CRYO-PRESERVATION OF THE PARASITIC PROTOZOA
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概要
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最近30年聞に発表された原虫の凍結保存に関する論文は， 200篇を越えている。そこでこの論文では，それらのうち主な論文を紹介するとともに凍結保存が可能ま原虫類の保存方法や保存期間などを総括し，さらに今後の問題点を論じた。また今までに十分な検討を加えずに用いられていた凍害保護剤について，特にグリセリンとDMSOの用い方，平衡時聞をどについて，著者の研究を中心に紹介した。原虫類の最適保存法及びこの論文の論旨は次の通りである。1) 原虫は，適当な保護剤を含む溶液あるいは培地中に撹拌し，試験管またはアンプルに分注する。2)保護剤の濃度は，グリセリンは10%前後， DMSOは， 7.5%前後が適当である。グリセリンの場合は，比較的高い温度(例えば37C)で30-60分平衡させる。高温に耐えない原虫は， 25C前後で60-90分平衡させる。DMSOは，低い温度(例えば0C)で加え，平衡時聞をおかず直ちに凍結する。3)凍結は2段階を用いる。すなわち， -30C前後のフリーザー中で約90分予備凍結し(この時冷却率は約1C，1分)，ついで保存温度へ移す。4)保存温度としては，液体窒素のような超低温が好ましいが， -75Cでも数カ月程度は保存可能である。5)凍結材料は，37～40Cの恒温槽中で急速融解し，融解後は，すみやかに動物あるいは培地へ接種する。6)原虫の種類によっては， もっと簡単に保存できる。原虫ごとに予備試験を行い，目的の保存温度に数日保存して高い生存率の得られる方法を採用するとよい。7) 今後の問題点としては，保存原虫の性質(薬剤耐性，抗原性，感染性など)の長期保存における安定性を検討することと純低温生物学的な立場から超低温下における細胞の生死のメカニズムを解明することである。前者については，多くの研究者が凍結保存による実験株の性質の変化は認められないと指摘している。8) 終りに数多くの実験株を保存し， 研究者に提供する低温保存センターの設置の必要性を提案した。In the present paper, about 200 literatures on the cryo-preservation of the parasitic protozoa have been surveyed, and the following problems have been discussed: cooling rate, storage periods at various temperatures, effects of cryo-protective substances in relation to equilibration time or temperatures, and biological properties before and after freezing. This paper is composed of three main chapters, and at first, the history of cryo-preservation is reviewed in details. In the second chapter, other literatures, which were not cited in the first chapter, are introduced under each genera or species of the protozoa. In the last chapter, various factors on cryo-preservation mentioned above are discussed by using the author's data and other papers in which various interesting problems were described. The following conclusions have been obtained in this study: Before preservation at the lowest storage temperature, it appears preferable that samples are pre-cooled slowly at the rate of 1 C per minute until the temperature falls to -25 to -30 C. The cooling rate might be obtained by the cooling samples for 60 to 90 minutes at -25 to -30 C freezer. For storage, however, lower temperatures as low as possible are better for prolonged storage of the samples. Many workers recommended preservation of the samples in liquid nitrogen or in its vapor, but the storage in a dry ice cabinet or a mechanical freezer is also adequate, if the samples are used within several weeks or at least several months. Cryo-protective substances such as glycerol or DMSO are highly effective to keep higher survival rate of the protozoa in frozen state. Most workers recommended to use 10% glycerol or 5 to 7.5% DMSO for this purpose. For the use of glycerol, at least 30 to 60 minutes of equilibration at temperatures as high as 37 C, is necessary to produce satisfactory results, because at 25 C or lower temperatures, the cryo-protective action of glycerol becomes insufficient. In DMSO, however, samples should be cooled as soon as possible after adding the substance into protozoan suspension. Prolonged equilibration with DMSO is apparently toxic to trichomonad. Many workers pointed out that biological properties of the protozoa such as infectivity, virulence, antigenicity, and drug resistance, were not changed by prolonged period of preservation at the low temperature. These aspects are greatly advantageous for cryo-preservation. By adopting the cryopreservation technique, furthermore, we save expenses for the maintenance through animal passages or in vitro culture, and thus we can store much more protozoan species or strains in the laboratory. Finally, the author proposed to build the cryo-preservation center of protozoan strains, because in this country we have no adequate center to deposit our strains. Even if nobody use a certain strain for experiment at the moment, yet we can preserve them for the future need. Other laboratories also have many strains, and some of them may be used frequently, but others are not. If all strains be collected in one center, and if the center supplies each strain in case of need, we could save a lot of expense to preserve strains in each laboratory.
- 1975-09-15
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