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

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.

The need to compare: assessing the level of agreement of three high-throughput assays against Plasmodium falciparum mature gametocytes

“…We utilized our established protocol for gametocytogenesis induction49, with modifications consisting in the maintenance of the cultures at 15% gametocytemia and 1% haematocrit (hct) post MACs column isolation on day 8 of gametocytogenesis and daily media exchange until the parasites were used for assays on day 12.…”

The development of sexual stage malaria gametocytes in a Wave Bioreactor

“…N-acetylglucosamine (GlcNac), at a concentration of 50 mM, was previously shown to stop the multiplication of asexual parasites over a 72 h period without harm to gametocyte development [26]. In our system, several initial tests with asynchronous and synchronous cultures allowed us to determine that GlcNac at a concentration of 20 mM for 48 h successfully inhibited asexual parasite growth in the Cellbag (Fig.…”

“…Even in parasite lines with relatively high conversion rates, gametocytes usually represent less that 1% of the parasitized cells within a given culture [6, 15] and, consequently, the amount of gametocyte material that can be isolated from in vitro systems is not conducive to large or high-throughput studies. Recently, Delves et al [25] and Duffy et al [26] reported detailed protocols for the routine culture of gametocytes in volumes between 0.2 and 200 ml.…”

Biological characterization of chemically diverse compounds targeting the Plasmodium falciparum coenzyme A synthesis pathway

“…Induction of P. falciparum gametocyte differentiation and gametocyte culturing were performed as previously described [62]. Compound activity against early stage gametocytes (covering the development from stage I to IIb/III) and late stage gametocytes (stage IV to V) of the recombinant P. falciparum line NF54 Pfs16 was assessed in concentration-response assays, in two experimental replicates consisting of two technical repeats each, at a highest concentration of 40 μM, as described earlier [30, 51].…”

“…Stage V gametocyte cultures at day 11 post-induction (24 h incubation) or day 12 post-induction (2 h incubation), obtained as previously described [62], were dispensed into 384 well plates (Axygen®) at 0.5% hematocrit and ~5% gametocytemia and incubated at 37 °C with 90% N 2 , 5% CO 2 , 5% O 2 with compounds for the indicated exposure durations. Compounds were tested in concentration-response assays at a final highest concentration of 40 μM, in two biological replicates, without technical repeats.…”