Goa Medical College and Hospital, Bambolim, Goa, 403202, India.
Departments of Chemistry and Global Health, University of Washington, Seattle, WA, 98195, USA.
Malar J. 2023 Aug 31;22(1):250. doi: 10.1186/s12936-023-04668-2.
Plasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning.
In order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either - 80 °C or liquid nitrogen were also compared.
Of the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P < 0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection of 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitaemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (> 20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 h. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average of 30.0% post-MACS parasitaemia and an average of 5.30 × 10 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 days) or long-term (7-10 years) storage at - 80 °C on parasite recovery, enrichment or viability was observed.
Here, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.
间日疟原虫是第二大常见疟疾病原体，但由于缺乏连续的体外培养系统，研究它仍然具有挑战性，这凸显了建立包含多次冻融的临床分离株生物库的必要性，以便用于功能测定。比较了不同的冷冻保存寄生虫分离物的方法，随后验证了最有前途的方法。量化早期和晚期寄生虫的富集和寄生虫成熟度，以方便测定计划。
为了比较冷冻保存方案，用四种基于甘油的混合物冷冻了 9 个临床间日疟原虫分离物。通过载玻片显微镜测量解冻后、经 KCl-Percoll 富集后和短期体外培养中的寄生虫恢复情况。通过磁激活细胞分选 (MACS) 测量晚期寄生虫的富集情况。还比较了 -80°C 或液氮中的短期和长期储存对寄生虫的影响。
在四种冷冻保存混合物中，一种混合物（甘油：血清：RBC 的比例为 2.5:1.5:1）导致寄生虫回收率提高，并在短期体外培养中显著（P<0.05）提高了寄生虫的存活率。随后使用该方案生成了一个寄生虫生物库，其中包含 106 个临床分离物，每个分离物有 8 个小瓶。通过测量 47 次解冻后的几个因素验证了生物库的质量：解冻后寄生虫血症的平均减少（25.3%）；经 KCl-Percoll 富集后的平均倍数（6.65 倍）；以及寄生虫的平均回收率（22.0%，取自 30 个分离物）。在短期体外培养中，在 48 小时内，60.0%的分离物中观察到环状期寄生虫向晚期阶段（>20%的滋养体、裂殖体和配子体）的成熟。通过 MACS 富集成熟的寄生虫阶段显示出良好的重现性，MACS 后寄生虫血症平均为 30.0%，每个小瓶平均有 5.30×10 个寄生虫。最后，测试了储存温度的影响，在 -80°C 下短期（7 天）或长期（7-10 年）储存对寄生虫回收、富集或活力没有明显影响。
这里展示了一种优化的间日疟原虫临床分离物冷冻方法，作为生成和验证用于功能测定的寄生虫生物库的模板。
