solution of the present invention").
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, "freezing damage" means negative effects such as cell damage, death, and functional deterioration caused by cell freezing and thawing. In addition, "mesenchymal stem cells" are stem cells having the ability to differentiate into cells belonging to the mesenchymal system, such as osteoblasts, fat cells, muscle cells, and chondrocytes. The mesenchymal stem cells include bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and the like, depending on the tissue to be collected. The origin does not matter. In fact, the cryoprotective solution of the present invention can exhibit 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 frost damage protective liquid of the present invention contains hydroxylethyl starch and dimethyl sulfoxide as active ingredients. The concentrations (final concentrations) of hydroxylethyl starch and dimethyl sulfoxide during cryopreservation of mesenchymal stem cells are not particularly limited as long as they have a protective effect on mesenchymal stem cells from frost damage. The concentration of hydroxylethyl starch is usually 4-8% (w / v), preferably 5-7% (w / v), and 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 frost-damaged protective solution of the present invention is mixed with the suspension of mesenchymal stem cells in an equal amount and frozen, the concentrations of hydroxylethyl starch and dimethylsulfoxide in the frost-damaged protective solution of the present invention are as described above. The concentration may be twice the final concentration. The frost damage protective liquid of the present invention can be prepared, for example, based on physiological saline by adding the above active ingredient.
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。 The present invention is a method for cryopreserving mesenchymal stem cells, which comprises suspending the mesenchymal stem cells in a solution containing hydroxylethyl starch and dimethylsulfoxide and freezing them (hereinafter, "the present invention"). The method of cryopreservation of the invention ") is provided.
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 dimethylsulfoxide is performed, for example, by adding the above frost damage protective solution to a cell suspension containing mesenchymal stem cells and mixing the two 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 mesenchymal stem cells during cryopreservation is not particularly limited, but is, for example, a concentration of 1x10 4 cells / mL to 1x10 8 cells / mL (for example, 1x10 5 cells / mL to 1x10 7 cells / mL). Can be adopted. Therefore, for example, when the frost damage protective solution of the present invention is mixed with the suspension of mesenchymal stem cells in an equal amount and frozen, the concentration of the mesenchymal stem cells in the cell suspension is 2 of the above final concentration. The concentration may be doubled. In the preparation of the cell suspension, for example, physiological saline or a medium can be used. As the medium, for example, RPMI1640 medium is suitable. Anticoagulants (eg, heparin, ACD solution, etc.) may be added to the cell suspension as appropriate.
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 at cryopreservation is usually 3 to 5% (w / v), preferably 4% (w / v). Serum albumin or serum can be added to the cell suspension either directly or indirectly through addition to the frost protection solution of the present 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 by, for example, slow freezing with a program freezer or simple freezing with an ultra-low temperature freezer. In slow freezing with a program freezer, cooling can be performed at a planned rate of, for example, -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 container or a styrofoam box, placed in a freezer, and slowly frozen at the same speed as described above. As the freezing tube and the freezing treatment container, for example, a commercially available product such as BICELL (Japan Freezer Co., Ltd.) can be used. In the cryopreservation method of the present invention, the simple freezing method can be used for simple and efficient work. The set temperature of the freezer in the freezing step can be −80 ° C., but may be higher temperature (for example, −75 ° C., −70 ° C., −65 ° C., −60 ° C.). Further, 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 dimethylsulfoxide, and the frozen cells in the transport thereof. Provided is a method of setting the temperature to a temperature higher than −80 ° C. (hereinafter referred to as “transport method of the present invention”).
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 temperature, but for example, dry ice or various cooling devices can be used. Dry ice is particularly preferable 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 transport method of the present invention, for example, a tube containing frozen mesenchymal stem cells is placed in a suitable transport container (for example, a styrofoam box), and dry ice is packed around the tube in a frozen state. Can be transported while maintaining. The temperature of the dry ice itself is -79 ° C, but the temperature of the frozen cells can rise depending on the surrounding environment during transportation. However, according to the present invention, it is possible to maintain the survival and proliferative ability of mesenchymal stem cells for a long time even when the temperature of the frozen cells becomes higher (for example, −78 ° C. to −60 ° C.). It is also possible to transport a wide range of products overseas. The transportation 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。 At the stage of using mesenchymal stem cells, for example, it is preferable to rapidly thaw the frozen cells in the tube in a constant temperature bath at 37 to 40 ° C. It is preferable to shake the tube during rapid melting. The thawing time is preferably completed within 5 minutes, more preferably within 3 minutes. After thawing, mesenchymal stem cells can be seeded in flasks at an appropriate density and cultured in an incubator under normal conditions (eg, 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 in more detail based on 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 fibers Stem 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 human adipose tissue-derived mesenchymal stem cells. DMEM (L) (Thermo Fisher Scientific, ref: 11885084) was used for culturing 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 damage protection liquid The frost damage protection liquid used is as follows (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)
It was used by setting it to -80 ° C or -60 ° C (the actual temperature is about ± 2 ° C as the 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 (Nippon Freezer BICELL)
It was used in a state of 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 a 500 μL sample with the trypan blue staining method, the sample was measured in 50 steps, and the average value of the viable cell density and the survival rate 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, starting culturing, and performing several passages, cells in the logarithmic growth phase were collected and CP-1 or control (10% DMSO, 10% DMSO,) at a density of 1x10 6 cells / mL. Frozen at 4% HSA). BICELL manufactured by Nippon Freezer Co., Ltd. was used for freezing, and slow freezing was carried out in a -80 ° C freezer. After 24 hours of slow freezing, the cells were moved to an LN2 tank, stored as they were in the LN2 tank for 1 week or longer, and then the cells were moved to a freezer (-80, -60 ° C.) set at each temperature. After storing in a freezer for a certain period (for example, 1 week), each cell was thawed, and the survival rate immediately after thawing, the survival rate after 24 hours, the adhesion rate to the incubator, etc. 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) Thaw 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 water bath at 37 ° C. The entire amount of thawed cells was added to the washing medium and centrifuged (500 g, 5 minutes at room temperature). The supernatant was aspirated and the pellet was tapped. 5 mL of medium (w / 10% FBS) was added to the pellet and cell-counted with Vi-CELL XR (using 550 μL). The cells were seeded in flasks at an appropriate density and cultured in an incubator at 37 ° C. and 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) Subculture (adhesive cells)
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 cell detachment under a microscope. The flask was tapped to detach the cells from the flask and an appropriate amount of medium (w / 10% FBS) was added. The entire 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 cell counting was performed with Vi-CELL XR (using 550 μL). The cells were seeded in flasks at an appropriate density and cultured in an incubator at 37 ° C. and 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 (adhesive cells)
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 cell detachment under a microscope. The flask was 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 medium (w / 10% FBS) was added to the empty flask and washed. The entire amount of cells was collected in a centrifuge tube and cell-counted (using 550 μL). The amount of cells required for freezing (1x10 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 pellets to a density of 1x10 6 cells / mL. The cells were dispensed 1 mL each 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. After 24 hours, 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 preservation test (-60 ° C)
The cells stored in the LN2 tank for 1 week or more were transferred to a freezer set at −60 ° C. and allowed to stand for 1 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 of time were thawed by shaking in a warm water bath at 37 ° C. The entire amount of thawed cells was added to the washing medium, and centrifugation (500 g, 5 minutes, room temperature) was performed. The supernatant was aspirated and the pellet was tapped. 5 mL of medium (w / 10% FBS) was added to the pellet and cell-counted with Vi-CELL XR (using 550 μL). The cells were seeded in flasks at an appropriate density and cultured in an incubator at 37 ° C. and 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 microscope-connected camera. The medium in the flask was aspirated, 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 cell detachment under a microscope. The flask was tapped 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 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. Medium (w / 10% FBS) was added to the pellet and cell-counted with Vi-CELL XR. The survival rate, the number of viable cells, and the 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 In both CP-1 and control-frozen conditions, the survival rate did not decrease sharply after storage in a freezer set at -60 ° C compared to storage in an LN2 tank (Table). 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 the cells were cryopreserved in a -60 ° C freezer for 1 week and then cultured, almost all the cells frozen in CP-1 adhered to the flask the day after thawing, whereas in the cells frozen in the control, the cells adhered to the flask. Many cells that did not adhere to the flask were observed (Figs. 1 to 4). In addition, bone marrow-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells had a significantly higher adhesion rate the day after thawing than human neonatal skin 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 period of time, frost damage is suppressed and survival and proliferation ability after thawing are maintained. However, it has become possible to transport mesenchymal stem cells to dry ice both in Japan and overseas. Since mesenchymal stem cells have been applied to regenerative medicine, the present invention can make a great contribution to the field of regenerative medicine in particular.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009521949A (en) | 2006-01-04 | 2009-06-11 | ドゥ−コープ テクノロジーズ リミテッド | Cryoprotective composition and method of use thereof |
| WO2011021618A1 (en) | 2009-08-19 | 2011-02-24 | タカラバイオ株式会社 | Cell preservation method |
| WO2013187077A1 (en) | 2012-06-15 | 2013-12-19 | 極東製薬工業株式会社 | Stem cell preservation medium, stem cell preservation method, and stem cell preservation system |
| JP2017525340A (en) | 2014-07-07 | 2017-09-07 | ターガザイム，アイエヌシー． | Production and cryopreservation of fucosylated cells for therapeutic use |
| WO2017172679A1 (en) | 2016-03-28 | 2017-10-05 | Cook General Biotechnology Llc | Viable cell compositions, and methods related to same |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009521949A (en) | 2006-01-04 | 2009-06-11 | ドゥ−コープ テクノロジーズ リミテッド | Cryoprotective composition and method of use thereof |
| WO2011021618A1 (en) | 2009-08-19 | 2011-02-24 | タカラバイオ株式会社 | Cell preservation method |
| WO2013187077A1 (en) | 2012-06-15 | 2013-12-19 | 極東製薬工業株式会社 | Stem cell preservation medium, stem cell preservation method, and stem cell preservation system |
| JP2017525340A (en) | 2014-07-07 | 2017-09-07 | ターガザイム，アイエヌシー． | Production and cryopreservation of fucosylated cells for therapeutic use |
| WO2017172679A1 (en) | 2016-03-28 | 2017-10-05 | Cook General Biotechnology Llc | Viable cell compositions, and methods related to same |
Non-Patent Citations (1)
| Title |
|---|
| Naaldijk, Y. et.al.，Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide，BMC biotechnology，2012年，vol.12, no.49，pp1-10 |
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