Dash Rashmi, Skillman Kristen M, Pereira Ligia, Mascarenhas Anjali, Dass Sheena, Walke Jayashri, Almeida Anvily, Fernandes Mezia, Gomes Edwin, White John, Chery-Karschney Laura, Khandeparkar Anar, Rathod Pradipsinh K, Duraisingh Manoj T, Kanjee Usheer
bioRxiv. 2023 Mar 18:2023.03.17.533128. doi: 10.1101/2023.03.17.533128.
is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous 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 isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term 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 culture. A parasite biobank was subsequently generated using this protocol resulting in a collection with 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 parasitemia 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 culture, robust maturation of ring stage parasites to later stages (>20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 hours. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average 30.0% post-MACS parasitemia and an average 5.30 × 10 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 day) or long term (7 - 10 year) storage at -80°C on parasite recovery, enrichment or viability was observed.
Here, an optimized freezing method for clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.
是疟疾的第二大常见病因，但由于缺乏连续培养系统，对其研究仍具有挑战性，这突出了建立一个临床分离株生物样本库的必要性，每个样本需多次冷冻以用于功能测定。比较了冷冻保存寄生虫分离株的不同方法，随后对最有前景的方法进行了验证。对早期和晚期寄生虫的富集以及寄生虫成熟情况进行了量化，以方便测定计划的制定。
为了比较冷冻保存方案，用四种基于甘油溶液的混合物冷冻了九种临床分离株。通过玻片显微镜检查测量解冻后、氯化钾-聚蔗糖梯度离心富集后以及短期培养后的寄生虫回收率。通过磁珠分选法（MACS）测量晚期寄生虫的富集情况。还比较了寄生虫在-80°C或液氮中的短期和长期储存情况。
在四种冷冻保存混合物中，一种混合物（甘油溶液：血清：红细胞，比例为2.5:1.5:1）提高了寄生虫回收率，并在短期培养中使寄生虫存活率有统计学意义的提高（P<0.05）。随后使用该方案建立了一个寄生虫生物样本库，共收集了106个临床分离株，每个分离株有8个冻存管。通过对47次解冻后的几个因素进行测量，验证了生物样本库的质量：解冻后疟原虫血症的平均降低率（25.3%）；氯化钾-聚蔗糖梯度离心后的平均富集倍数（6.65倍）；以及寄生虫的平均回收率（22.0%，从30个分离株中测量）。在短期培养中，60.0%的分离株在48小时内观察到环状体阶段的寄生虫强劲成熟为后期阶段（滋养体、裂殖体和配子体>20%）。通过MACS富集成熟寄生虫阶段显示出良好的重复性，MACS后疟原虫血症平均为30.0%，平均每管有5.30×10 个寄生虫。最后，测试了储存温度的影响，未观察到在-80°C下短期（7天）或长期（7 - 10年）储存对寄生虫回收率、富集或活力有重大影响。
本文展示了一种针对临床分离株的优化冷冻方法，作为生成和验证用于功能测定的寄生虫生物样本库的模板。
