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Synchronous culture of Plasmodium falciparum at high parasitemia levels

Nature Protocols volume 4, pages 1899–1915 (2009)Cite this article

Abstract

This protocol describes a method for preparing cultures of Plasmodium falciparum synchronized at any intraerythrocytic stage. Using this method, around 60% parasitized cells may be obtained. On the basis of Trager and Jensen's original continuous culture method, our approach relies on the use of fresh human blood not older than 2 weeks, a low hematocrit between 0.8 and 1.5%, a starting frozen inoculum of 10% ring-stage parasitemia, human serum replaced with AlbuMAX I and alternating sorbitol and Percoll synchronization methods to shorten the cycle window to 4–6 h and reduce sorbitol toxicity. From our synchronized high parasite density cultures, 3–5 ml of infected red blood cells can be obtained in 1 week, corresponding to 1.2 mg of total parasite protein per ml of harvested culture. On the basis of the variables parasitemia and packed cell volume, we provide an equation to accurately calculate the amount of complete medium required every 24 h corrected for the cycle stage and capacity of the culture flask. Ten days suffice to complete the protocol from a frozen stock of parasites.

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Figure 1: Flow diagram and growth curves showing the synchronous culture of Plasmodium falciparum at high parasitemia levels.
Figure 2: Staining cell smears.
Figure 3: State of the culture 48 h after thawing.
Figure 4: Synchronization of cultures using sorbitol.
Figure 5: Synchronization of cultures using Percoll 70%.
Figure 6: High-density synchronized cultures of Plasmodium falciparum.

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Acknowledgements

This work was supported by grants from the Spanish Ministry of Education and Science (BIO2006-12355 and BIO2007-67885) and by the Research Teams Consolidation Programme of the UCM (Research Team 920267 - Comunidad de Madrid). A.R. was on leave from The Pasteur Institute of Iran and holds a fellowship awarded by the AECI-Spanish Ministry of Foreign Affairs. D.M. and C.M. were supported by the Universidad de Cartagena (CO) and the Alban Programme of High Level Scholarships for Latin America, European Union scholarships Nos. E06D101036CO and E07D400516CO. M.L. holds an FPU fellowship from the Spanish Ministry of Education and Science. P. falciparum strains 3D7 and Dd2 were deposited at MR4 by D. Walliker and D.J. Carucci. J. Martinez and E. Albizua from the Haematology Service (University Hospital 12 Octubre, Madrid) kindly provided the patient isolate. Ana Burton read and commented on the manuscript.

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  1. Azar Radfar

    Present address: Present address: Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Ciudad Universitaria, Madrid, 28040, Spain

    Azar Radfar, Darío Méndez, Carlos Moneriz, María Linares, Patricia Marín-García, Antonio Puyet, Amalia Diez & José M Bautista

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Contributions

A.R., A.D. and J.M.B. conceived, designed and set up the initial protocol; A.R., D.M., C.M., M.L. and, P.M.-G. cultured the parasites; D.M., C.M., M.L. and P.M.-G. improved the protocol in the laboratory; D.M., C.M., M.L., P.M.-G., A.D. and J.M.B. designed experiments and controls; M.L. grew up the clinical isolate; A.D., A.P. and J.M.B. participated in the analysis and interpretation of the data; all authors contributed to writing different sections of the paper; A.D. and J.M.B. coordinated the laboratory work; J.M.B. wrote the final manuscript versions.

Corresponding author

Correspondence to José M Bautista.

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Radfar, A., Méndez, D., Moneriz, C. et al. Synchronous culture of Plasmodium falciparum at high parasitemia levels. Nat Protoc 4, 1899–1915 (2009). https://doi.org/10.1038/nprot.2009.198

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