Development of aPlasmodium vivaxbiobank for functionalex vivoassays

Abstract

AbstractBackgroundPlasmodium vivaxis the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuousin vitroculture 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.MethodsIn order to compare cryopreservation protocols, nine clinicalP. vivaxisolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-termin vitroculture 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.ResultsOf 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-termin vitroculture. 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-termin vitroculture, 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 × 105parasites/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.ConclusionsHere, an optimized freezing method forP. vivaxclinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.