JP6475242B2 - ホウ素添加細胞低温保存培地 - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Description
細胞毒性分析
五ホウ酸ナトリウム五水和物がヒト歯胚幹細胞に対する毒性作用を有するかどうかを示すために、5μg/ml〜700μ/mlの範囲の13の種々の濃度で細胞生存率分析を行った。保存溶液は、10mg/mlの五ホウ酸ナトリウム五水和物培地に溶かし、0.22μmフィルターを介してろ過することにより準備した。細胞に適用される中間の濃度を細胞培養培地中で調製し、ヒト歯胚幹細胞に適用した。
凍結プロセス中に、その標準プロトコールと異なり、20μg/ml NaBを細胞低温保存培地に添加した。対照群を標準低温保存培地により培養した。その細胞の凍結中に、20μg/ml NaBに加えて、種々の量のMe2SO(10%、7%、5%および3%Me2SO)を20% FBS(ウシ胎仔血清)および1% PSA(ペニシリン・ストレプトマイシン・アムホテリシン)を含有した培地に添加した。長期間(6か月)凍結した群を除いて、その他の細胞を4回の反復凍結解凍プロセスに付した。各凍結プロセス中に、100万個の細胞を1つの凍結用チューブに入れ、凍結タンクを用いて−80℃に凍結した。凍結用チューブは、1日後に−196℃の液体窒素蒸気に移した。各凍結プロセス後、細胞を37℃水浴中で急速に溶かし、生存率分析をトリパンブルーで行った。
長期間凍結した細胞を解凍し、標準手順とは異なる凍結プロセス中に用いた20μg/mlのNaBが、間葉細胞分化にいずれかの効果を有するかどうかを把握するためにその細胞を分化実験に付した。骨形成性、軟骨形成性および脂肪生成性の分化誘導細胞を染色(フォン・コッサ、アルシアンブルーおよびオイルレッド)および免疫細胞化学(Col I、Col II、オステオカルシン、FABP4)分析に付して、分化後に差異を形成しないことを示した。
統計分析は、スチューデントのt検定およびグラフパッドプリズムプログラムを用いて行った。
細胞生存率を完成させて、凍結実験の開始前に適用されるべき適当な五ホウ酸塩五水和物の濃度を決定した。毒性作用を200μg/mlおよびそれを超える濃度で3日間観察した(図1)。細胞生存率にいずれのネガティブな影響も有しなかった濃度のうち、20μg/mlのNaBを選択して、凍結実験に用いた。反復凍結した細胞および凍結し長期間貯蔵した細胞の細胞生存率分析の結果として、5%Me2SOの存在下の20μg/ml NaBの使用が、毒性作用を減少させることを観察した。かくして、低温保存培地中の5%Me2SOの使用は、細胞生存率を増加させることを可能にした。第2の凍結解凍プロセスの間およびそのプロセス後に細胞生存率が増加し始めることを観察した(図2)。5%Me2SOを適用した群における細胞は、長期間の凍結実験後に解凍し、それらの間葉系特性を特徴付けるために細胞に対して分化試験を行った。分化実験に続いて、増加を歯原性および軟骨形成性系統への細胞の分化において観察し、一方、わずかな減少を脂肪組織への幹細胞分化において観察した。染色および免疫細胞化学的分析によれば、細胞の分化能における有意な変化は存在しないことが判明した(図3〜4)。
本発明は、凍結解凍プロセス中に細胞および組織に生じ得る損傷を防ぐ凍結防止の凍結用(低温保存)培地である。本発明は、膵島、皮膚、角膜、心臓弁、静脈、血液および血液細胞、臍帯血ならびに移植療法に重要である組織、器官および組織片のごとき生体組織を保管するために用いる。加えて、本発明は、造血幹細胞、間葉系幹細胞、胚性幹細胞、iPS細胞(人工多能性幹細胞)のごとき組織再生および遺伝子治療に用いることができる幹細胞の長期保管に用いることもできる。本発明は、実験的研究に用いられる癌細胞、初代細胞系(繊維芽細胞、角化細胞等)および不死化細胞株を保管するために用いることができる。本発明は、体外受精目的の使用のために保管できるヒトおよび動物の***、卵子、精巣および卵巣の組織の保管に用いることができる。
Buchanan, S. S., Gross, S. A., Acker, J. P., Toner, M., Carpenter, J. F.およびPyatt, D. W. (2004). Cryopreservation of stem cells using trehalose:evaluation of the method using a human hematopoietic cell line. Stem cells and development, 13(3), 295-305.
Claims (1)
- − 20μg/ml 五ホウ酸ナトリウム五水和物(NaB)、
− 20% ウシ胎仔血清(FBS)、
− 1% ペニシリン・ストレプトマイシン・アムホテリシン(PSA)、および
− 10%、7%、5%または3% ジメチルスルホキシド(Me2SO)
を含む歯胚幹細胞用の低温保存培地であって、
保管される細胞を主成分としてコラーゲンを含む担体に付着させる場合に、用いられないことを特徴とする前記培地。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TR201309923 | 2013-08-20 | ||
TR2013/09923 | 2013-08-20 | ||
PCT/TR2014/000316 WO2015026307A1 (en) | 2013-08-20 | 2014-08-13 | Boron added cell cryopreservation medium |
Publications (2)
Publication Number | Publication Date |
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JP2016527917A JP2016527917A (ja) | 2016-09-15 |
JP6475242B2 true JP6475242B2 (ja) | 2019-02-27 |
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JP2016536064A Active JP6475242B2 (ja) | 2013-08-20 | 2014-08-13 | ホウ素添加細胞低温保存培地 |
Country Status (9)
Country | Link |
---|---|
US (1) | US9943076B2 (ja) |
EP (1) | EP3035798B1 (ja) |
JP (1) | JP6475242B2 (ja) |
DK (1) | DK3035798T3 (ja) |
ES (1) | ES2695223T3 (ja) |
HU (1) | HUE041896T2 (ja) |
PL (1) | PL3035798T3 (ja) |
PT (1) | PT3035798T (ja) |
WO (1) | WO2015026307A1 (ja) |
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CN109706119B (zh) * | 2018-04-13 | 2020-11-27 | 诺未科技(北京)有限公司 | 扩增造血干细胞的培养体系、方法及其用途 |
KR102542183B1 (ko) * | 2019-12-31 | 2023-06-14 | (주)두다 | 치아 유래 줄기세포의 분리를 위한 치아의 보존 방법 |
CN113040133B (zh) * | 2021-03-22 | 2022-03-11 | 北京贝来药业有限公司 | 作为干细胞来源的脐带/胎盘组织采集试剂盒及方法 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
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JPH02129127A (ja) | 1988-11-04 | 1990-05-17 | Sumitomo Seika Chem Co Ltd | 改良された赤血球保存液 |
CA2146098A1 (en) * | 1995-01-12 | 1996-07-13 | Ray V. Rajotte | Bulk cryopreservation of biological materials and uses for cryopreserved and encapsulated biological materials |
US5580714A (en) | 1995-03-08 | 1996-12-03 | Celox Laboratories, Inc. | Cryopreservation solution |
FR2778413B1 (fr) * | 1998-05-07 | 2000-08-04 | Immunotech Sa | Nouveaux reactifs et methode de lyse des erythrocytes |
JP2004350615A (ja) * | 2003-05-30 | 2004-12-16 | Univ Nihon | 単球株化細胞の凍結保存方法及び該方法に利用される培地 |
EP2221362A1 (en) * | 2009-02-19 | 2010-08-25 | Naturin GmbH & Co | Method for the cryopreservation of cells, artificial cell constructs or three-dimensional complex tissues assemblies |
US8519125B2 (en) | 2009-05-11 | 2013-08-27 | Biomatrica, Inc. | Compositions and methods for biological sample storage |
CN101983562B (zh) | 2010-05-26 | 2013-04-17 | 赛业(广州)生物科技有限公司 | 一种非程序细胞冻存液 |
EP2471359B1 (en) * | 2010-12-30 | 2016-03-30 | Cellulis S.L. | Method of freezing cells |
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WO2015026307A1 (en) | 2015-02-26 |
HUE041896T2 (hu) | 2019-06-28 |
EP3035798A1 (en) | 2016-06-29 |
PT3035798T (pt) | 2018-11-22 |
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