Abstract
BackgroundFiltration of leukocytes (WBCs) is a standard practice of malaria ex vivo cultures. To date, few studies have considered the effect of filtration or the lack thereof on the survival of Plasmodium vivax ex vivo cultures through one cycle of maturation. This study investigates the effect of WBC filtration and culture media supplementation on the survival of 48–72 h ex vivo cultures.MethodsUsing parasitaemia density, the study compares the survival of Plasmodipur® filtered, filter-retained or washed ex vivo cultures, maintained with McCoy’s5A medium supplemented with 25% serum alone or 20% in combination with 5% chemically defined lipid concentrate (CDLC), and in washed ex vivo cultures plus GlutaMAX™, benchmarked against IMDM™ or AIM-V™ media; also, assessed the survival of ex vivo cultures co-cultivated with human red blood cells (hRBCs).ResultsAfter 48 h of incubation a statistically significant difference was detected in the survival proportions of filtered and the filter-retained ex vivo cultures supplemented with serum plus CDLC (p = 0.0255), but not with serum alone (p = 0.1646). To corroborate these finding, parasitaemias of washed ex vivo cultures maintained with McCoy’s5A complete medium were benchmarked against IMDM™ or AIM-V™ media; again, a statistically significant difference was detected in the cultures supplemented with CDLC and GlutaMAX™ (p = 0.03), but not when supplemented with either alone; revealing a pattern of McCoy’s5A medium supplementation for Aotus-derived P. vivax cultures as follows: serum < serum + GlutaMAX™ < serum + CDLC < serum + CDLC + GlutaMAX™; confirming a key role of CDLC in combination with GlutaMAX™ in the enhanced survival observed. Lastly, results showed that co-cultivation with malaria-naïve hRBCs improved the survival of ex vivo cultures.ConclusionsThis study demonstrates that WBC filtration is not essential for the survival of P. vivax ex vivo cultures. It also demonstrates that McCoy’s5A complete medium improves the survival of Aotus-derived P. vivax ex vivo cultures, with no significant difference in survival compared to IMDM and AIM-V media. Finally, the study demonstrates that co-cultivation with hRBCs enhances the survival of ex vivo cultures. These findings are expected to help optimize seeding material for long-term P. vivax in vitro culture.
Highlights
Filtration of leukocytes (WBCs) is a standard practice of malaria ex vivo cultures
Filtration of leukocytes is not essential for the survival of Plasmodium vivax Aotus‐derived ex vivo cultures The observation that P lasmodipur® filter-retained iRBCS ex vivo cultures, survived longer than filtered Infected Red Blood Cells (iRBCs) during attempts to grow Aotus-derived P. vivax parasites in vitro, prompted the design of an experiment to assess the effect of filtration on the survival of filtered versus retained P. vivax SAL-1 Aotus-derived iRBCs ex vivo cultures, maintained with McCoy’s5A culture medium supplemented with either 25% serum alone or 20% in combination with 5% chemically defined lipid concentrate (CDLC), determining their endpoint parasitaemias at 48 or 72 h of incubation (Fig. 1)
The serendipitous finding that a P. vivax-positive clinical sample inoculated into an Aotus monkey remained viable after being left at room temperature for three days, prompted the hypothesis of this study, that filtration of White Blood Cells (WBCs) was not essential for the survival of P. vivax ex vivo cultures, but that leukocytes may play a role at maintaining a homeostatic milieu mimicking whole blood conditions ex vivo
Summary
Few studies have considered the effect of filtration or the lack thereof on the survival of Plasmodium vivax ex vivo cultures through one cycle of maturation. This study investigates the effect of WBC filtration and culture media supplementation on the survival of 48–72 h ex vivo cultures. Plasmodium vivax has proven difficult to maintain in continuous in vitro culture beyond 48–72 h of incubation, due mainly to its special culture conditions [17], its preference at invading young reticulocytes that are restricted to the bone marrow [18,19,20,21,22,23] and a plethora of other factors that may impact the fitness of the parasite [24]. A clear understanding of the culture conditions and nutrient media supplementation requirements for the survival of Aotus-derived ex vivo cultures during the critical first 48–72 h of incubation is essential for the establishment of a long-term in vitro culture [21]
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