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Large-scale production of Plasmodium falciparum gametocytes for malaria drug discovery

Nature Protocols volume 11, pages 976–992 (2016)Cite this article

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Abstract

The tightly controlled induction of Plasmodium falciparum gametocytes in large-scale culture is a fundamental requirement for malaria drug discovery applications including, but not limited to, high-throughput screening. This protocol uses magnetic separation for isolation of hemozoin-containing parasites in order to (i) increase parasitemia, (ii) decrease hematocrit and (iii) introduce higher levels of young red blood cells in a culture simultaneously within 2–4 h. These parameters, along with red blood cell lysis products that are generated through schizont rupture, are highly relevant for enabling optimum induction of gametocytogenesis in vitro. No other previously published protocols have applied this particular approach for parasite isolation and maximization of fresh red blood cells before inducing gametocytogenesis, which is essential for obtaining highly synchronous gametocyte classical stages on a large scale. In summary, 500–1,000 million stage IV gametocytes can be obtained within 16 d from an initial 10 ml of asexual blood-stage culture.

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Figure 1: Intra-erythrocytic life cycle of P. falciparum.
Figure 2: Gametocyte induction workflow.
Figure 3: Representative images of 100× oil-immersion bright-field micrographs of Giemsa-stained thin blood smears.
Figure 4: High-throughput late-stage gametocyte confocal imaging assay performance.
Figure 5: VarioMACS preparation for PROCEDURE Steps 14–18, 24A(iv–vi), 24B(iii), 25 and 26.

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Acknowledgements

The authors thank and acknowledge the Australian Red Cross Blood Bank for the provision of fresh red blood cells, without which this research could not have been performed. We thank Medicines for Malaria Venture (MMV) for the supply of the MMV Malaria Box. This work was supported by the Australian Research Council (LP120200557 to V.M.A.) and MMV.

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Authors and Affiliations

  1. Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland, Australia

    Sandra Duffy, Sasdekumar Loganathan, John P Holleran & Vicky M Avery

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Contributions

S.D. conceived, developed and performed the majority of the experimental research involved in optimizing this protocol. S.D. contributed significantly to troubleshooting and undertook the studies using the product of the protocol. S.D. wrote the manuscript and revisions. J.P.H. contributed to writing of the first draft and revision of the final manuscript, and assisted with troubleshooting the protocol. S.L. contributed to the final manuscript, provided technical assistance during the development and optimization of the induction protocol and formatted images. S.L. also designed Figure 2. V.M.A. supervised the drug discovery program, provided guidance and contributed to the interpretation of data and writing of the manuscript.

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Correspondence to Vicky M Avery.

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Duffy, S., Loganathan, S., Holleran, J. et al. Large-scale production of Plasmodium falciparum gametocytes for malaria drug discovery. Nat Protoc 11, 976–992 (2016). https://doi.org/10.1038/nprot.2016.056

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