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Routine in vitro culture of P. falciparum gametocytes to evaluate novel transmission-blocking interventions

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Abstract

The prevention of parasite transmission from the human host to the mosquito has been recognized as a vital tool for malaria eradication campaigns. However, transmission-blocking antimalarial drug and/or vaccine discovery and development is currently hampered by the expense and difficulty of producing mature Plasmodium falciparum gametocytes in vitro—the parasite stage responsible for mosquito infection. Current protocols for P. falciparum gametocyte culture usually require complex parasite synchronization and addition of stimulating and/or inhibitory factors, and they may not have demonstrated the essential property of mosquito infectivity. This protocol details all the steps required for reliable P. falciparum gametocyte production and highlights common factors that influence culture success. The protocol can be completed in 15 d, and particular emphasis is placed upon operating a gametocyte culture facility on a continuous cycle. In addition, we show how functionally viable gametocytes can be used to evaluate transmission-blocking drugs both in a field setting and at high throughput (HTP) for drug discovery.

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Figure 1
Figure 2: The developmental progression of gametocyte cultures using this protocol.
Figure 3: Historical culture data showing how the efficiency of functionally mature gametocyte production declines for NF54 and 3D7 strain P. falciparum parasites the longer a parasite line is kept in continuous asexual culture.
Figure 4: Human serum is a critical determinant of gametocyte culture success.
Figure 5: Human blood supports gametocyte culture growth for 14 d after collection.
Figure 6
Figure 7: The protocol workflow relies on continuous culture of asexual feeder cultures that are periodically split; excess parasite biomass is then used to set up gametocyte cultures at 1% ring-stage parasitemia and 4% hematocrit (HCT).
Figure 8: Areas of Neubauer chamber to count to accurately calculate exflagellation.

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Change history

  • 09 March 2017

    In the version of this article initially published, an author was missing from the author list; this has been corrected to include Alexandre C. Dufour as an author. His affiliations were added as follows: "Department of Life Sciences, Imperial College London, London, UK" and "Bioimage Analysis Unit, Institut Pasteur, Paris, France." And the Author Contributions statement was amended to include the following: "A.C.D. was responsible for developing the automated image analysis algorithm used to detect male and female gametes." The error has been corrected for the PDF and HTML versions of this article.

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Acknowledgements

We acknowledge the financial support provided by the Medicines for Malaria Venture (grant RD/08/2800 to R.E.S., J.B. and M.J.D.) and the Bill and Melinda Gates Foundation (grant OPP1043501 to R.E.S., J.B. and M.J.D.) that assisted this work.

Author information

Authors and Affiliations

Authors

Contributions

M.J.D., U.S., A.R., C.M.-B. and S.M. all contributed to the development and standardization of the protocol. U.S. provided parasite culture support and supplied primary data for the manuscript. J.B. and R.E.S. guided the process. M.J.D. compiled and analyzed the primary data and wrote the draft manuscript, with all authors contributing to the final manuscript. A.C.D. was responsible for developing the automated image analysis algorithm used to detect male and female gametes.

Corresponding author

Correspondence to Michael J Delves.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 P. falciparum Dual Gamete Formation Assay – analysis of exflagellation

Exflagellation is detected and quantified from x6 objective 10 frame timelapse images. Exflagellation centres are visualised indirectly as areas of disturbance of the RBC monolayer that can be identified using ICY Bioimage Analysis program (http://icy.bioimageanalysis.org/). Scale bar = 1000 µm.

Supplementary Figure 2 P. falciparum Dual Gamete Formation Assay – analysis of female gamete formation

Female gamete formation is detected and quantified from x6 fluorescence microscopy images. Female gametes are visualised by live staining with Cy3-conjugated anti-Pfs-25 antibody and identified using ICY Bioimage Analysis program (http://icy.bioimageanalysis.org/). Scale bar = 1000 µm.

Supplementary information

Supplementary Information

Supplementary Figures 1 and 2 (PDF 292 kb)

41596_2016_BFnprot2016096_MOESM11_ESM.avi

Two exflagellating cells (red arrows) 20 min post induction, imaged at a 100× objective with differential interference contrast filters (AVI 1222 kb)

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Delves, M., Straschil, U., Ruecker, A. et al. Routine in vitro culture of P. falciparum gametocytes to evaluate novel transmission-blocking interventions. Nat Protoc 11, 1668–1680 (2016). https://doi.org/10.1038/nprot.2016.096

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