Cultivation of the erythrocytic stages of Plasmodium parasites, specifically the most important and deadly for humans, Plasmodium falciparum, has required a lot of effort and time in order to develop a continuous in vitro culture. Moreover, the development of methods to synchronize P. falciparum parasites (which grow asynchronously in vitro) has become an essential tool in research to study different immulogical, biochemical or physiological aspects of the parasite. We have compared two different synchronization methods, one based on differential permeability of the membrane of parasitized erythrocytes, and the other on the sedimentation behavior in gelatin solution. An optimized method has been established which allows for maintaining a healthy, highly synchronous culture for longer periods of time. Asexual erythrocytic stages of a complete P. falciparum cycle have been obtained, which is the starting point of the stage-specific assays of the activity of new antimalarial drugs.
Malaria is the most important parasitic infection in humans. Globally, 3.2 billion people in 97 countries and territories are at risk of being infected with malaria and developing disease. In 2014, 198 million cases of malaria occurred, causing 584,000 deaths, especially in sub-Saharan African countries, where children under 5 years were the most affected population group [
Malaria is caused by five species of parasites belonging to the genus Plasmodium. Four of these (P. falciparum, P. vivax, P. malariae and P. ovale) are human malaria species, which are transmitted from one person to another by female mosquitoes of the genus Anopheles [
In human host, P. falciparum exhibits a synchronous life cycle for approximately 48 hours. A blood sample taken at any time from an infected host will show a parasite population at the same stage of the developmental cycle, e.g. mostly ring stages or schizonts [
Various synchronization methods have been reported considering different properties such as temperature cycling [
This study compares the synchronization method described by Lambros and Vanderberg based on sorbitol treatment [
RPMI 1640 with 25 mM HEPES, sodium bicarbonate and glutamine (GIBCOTM ref: 52400-025), supplemented with 10% of pooled human sera AB (14-490 E Cambrex) and 0.15 mM of hypoxanthine (from HT supplement ×50, GIBCOTM ref: 41065). Human sera are decomplemented 30 minutes at 56˚C, aliquoted and stored frozen at −20˚C until use. Complete medium is usually prepared fresh just before use and pre-warmed at 37˚C.
Red blood cells stock suspensions are prepared from whole blood bags coming from incomplete blood donation, provided by the Spanish Red Cross (<25 days after sampling). Whole blood is aliquoted and stored at 4˚C. To prepare red blood cells, whole blood is centrifuged and washed 3 times with serum-free RPMI by centrifugation (10 minutes at 2000 rpm). The upper phase, containing white blood cells, is removed. Washed red blood cells are kept as a 50% suspension in complete medium, and stored for a maximum of 4 days at 4˚C. The human biological samples were sourced ethically, and were used for research in compliance with the terms of the informed consents.
P. falciparum 3D7 strain was obtained from the MR4 Resource Center (ATCC) and was maintained in complete medium at a hematocrit of 5% in continuous culture using a method adapted from Trager and Jensen [
To establish synchrony, two different methods were considered: Sorbitol treatment (isolation of ring stage) and Plasmion treatment (isolation of trophozoite and schizont stage). Sorbitol solution: 5% (w/v) of sorbitol (Sigma S 6021) in cell culture grade water (Sigma W3500). Plasmion solution (Laboratoire Fresenius Kabi, France).
Sorbitol treatment to synchronize cultures: This synchronization method, described by Lambros and Vanderberg [
Plasmion treatment to synchronize cultures: A method based on sedimentation behavior in gelatin solution which also selects the phenotype of knobs+ parasites. In a Plasmion solution, the knob+ stages float, while the younger stages (rings) sediment like uninfected RBCs. This property is used to select mature parasites stages (trophozoites and schizonts). Pre-warm an aliquot of Plasmion. Remove most old medium from the flask culture and transfer to a 15 mL tube. Centrifuge 5 minutes at 18000 rpm at room temperature, discard culture medium and read the pellet volume. Add 1.4 mL of complete medium and 2.4 mL of Plasmion solution for each mL of cell pellet and mix gently. After incubating for 30 minutes at 37˚C without any movement to avoid disturbing sedimentation, take the supernatant (containing the older stages of the parasite, trophozoites and schizonts) and use it to make a dilution or to establish the specific culture conditions needed.
Method described by Lambros and Vanderberg (1979) with some modifications [
Method described by Ter Kuile et al. (1993) with some modifications [
Percentage of parasitemia and predominant stage were evaluated by Giemsa staining every 24 hours. After 2 and 6 hours of Plasmion treatment, the culture was also evaluated (total time of culture: 168 hours). A Giemsa smear was also made after each synchronization treatment in order to measure the effect of the specific method. This synchronization method was made twice with three replicates each time.
Different sorbitol and plasmion cycles were used to establish an optimized synchronous method. Two differerent synchronization methods, one based on differential permeability (Sorbitol 34 hours) and the other on sedimentation behavior (Sorbitol 6 hours), were compared.
This synchronization method started with a sorbitol treatment after which the culture was adjusted to 1% parasitemia and 5% hematocrit (
Vanderberg results [
This synchronization method was based on the properties of the Plasmion solution (that allows for purification of mature parasites without affecting their viability) combined with three sequential sorbitol treatments (two each 48 hours of culture and the third one 6 hours after Plasmion treatment). Results of this method are illustrated in
Plasmion treatment. The ring stage was significantly enriched after sorbitol (83%), but percentage of parasitemia was not so high (0.11%) due to the initially low proportion of rings at this moment (24%). Parasites grow until 168 hours of culture, although the predominant stage at this time was the opposite as expected (rings instead of trophozoites). Parasitemia also decreased (from 3.20 to 3.06; no gametocytes observed), showing that the culture was not so healthy due to the dramatic decrease in parasitemia occurring after the third sorbitol treatment (0.11%).
These synchronizations methods (sorbitol and Plasmion) are essential tools to study different aspects of malarial parasites, such as inmunological, biochemical, and physiological differences between the different stages of parasite development. In order to obtain healthy parasites to guarantee their survival during such studies, some factors have been considered. Based on our results, sorbitol treatment is a more aggressive method than Plasmion treatment: gametocytes stages were observed during sequential sorbitol protocols (more than two sorbitol treatments in less than 48 hours), while no gametocytes were observed during Plasmion treatment. For sorbitol treatment, a very important limiting factor is the proportion of rings at the start of the process. For example, a 29% of parasitemia reduction (from 6.75% before sorbitol to 4.77% after sorbitol) was observed, when the proportion of rings at the initial time was 99%. By contrast, if the proportion of rings was 40%, parasitemia reduction increased to 93% (from 4.80% before sorbitol to 0.36% after sorbitol). Ring age may also influence development of the treatment: late rings (16 - 26 hours) are more susceptible to sorbitol (due to permeability changes in these erythrocytes parasitized by rings), so that at this point, age of the rings may have influenced sorbitol efficiency. Treatment with Plasmion allows for achieving a higher concentration of mature stages (trophozoites and schizonts) very easily and quickly without affecting parasite viability. An important factor to consider is the reinvasion capacity of the mature stages, which may be compromised at high parasitemia levels.
Two different assays were performed to establish the best conditions for reinvasion (with rings or trophozoites at initial time of culture) (
Considering all these conditions (percentage of predominant stages before synchronization treatment, initial
parasitemia, growth factor, etc.) a method for optimal synchronization of cultures was established (
This method combines two sequential sorbitol treatments (each 48 hours) and a Plasmion treatment at 24 hours of culture (
Time (h) | CULTURE | TREATMENT | OBSERVATIONS | |
---|---|---|---|---|
0 | Asynchronous | Sorbitol | ||
5% - 7%p | At least 1.5% parasitemia after treatment | |||
90% rings | Consider decrease in parasitemia (80%) | |||
5% Hematocrit | ||||
24 | Mature stages | Plasmion | Consider mature stage reinvasion (×4 up to ×9 when synchrony increases) | |
48 | Ring stages | Sorbitol | Consider age of rings | |
(Rings of 0 - 16 h, best results) | ||||
72 | Mature stages | If %p > 2: Plasmion | High parasitemia in mature stages | |
If %p < 2: dilution to adjust to 1% parasitemia | may compromise viability | |||
96 | Rings stages | If %p > 3 dilution: to adjust to 3% parasitemia | The next day will be the same %parasitemia with mature stages | |
120 | Mature stages | Medium change/Plasmion | Depending on degree of synchrony: | |
Plasmion if %mature stages < 90% | ||||
Medium change if synchrony is high | ||||
144 | Ring stages | Sorbitol/Dilution | Sorbitol if there are still mature stages Dilution to keep the culture until assay |
Plasmion treatment is the same as reported by other authors [
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 [
Ring stage was observed 6 hours after Plasmion treatment (57%), and the highest percentage of rings (95%) was found 10 hours after Plasmion treatment. The “0-hour start time”, for a synchronous complete cycle of 48 hours of the erythrocytic stages has been defined at this point. 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 [
In agreement with other authors [
The new synchronization method has been well characterized and allows for obtaining the specific asexual erythrocytic stages of a complete 48-hours cycle of P. falciparum parasites. In most studies, the establishment of highly synchronized cultures of Plasmodium falciparum has become essential. These being isolation of early rings, late rings, early trophozoites, late trophozoites, early schizonts and late schizonts [
Standard synchronization methods of P. falciparum cultures (sorbitol and Plasmion) were compared to assess the advantages of each of them (obtention of early stages (rings) with sorbitol treatment and late stages (trophozoites and schizonts) with Plasmion treatment). An optimized method was established that maintains the high degree of synchrony of the parasites and optimal culture conditions for long periods of time. The specific asexual erythrocytic stages of a complete 48-hour cycle of P. falciparum parasites have been defined, and their specific time of emergence has been determined, in order to be used for stage specificity assays to characterize the stage-specific activity and rates of action of novel antimalarial drugs.
The authors are indebted to our partners at the Medicines for Malaria Venture, who not only helped to support this work financially but also provided advice and facilitated collaborations within the malaria scientific community.