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首页> 外文期刊>Journal of Parasitology >Long-Term Storage of Cryptosporidium parvum for In Vitro Culture
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Long-Term Storage of Cryptosporidium parvum for In Vitro Culture

机译：用于体外培养的密码孢子座的长期储存

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The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.
机译：密码孢子率终生阶段的长期储存是寄生虫体外培养的先决条件。在感染的宿主细胞内，在液氮中储存密码孢子虫卵囊，孢子和细胞内形式，利用不同的冷冻保护剂（二甲基亚甲醚[DMSO]，甘油和胎牛血清[FCS]），然后在体外培养。通过估计3-和7天血液培养物中的定量聚合酶链反应（QPCR）的总密集孢子率拷贝数来定量体外性能。尽管从储存的卵囊或受感染的宿主细胞中回收了很少的寄生虫，但是储存在液氮中的孢子中成功回收液氮中。将寄生虫DNA的更多副本从培养那些孢子比在4℃保持在4℃的同一时期的那些孢子中培养。储存在Roswell Park Memorial Institute（RPMI）培养基中的孢子和5％DMSO的孢子中孢子的最佳表现，其产生240％和330％的寄生虫DNA拷贝（在第3天和感染后70％） ）与对照相比。在液氮中储存在液氮中的储存比4℃的卵囊储存更有效，并且代表了用于体外培养的可行感染密码孢子脒等分试样的更一致的方法。

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