Synchronous culture of Plasmodium falciparum at high parasitemia levels

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.

ThePlasmodium falciparumblood stages acquire factor H family proteins to evade destruction by human complement

“…The cells were washed once with cell culture medium to remove the sorbitol, diluted to 5 vol% hematocrit with cell culture medium and further cultivated as described in the preceding texts. Schizont‐iRBCs were purified using Percoll gradient centrifugation as described (Radfar et al , 2009). To gain access to live MZ, schizont‐iRBCs were either treated with 0.05% saponin in incomplete cell culture medium (without serum) for 5 min at 37°C or the MZ were purified using a published protocol (Boyle et al , 2010).…”

Leucas mollissima, a Source of Bioactive Compounds with Antimalarial and Antimycobacterium Activities

“…(−)epi-marmelo lactone (1) Biological screenings: Antimalarial screening: A primary screening for compounds 1-9 was done as per standard protocols [18] at the 10 µM concentration, and for the crude mixture, a 1 µg/mL concentration was used. Protocol details are given in Supporting Information.…”

<i>In Vitro</i > Culture of <i>Plasmodium falciparum</i>: Obtention of Synchronous Asexual Erythrocytic Stages

“…This method may be used to synchronize cultures for months, thus ensuring survival and a high degree of synchrony of parasites. As compared to other methods described using low hematocrit conditions [15], which are labor-intensive (requiring culture medium changes at any time for at least two weeks), our optimized method is simple to develop, maintains culture synchrony, and avoids stress to the parasites. This procedure (SorbitolPlasmion-Sorbitol) may be repeated every week taking into account the observations in Table 1, achieving a highly synchronous culture.…”

“…It is very important to consider the maturity of trophozoite stages and, even more, the presence of early schizonts (two or three nuclei) when Plasmion treatment is applied, because the greater degree of maturity of trophozoites and even more schizonts may influence determination of the "0-hour start time" (95% of early rings obtained), which will be earlier when these stages (trophozoites and schizonts) are very mature. Another advantage of Plasmion treatment is that early and late schizonts are obtained without the need to combine a percoll gradient with sorbitol treatment [15]. Some authors [10] [15] recently reported a new synchronization method based on the combination of sorbitol treatment with percoll gradients and magnetic column purification to achieve high parasitemia levels (up to 40%).…”