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

• Published in: Nature protocols Vol. 11; no. 5; pp. 976 - 992
• Main Authors: Duffy, Sandra, Loganathan, Sasdekumar, Holleran, John P, Avery, Vicky M
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.05.2016; Nature Publishing Group
• Subjects: 14/19; 14/34; 14/56; 49/47; 49/56; 631/154/1435/2163; 631/1647/2230; 631/250/255/1629; 631/326/417/1716; Analytical Chemistry; Biological Techniques; Blood; Care and treatment; Cell culture; Computational Biology/Bioinformatics; Development and progression; Drug discovery; Drug therapy; Erythrocytes; Erythrocytes - parasitology; Gametocytes; Genetic aspects; Germ Cells; Hematocrit; Hemozoin; High-throughput screening; High-Throughput Screening Assays - methods; Humans; Immunomagnetic Separation - instrumentation; Immunomagnetic Separation - methods; Life Sciences; Liver; Lysis; Magnetic separation; Malaria; Microarrays; Microbiological Techniques - methods; Morphology; Mosquitoes; Optimization; Organic Chemistry; Parameters; Parasitemia; Parasites; Physiological aspects; Plasmodium falciparum; Plasmodium falciparum - isolation & purification; Protocol; Vector-borne diseases; Workflow; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.056

This protocol for the induction and isolation of Plasmodium falciparum gametocytes combines seven parameters that have been shown to facilitate the optimum induction of gametocytogenesis in vitro to obtain highly synchronous gametocyte stages on a large scale. 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.