synchronous culture of plasmodium falciparum at high

nature protocols | VOL.4 NO.12 | 2009 | 1899

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IntroDuctIonThe successful culture of malaria parasites is crucial for gaining insight into their biology, biochemistry, immunology and pharma-cology1. Unfortunately, recent developments in omics sciences have not been matched by significant progress in culturing techniques for malaria parasites. In static cultures, parasite growth and survival are dramatically hindered at high parasitemias (above 10%; see Box 1 for definitions)2, and only a few adapted laboratory strains of the species Plasmodium falciparum can be easily cultured using stand-ard methods to yield parasitemia levels of up to 30% (ref. 3). Thus, although several methods have been developed, the technique used for the in vitro culture of the intraerythrocytic stages of P. falciparum remains essentially the same as that originally described by Trager and Jensen4. The protocol described by these authors was based on the use of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-buffered RPMI tissue culture medium supplemented with human serum, erythrocytes and sodium bicarbonate. Since then, efforts and time have been invested in trying to improve the in vitro growth of the asexual stages of the Plasmodium life cycle (see Box 2), as these stages give rise to the most common patho-logical conditions of malaria and are thus a main target for research into antimalarial drugs1,5.

For reasons including cost, reproducibility and possible pres-ence of inhibitory immune factors and antimalarial drugs, initial research was focused on trying to find types of mammalian sera other than human or on developing serum-free media for parasite culture6,7. Despite testing cow, monkey, horse, goat, sheep, rabbit and pig sera, none have proven to be as good as human serum6,7, and several chemical preparations have been found to be just as efficient as human serum for culturing purposes. These new culture media span from freshly prepared human high-density lipoprotein fraction8 to commercial serum replacements, such as Nutridoma-SR (4%)8,9, AlbuMAX I9 and AlbuMAX II10, although the latter require supplementation with hypoxanthine as a source of purines11,12. AlbuMAX I has been reported to improve growth, yielding more than twofold parasites at any stage of the growth

cycle, and is suitable for in vitro antimalarial screening as in vitro drug susceptibility assays have shown the similar sensitivity to anti-malarials and natural product extracts of parasites grown in this medium and human serum–supplemented media13.

Two main culture methods were initially reported: the Petri dish or candle jar technique, and a continuous flow method14. The latter procedure requires sophisticated equipment and is expensive to maintain in terms of the fresh blood, media and serum required15. Using the former method, cultures with parasitemia levels of up to 85% have been achieved after 7 d, but only asynchronous cultures11. So far, synchronized cultures of P. falciparum containing up to 40% parasitized cells have been obtained only through the use of diluted red blood cell (RBC) suspensions and daily replacement of culture medium16. Erythrocyte reinvasion by schizont progeny can be enhanced by diluting cultures to a 1% hematocrit (see Box 1 for definitions) immediately after synchronization with Percoll17.

Highly synchronized high parasitemia cultures are needed for comparative studies on global gene expression at specific parasite stages based on proteomic or microarray analysis13. Low hematocrit cultures with only small proportions of uninfected RBCs consid-erably reduce interference in proteomic studies16,18. Also desirable is that the 23-Mb genome of P. falciparum with ~5,800 protein-encoding genes19 should be amenable to accurate functional ana-lysis by proteomics of different pathogenic patient isolates. In effect, new insights into diseases have been recently provided by com-paring genomes across different cell phenotypes20, and proteomic studies on the most experimentally accessible stages of P. falciparum clones have made considerable progress in characterizing para-site subproteomes18,21,22. However, although these proteome-wide studies have identified approximately half the 5,800 predicted genes of the genome sequence of P. falciparum, we still need to iden-tify less abundant proteins23 arising from different experimental conditions across the asexual cycle over a shorter window than the duration of the stage’s rings, trophozoites or schizonts. Efforts targeted at developing an attenuated malaria vaccine, discovering

Synchronous culture of Plasmodium falciparum at high parasitemia levelsAzar Radfar1, Darío Méndez, Carlos Moneriz, María Linares, Patricia Marín-García, Antonio Puyet, Amalia Diez & José M Bautista

Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Ciudad Universitaria, Madrid 28040, Spain. 1Present address: Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. Correspondence should be addressed to: J.M.B. ([email protected]).

Published online 19 November 2009; doi:10.1038/nprot.2009.198

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.

1900 | VOL.4 NO.12 | 2009 | nature protocols

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new antimalarial targets against different blood parasite stages or hemozoin-based studies will also require a method of easily pro-ducing large amounts of short window synchronized parasites. Moreover, it should be possible to generate whole-parasite anti-gens of a given stage from highly synchronized cultures and soluble antigens by the lysis of parasites or culture supernatants.

Recently, using this protocol to produce high parasitemia syn-chronous cultures in low hematocrit conditions18, we were able to obtain sufficient protein to compare the effects of chloroquine on the oxidatively modified proteome across the parasite’s intraer-ythrocytic stages, providing data on the early molecular effects of the drug, as well as on the normal protein-oxidation modifications that occur during the parasite cycle18. Other potential applications of this protocol include immunoproteomic analysis using local clinical isolates, hemoglobin uptake pathway research and puri-fication of large protein assemblies for structural biology analy-ses (e.g., mitochondrial cytochrome bc1 complex, as the target of the antimalarial agent atovaquone). Although our protocol has proven benefits for the medium- and large-scale preparation of parasitized red cells, one of its drawbacks is the technical attention required, in that culture medium changes may be required any time during the day or night to avoid parasite stress. Finally, it should be mentioned that a limitation of our method is that no positive growth control can be run as any problems arising could as much be related to the inoculum or the parasite strain as to the technique, and thus in either case propagation at any stage would be affected (see Experimental design).

There are several methods available to obtain similar results to our protocol4,14–17, some of which provide theoretical estimates of the hematocrit needed to achieve a given level of parasitemia24. All these methods, including this protocol, are based on the origi-nal Trager and Jensen technique4,14. As described later by Jensen15, current laboratory methods (excluding the most recent bioreactor culture procedures25 that do not yield high parasitemias) indicate that high parasitemias can be achieved but no precise details on the daily follow-up of the cultures are given. Although some protocols also recommend the use of fresh blood15,16, after comparing the use of different erythrocyte age controls, we have observed that para-sitemia is seriously hindered in red cells older than 2 weeks. Other methods have tested15,16 or theoretically suggested24 low hematocrit values to achieve high parasitemias, showing the importance of diminishing the relative proportions between the number of red cells and parasites as growth proceeds. Thus, although plots of hematocrit versus parasitemia in culture have been provided by other authors24, we present an equation based on this relationship and corroborated by our own experimental data (data not shown) to calculate the volume of culture medium required at a given developmental stage of the parasite for the desired parasitemia

in a specified volume of red cells. Culture improvement through the use of AlbuMAX (I or II) instead of human serum has also been described by others11–13,15, and based on our observations, this opens the possibility of using human red cells from any AB0 group. The use of sorbitol alone has been found not to achieve suitable synchrony, especially at high parasitemias15 and thus the use of alternatives has been proposed, including Percoll in different pro-portions17,24,26–28, gelatine14,29, Percoll/sorbitol gradient30 and also a combination of sorbitol and gelatine21. Our protocol recommends alternating sorbitol and Percoll 70% synchronization at defined stages of development during culture to provide a short-cycle window and diminish parasite stress.

Experimental designModifications to original culture method. Here we describe the protocol for the culture of P. falciparum in sufficient detail for a nonspecialist to obtain 1.2 mg of total parasite protein per ml of harvested culture. Short window synchrony is achieved by intro-ducing the following crucial modifications to the original Trager and Jensen method4:

(1) Only fresh human blood from healthy donors is used. This is added to the culture medium when the parasites reach the late trophozoite stage, just before RBC invasion. Whole blood is stored at 4 °C in the blood preservative citrate-phos-phate-dextrose-adenine (CPDA). Although CPDA has been reported to maintain erythrocyte fitness for malaria cultures for up to 35 d (ref. 15), invasion susceptibility also decreases with RBC age31. Experimentally, high invasion rates are con-sistently recorded when fresh whole blood for use in cultures is stored < 15 d or washed RBC are kept for up to 5 d. The use of RBC outside this age range reduces invasion to below twofold.

(2) The hematocrit of the culture medium is kept low at 1.0% for first invasion and then increased to 1.5% for the last invasion. However, when the calculated volume of culture medium for parasite sustainability over 24 h exceeds 140 ml (maximum capacity of the culture flask used), the hematocrit may be reduced to 0.8%. Using this option, the medium needs to be replaced only once a day. The hematocrit is critical in that the volume of culture medium required for each change depends on the parasitemia and the total volume of RBCs14. The general recommendation for Petri dish or candle jar laboratory cultures is a 5% hematocrit5,32–34. Under these conditions, culture is labor-intensive, requiring constant attention as 2–3 medium changes are required daily to maintain parasite growth, in particular for parasitemias above 20%. Furthermore,

Box 1 | DEFINITIoNS parasitemia. Used here to indicate the percentage of parasite-infected red blood cells with respect to the total number of red blood cells in culture.Hematocrit. Used here to indicate the percentage of the volume of the culture medium that is composed of red blood cells.synchronization window. Used here to indicate the time span within the intraerythrocytic developmental life cycle that presents more than 85% of the cultured parasites.short window synchronization. Used here to indicate that more than 85% of the cultured parasites have a < 6 h difference in their life-cycle developmental stage (see Box 2).Invasion window. Used here to indicate the time range at which the maximum percentage of parasites invades new erythrocytes.

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schizont development and merozoite invasion are impaired by the toxicity of by-products of a high metabolic activity including that of noninfected erythrocytes35,36. Thus, the volume of culture medium should be calculated in terms of its role in diluting metabolic waste as well as producing a given hematocrit. We provide an equation to calculate this volume (Box 3).

(3) A synchronized young ring-stage frozen inoculum is used with at least a 10% level of parasitemia to start the culture. It has been reported that cultures initiated from frozen inocula require about 1 week, i.e., 1–2 subcultures, before they achieve normal growth rates1,5,9,11,15. After this period, these cultures are synchronized by the sorbitol or Percoll 70% methods. Sorbitol frequently gives an 18-h synchrony window, i.e., 85% of the cultured parasites have around 18 h difference in their life-cycle developmental stage (see Box 2)15. According to this protocol, using a synchronized frozen inoculum, 10% parasitemia is achieved 48 h after thawing and the synchrony window obtained after sorbitol addition is reduced to around 10 h.

(4) Both sorbitol37 and Percoll 70% (ref. 38) are used to synchro-nize the parasites in a continuous subculture system. Initial synchronization with sorbitol improves subsequent Percoll 70% synchronization, shortening the synchrony window to only 4 h. In this protocol, Percoll 70% solution is supple-mented with sorbitol. This procedure is based on the differential permeability and density of erythrocytes harbor-ing parasites at different stages of development39. Cells that are highly permeable (e.g., trophozoites and schizonts) take up sorbitol to a greater extent than those of lower perme-ability (e.g., ring stage), and swell accordingly following the osmotic entry of water. Thus, cells containing increasingly

mature stages are less dense because of their increased permeability and decreased intrinsic density, and therefore settle in the less-dense layers of the Percoll cushion40,41.

(5) A standard CO2 incubator is used instead of gassing the

culture flasks with a gas mixture11 at each passage or changing the medium. This reliable low-cost alternative has no detrimental effects on parasitemia or viability. Experimentally, growth rates, parasitemia levels or viability fail to differ whether a gas mixture or CO

2 is used.

Overview of protocol stages. An overview of the protocol’s stages is given in Figure 1.

The above modifications are strictly applied here to two labo-ratory P. falciparum strains (3D7 and Dd2) and to a clinical iso-late that have been successfully grown in vitro in different human blood group erythrocytes. We describe the culture of parasites in RPMI1640 medium supplemented with 25 mM HEPES, 0.5% AlbuMAX I, 1.77 mM sodium bicarbonate, 100 µM hypoxanthine and 12.5 µg ml − 1 gentamicin sulfate, at a pH of 7.2 in 5% CO

2 or a

gas mixture of 96% nitrogen, 3% carbon dioxide and 1% oxygen. The culture medium is prepared weekly without sodium bicarbo-nate (incomplete medium) and stored at 4 °C. To avoid increas-ing the pH, sodium bicarbonate is added freshly after warming to 37 °C before use.

The parasites are cultured in 150-cm2 culture flasks and allowed to settle in a horizontal position such that the medium covers the erythrocyte monolayer. The maximum volume of culture medium used is a 140-ml per flask and the parasites are normally subcul-tured at intervals of 2 d. RBCs are prepared from fresh whole blood obtained in CDPA from a healthy donor and stored at 4 °C for at least 1 d. The upper serum layer is removed and erythrocytes are

Box 2 | INTraEryThrocyTIc BlooD STagES oF Plasmodium falciParum IN culTurE Malaria parasites show several intraerythrocytic developmental stages. In all of these stages, the same parasite structures stain the same color regardless of the Giemsa or Wright’s dye used. Thus • Chromatin is usually round in shape and red or dark stained. • The cytoplasm occurs in different forms, from a ring shape

to an irregular shape, but always stains blue.The P. falciparum intraerythrocytic development stages observed every 4 h after invasion during our continuous culture protocol may be seen below (stained with Wright’s). The ring stage is observed between 6 and 22 h, the trophozoite stage is observed between 22 and 38 h, the schizont stage is observed between 38 and 48 h and the merozoites are observed at 48 h, just before invasion. To identify the synchronization window at high parasitemia, consider the maximum percentage of parasite forms (always >85%) at a given stage and their time range estimated according to the figure above. For example, Figure 6c shows a synchrony of 90% trophozoites in a 4-h window. Here, we can observe 90% of infected red blood cells with medium age trophozoites of 30–34 h along with 10% other forms. The invasion window can also be approximated by estimating the synchronization window. Thus, the invasion window is the same time range observed in the culture smears but displaced in time to reach the 48-h segmented schizont stage. For example, a synchrony window in a smear of 38–42 h will predict an invasion window of 10–6 h from the present.

6 h 10 h 14 h 18 h

22 h 26 h 30 h 34 h

38 h 42 h 46 h 48 h

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treated with Lymphoprep and washed so that the RBCs are ready for use and free of white blood cells.

To obtain intraerythrocytic parasite stages, cultures are sorbi-tol- and Percoll-synchronized as previously described37,38. It should be emphasized that the parasites are synchronized when needed, a maximum of two (mostly) or three (rarely) times in a period of 2 weeks. Sequential treatment with sorbitol is avoided as this weakens the RBCs and consequently reduces parasitemia. During parasite culture and before proceeding with any procedure steps, a smear should serve to determine the progress achieved and help in further decision making. Wright’s eosin methylene or Giemsa staining can be used to determine parasitemia and parasite stage during the culture process. Wright’s eosin methylene blue solu-tion stains RBCs on a red background (Fig. 2a), whereas Giemsa solution stains RBCs a pinkish or greyish color (Fig. 2b,c) depend-ing on whether the regular or rapid method is used. Note that although Giemsa 10% staining produces a greyish background in the usual staining time, with Wright’s stain the incubation time must be adjusted according to the environmental temperature. Thus, if the smear is too dark (Fig. 2d,e), it can be lightened by washing with methanol (Fig. 2f,g). Despite differences between stains, both staining techniques are appropriate to estimate para-sitemia in the cultures by microscopy observation. However, rapid staining methods such as Wright’s or Giemsa 10% staining42,43 are

able to reveal the parasite stage and RBCs are needed to allow for quick decision making, such as the best timing for synchronization, harvesting or freezing.

Increased yields are obtained by subculturing every 2 d for 6–8 h before invasion. This is done by equally dividing the contents of each flask into two or more flasks and quickly restoring the hema-tocrit and medium volume in each of these two flasks with fresh erythrocytes and fresh medium to keep the hematocrit between 1 and 1.5% in the required volume (maximum 140 ml) of culture medium. This ensures the parasites have access to fresh intact RBCs for the next invasion. By applying the above-mentioned critical points to improve the in vitro growth conditions, cultures will show 20–30% parasitemia 4 d after launching. At this time point, the cultures are sorbitol-synchronized, and 48 h later a synchronous culture of up to 50% parasitemia is obtained (thawing-to-stand-ard growth rate, Fig. 1a). Next, continuous subculture induces a stationary phase of growth following either one of the two optional Steps 43A or 43B for synchronization to culture harvesting (Fig. 1b,c).

The purification and concentration of mature-stage parasites (tro-phozoites and schizonts) by sedimentation of knob-infected erythro-cytes in gelatine44 or Physiogel45 have been reported elsewhere. As the procedure described here achieves a very high level of invasion ren-dering high parasitemias of any erythrocytic stage of knobless strains

Box 3 | chaNgE oF culTurE mEDIum adjusting medium changesDespite the slightly different growth rates reported for different clones47, strains Dd2 and 3D7 and the clinical isolate tested in this protocol were treated similarly in terms of culture medium requirements to give close results in parasitemia increases per day. It is considered that 50 µl of packed cells in culture (pellet) require 2.5 ml of culture medium at 10% parasitemia24. Thus, per 1 µl of infected red blood cells alone (µl pellet × % parasitemia/100), 500 µl of fresh culture medium are required; this is the amount that is estimated to sustain viability for ~24 h. To calculate the volume of culture medium needed for each 24 h change, based on the above, we devised the following equation, which has been experimentally confirmed: V(ml)/24 h = 0.005 (µl RBC pellet)(% parasitemia).This equation helps calculate the approximate volume of culture medium needed when working with microplates, Petri dishes or culture flasks, to ensure that the infected red blood cells have sufficient medium to survive 24 h. For example, if one wishes to culture a 1,200-µl RBC pellet with a young-stage parasitemia of 20% in a 150-cm2 flask, the volume of medium needed using the above equation will be 120 ml/24 h. crItIcal However, for a 1,200-µl pellet containing mature forms, consider the invasion process will occur before 24 h and para-sitemia will exceed 20%. Hence, a higher volume of culture medium far from the calculated 120 ml will be required. To deal with this particular situation of departing from mature stages, one may wish to equally distribute the packed culture from each flask into two flasks using fresh erythrocytes and medium to keep a 1% hematocrit in each 120 ml of culture medium, without having to replace the medium twice in 24 h. Optionally, the culture medium should be changed twice in 24 h. crItIcal This option can distress the next invasion as part of merozoites could be lost. It should be noted that the culture of a ring-stage 1,200-µl pellet to achieve 40% parasitemia might not require dividing the flask contents into two as no invasion will take place within the following 24 h. Nevertheless, given that in this case the 150-cm2 flask has insufficient capacity for the 240 ml of culture medium required according to the equation, choose between changing the culture medium twice in 24 h without adding fresh red blood cells or dividing the culture into two 150-cm2 flasks, also without adding fresh red cells, and diminishing the hematocrit value to 0.8%. crItIcal It should be emphasized that keeping the culture in the appropriate volume of medium avoids parasite toxicity and helps preserve the viability and synchronization of erythrocytic stages of Plasmodium falciparum.changing medium proceduresUse a suction pump to extract the old medium without disturbing the RBC.Occasionally, when pooling from more than one flask is required, to minimizing the parasite lost, proceed as follows: (i) Mix culture flasks together and aliquot into 50 ml Falcon tubes. (ii) Centrifuge at 250g for 5 min at 37 °C and discard the supernatant. (iii) Add new medium and distribute evenly into new culture flasks.


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Driving cultures to synchrony and standard growth rates from frozen stock

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Subculture the flask by dividing its contents

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hematocritand 17–20%parasitemia

Three flasks showing ~60% parasitemia are obtained.

Harvest two flasks(~2 ml infected RBC)

and divide the contents of theother one into three new flasks

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Figure 1 | For caption see next page.

of P. falciparum (3D7 and Dd2), specific stage enrichment with sup-plementary chemicals is not required. It should be emphasized that the protocol described provides any short window in the stage of rings for which enrichment techniques have not been yet developed. Intact and healthy P. falciparum are obtained in high synchrony at any asexual stage, in large amounts and over a short period of time. The procedure can be performed in any modest laboratory in studies designed to develop vaccines and new drugs against malaria.

Strains and isolates. Although this protocol was designed using laboratory strains, its use on a clinical isolate from a patient with malaria admitted to the University Hospital 12 de Octubre (Madrid) (unpublished data) is also examined. The blood sample

was maintained for 24 h at 5 °C and the protocol described here was followed to adapt the strain in the absence of human serum. After 16 d of continuous culture, this wild-type isolate reached a parasitemia level of 40% and was maintained for 22 d and then cryopreserved. After thawing, the isolate has been in vitro cultured for months (long term), yielding parasitemias in the range of 50–60%. After freezing and thawing several times, the isolate has preserved its capacity to grow in the absence of human serum.

Controls. As a positive control, it should be considered to run a parallel culture of parasites that is continuously maintained grow-ing in the same laboratory. Nevertheless, within our experimental


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a b c d

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greyish. (d) While Giemsa produces a greyish background, (e) Wright’s staining for more than 5 min in temperate climates yields dark smears. (f) The pinkish background of smears stained with Giemsa 10% and (g) the red cell background of smears stained with Wright’s can be recovered by washing in methanol.

Figure 2 | Staining cell smears. Cultures containing up to 50% ring or early trophozoite stage parasites stained with Wright’s (Step 22A) or Giemsa (Step 22B) methods are both adequate to estimate parasitemia levels under the microscope. Scale bar = 10 µm. (a) Parasites stained with Wright’s eosin methylene blue solution are clearly detected on a red cell background. (b) Giemsa 3% staining of the same preparation gives rise to a pale pinkish background when the regular method is employed. However, the staining time is 3–4 times longer than Wright’s staining. (c) Using the rapid method with Giemsa 10%, the difference in staining time is reduced, but the RBC background varies from reddish to

OPTION B: Cultures for short window synchronization

Step 43B(i-iv)

Subculture the flask by dividing its

contents intotwo flasks.

Do not add RBC

Synchronize withPercoll 70%

Change culturemedium

Subculture by dividingthe contents of eachflask into two flasks.

Four flasks of highly synchronized cultures

showing over 60% parasitemia are obtained.

Harvest three flasks(~3 ml infected RBC) and

divide the other one into three.

Harvest, freeze orsubculture and

synchronize. Hereafter, up to 5 ml of infected RBC

can be obtainedper week

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Figure 1 | Flow diagram and growth curves showing the synchronous culture of Plasmodium falciparum at high parasitemia levels. Parasitemia levels at each time point were determined by counting 1,200 cells. (a) Overview of a parasite culture from thawing to the time at which a synchronous culture showing standard growth rate is obtained (Steps 9–42). The experimental growth curve shows the daily parasitemia of the parasite culture procedures during the first 6 d after thawing (Steps 9–20). Surviving parasites after thawing are estimated 24 h later (corresponding to day 1 in the growth curve). With respect to the parasitemia on day 1, approximately, a fivefold increase is obtained after invasion by day 2 (Steps 21–23). Owing to the addition of fresh RBCs on the next day (corresponding to day 3 in the growth curve: Step 24), parasitemia decreases to around 6%, yet rises 20–30% on day 4. At this time point, the culture is synchronized by sorbitol treatment (Steps 26–39). On day 5, the parasitemia is adjusted 17–20% by the addition of fresh RBCs to give rise to a culture on day 6 with up to 50% parasitemia with a synchrony of 87 ± 3.4% late rings, 11.8 ± 3.2% early trophozoites and 1.1 ± 0.3% schizonts (Steps 40–42). Each point shown is the mean value of six independent experiments. From this step, two options can be followed (Step 43A or 43B) as depicted in the following panels. (b) Diagram depicting the procedures to achieve steady stationary growth of synchronous cultures at high parasitemia following optional Step 43A. From day 6 (Step 42), steady stationary growth can be achieved by subculturing one flask into three flasks (Step 43A(i)). This yields 3–5 ml of infected RBC showing around 60% parasitemia per week. The growth curve shows the daily parasitemia as subcultured every 48 h from day 6. By day 8 and 10 (Step 43A, v-vi), two culture flasks with ~ 50% parasitemia are harvested (Steps 51–56) and one is subcultured into three new flasks. The addition of fresh RBC to obtain 1–1.5% hematocrit and 17–20% parasitemia on days 7 and 9 (Step 43A(iii)) result in synchronous cultures with 50–60% of parasitemia on days 8 and 10. In the growth curve (from three independent experiments) 88 ± 1.6% late rings and 12% ± 3.3 early trophozoites were obtained at day 8, and 88.7 ± 1.9% early trophozoites, 10 ± 2.5% late rings and 1.3 ± 3.3% schizonts at day 10. (c) Diagram depicting the procedures to achieve steady stationary growth of highly synchronous cultures at high parasitemia following optional Step 43B. Two flasks from subculturing in day 6 (Step 43B(i)) are further synchronized by Percoll 70% (Step 43B, v-xiv). Hereafter, ~ 3–5 ml of infected red blood cells with a short window synchrony at around 60% parasitemia is obtained per week after subculturing. The curve shows steady stationary growth achieved by subculturing every 2 d from day 9 (Step 43B(xvi)). In the growth curve (from three independent experiments) at day 8 (24 h after Percoll treatment), the cultures exhibit the following synchrony: 89.3 ± 1.4 early trophozoites and 10.1% ± 1.3 late rings. By day 10, 89.8% ± 2.9 early trophozoites and 7.6% ± 2.3 late rings were obtained and by day 12, the cultures contained 91.9% ± 2.8 early trophozoites and 4.2% ± 2.0 late rings.


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design as well as in the other malaria parasite culture methods reported4,14,16,17,24,25, it is not feasible to run a positive control, given that usually a continuous culture is not maintained for months or years. The use of cultures starting from frozen stock in the absence of a positive control from the same laboratory is a limitation of the protocol. Nevertheless, as the protocol is designed to obtain highly

synchronous parasites at high parasitemia levels, large deviations from the standard propagation defined in this protocol should warn of the existence of a problem and the troubleshooting table should be checked. Conversely, the continuous growth of the cul-ture as reported here may be taken to indicate the optimal viability of the stored inoculum.

MaterIalsREAGENTS

Human blood from healthy adult donors ! cautIon All human samples should be handled following the biological guidelines for human samples: investigators should work with a class 2 safety cabinet, wear gloves and all materials should be disinfected with bleach before discarding.P. falciparum lines Dd2 (MRA-150) and 3D7 (MRA-102) from MR4 (ATCC)Gentamicin sulfate (Sigma, cat. no. G-1264)Hypoxanthine (Sigma, cat. no. H9636)NaOH (Panreac, cat. no. 131687)NaHCO

3 (Sigma, cat. no. S 5761)

RPMI 1640 (1 liter; Sigma, cat. no. R6504)AlbuMAX I (Gibco, cat. no. 11020-021)HEPES 1 M (Sigma, cat. no. H0887-100 ml)NaCl (Panreac, cat. no. 131494)Na

2HPO

4 (Panreac, cat. no. 131679)

NaH2PO

4.H

2O (Panreac, cat. no. 131965)

Phosphate buffered saline tablets (PBS) (OXOID, cat. no. BR0014G)Sorbitol (Sigma, cat. no. S3889)Percoll (vol/vol) (GE Healthcare, cat. no. 17-0891-01) ! cautIon This compound is carcinogenic and should be handled with gloves.CO

2 gas for incubator (Air Liquide)

Liquid nitrogen (Air Liquide—Charge 1000)Wright’s eosin methylene blue solution (Merck, cat. no. 1.01383.0500) ! cautIon It is toxic and should be handled with gloves.Giemsa’s azur eosin methylene blue solution (Merck, cat. no. 1.09204.0500) ! cautIon It is toxic and should be handled with gloves.Lymphoprep (AXIS-SHIELD, cat. no. 1114545)Glycerine (Panreac, cat. no. 131339)

EQUIPMENTWinged blood sampling set, e.g., TERUMO (TERUMO Europe, cat. no. MN-SVXX) and blood collection tubes coated with CPDA anticoagulant, e.g., VACUETTE (9 ml CPDA, cat. no. 455056)Plastic band, scissors, gauze and plasterMicroscope slides, e.g., Thermo Scientific microscope slides 76 × 26 mm (cat. no. DXD-10143562)CO

2 incubator, e.g., HERAcell 150 (Heraeus)

Laminar flow cabinet, e.g., MICROFLOW Laminar flow cabinet (MDH), to maintain media and cells in sterile conditionsDistilled water system, e.g., ELIX Progard 2 (Millipore, cat. no. PROG00002)Centrifuge, e.g., Beckman J2-21 Centrifuge (Beckman)Incubator shaker (Aerotron, Infors HT)Nikon ALPHAPHOT-2 YS2 Microscope (Nikon)Digital camera Moticam 2000 2.0 M pixel (Motic)PlasmoScored 1.3 software46

Syringes: 50 ml (BD Plastipak, BD, cat. no. 300866), 20 ml (BD Plastipak, BD, cat no. 300613) and 10 ml (BD Plastipak, BD, cat. no. 300014)Sterile E0 syringe filter Minisart (pore Ø = 0.20 µm, Sartorius AG, cat. no. 16534)Vacuum-driven disposable filtration system with 500 ml micropore bottles (Millipore, cat. no. SCGPU05RE), to filter all culture mediaAutoclave P-Selecta, Autester EThermostatic bath, B.A. BunsenPlastic tubes: 15-ml tube (Falcon, cat. no. 352196), 50-ml tube (Falcon, cat. no. 352070), 50-ml centrifuge tubes (Iwaki, cat. no. 2343-050) and 1.5-ml tube (Eppendorf, cat. no. 3810)Sterile disposable plastic pipettes: 25-ml pipettes (Sterilin, cat. no. 40125), 10-ml pipettes (Sterilin, cat. no. 900036), 5-ml pipettes (Sterilin, cat. no. 900034) and 1-ml pipettes (Sterilin, cat. no. 900030)Brand Sterile Pasteur pipettes (cat. no. 747720)Tissue culture flasks: 75-cm2 flask with vented cap (Iwaki, cat. no. 3123-075), 150-cm2 flask with vented cap (Iwaki, cat. no. 3133-150)

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REAGENT SETUPCultures All cultures should be manipulated in a laminar flow cabinet using standard aseptic techniques. Gloves must be worn at all times. All materials must be sterile and media should be filtered before use.NaOH 1 M solution Dissolve 4 g NaOH in distilled water and make up to 100 ml. It can be stored at room temperature (~25 °C) for 6 months.Stock hypoxanthine 0.1 M solution (0.1 M hypoxanthine, 1 M NaOH) Dissolve 1.361 g of hypoxanthine in 1 M NaOH and make up to 100 ml. It can be stored at 4 °C for 3 months.Stock gentamicin sulfate solution 50 mg (bottle) in 1 ml distilled water. It can be stored at 4 °C for 3 months.Incomplete medium (RPMI 1640 10.4 g liter − 1, HEPES 25 mM, AlbuMAX I 0.5% (wt/vol), hypoxanthine 100 µM, gentamicin 12.5 µg ml − 1) To prepare, mix one sachet of RPMI 1640 with 25 ml of 1 M HEPES, 1 ml hypoxanthine from the 0.1-M stock solution, 5 g AlbuMAX and 250 µl gentamicin (from 50 mg ml − 1 stock). Once well mixed, make up to 1 liter with Milli-Q water and filter the culture medium into sterile 500-ml micropore bottles. Store at 4 °C for 15–20 d.Complete medium (RPMI 1640 10.4g liter − 1, HEPES 25 mM, AlbuMAX I 0.5% (wt/vol), hypoxanthine 100 µM, gentamicin 12.5 µg ml − 1, sodium bicarbonate 1.77 mM) This medium is prepared just before use by adding 3 ml of 5% (wt/vol) sodium bicarbonate to 100 ml of incomplete medium. crItIcal Optionally, it can be stored at 4 °C for 7 d.Washing medium (RPMI 1640 10.4g liter − 1, HEPES 25 mM, hypoxanthine 100 µM, gentamicin 12.5 µg ml − 1) It is prepared in the same way as the incomplete medium but without adding AlbuMAX I, and it can be stored at 4 °C for 15–20 d.PBS for staining Dissolve a PBS tablet in 100 ml distilled water. It is stable at 25 °C for a long time.PBS solution for Percoll To prepare a PBS solution (5 mM sodium phosphate, 160 mM NaCl, ph 7.4), dissolve 1.40g of disodium hydrogen phosphate anhydrous (Na

2HPO

4), 0.04 g of sodium dihydrogen phosphate

monohydrate (NaH2PO

4) and 9.35 g of NaCl in 1 liter of distilled water, and

then adjust the pH to 7.4 with HCl. Finally, filter the solution with a syringe and it can be stored at 4 °C for 3 months.Sorbitol solution for culture synchronization To prepare the sorbitol solu-tion, dissolve 5g of sorbitol in 80 ml of distilled water by shaking for 5–10 min. Then make the volume up to 100 ml and filter the solution with a syringe. This solution can be stored at 4 °C for 3 months.Percoll 90% solution To prepare a 90% Percoll (vol/vol) solution, add 180 ml of Percoll to 20 ml of 10× RPMI 1640 sterile (one sachet of RPMI powder in Milli-Q water in a final volume of 100 ml and filter the solution using a syringe). It must be prepared fresh.PBS/sorbitol solution To prepare this medium containing 13.33% sorbitol, dissolve 7.99g of sorbitol in 60 ml of PBS solution for Percoll (see above) and filter using a syringe. It can be stored at 4 °C for 3 months.Percoll 70% solution for culture synchronization To prepare a Percoll 70% (vol/vol) solution, mix 39 ml of Percoll 90% (vol/vol) solution with 11 ml PBS/sorbitol solution, both prepared as indicated above. Then, filter the solution using a syringe and prepare 3 ml aliquots. This solution is stabilized for 6 months when stored at − 20 °C.Thawing solutions Solution A: Sterile 12% (wt/vol) NaCl.

Solution B: Sterile 1.6% (wt/vol) NaCl.Both of them can be stored at 4 °C for 6–9 months and they must be

warmed to 37 °C before use.Freezing solution To prepare 100 ml freezing solution, mix 0.65g of NaCl, 3.02g of sorbitol and 28 ml of glycerine. Filter the solution using a syringe and store at 4 °C for 6 months.


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proceDurepreparation of materials and rBcs ● tIMInG ~5 h1| Prepare the sterile material, solutions and media, and obtain blood samples from healthy donors. Human blood of all ABO groups is suitable for the growth of P. falciparum.

2| Wash the RBC when required for culture and only the necessary amount by firstly centrifuging 10-ml whole blood at 450g for 10 min at 20 °C and discard supernatant. crItIcal step Always remove the top layer of RBC. Optionally, whole blood can be deposited overnight at 4 °C before proceeding to Step 3. Whole blood should not be stored for more than 2 weeks.

3| Mix 5 ml of RBCs with 5 ml of washing medium in a 15-ml tube.

4| Add the mixture carefully to the 15-ml tube containing 5 ml of Lymphoprep.

5| Centrifuge at 450g for 20 min at 20 °C. Discard the supernatant.

6| Wash RBCs with 10 ml of washing medium.

7| Centrifuge at 450g for 5 min at 20 °C. Discard the supernatant.

8| Store the RBC with a 50% hematocrit at 4 °C by diluting the packed RBCs with an equal volume of incomplete culture medium. pause poInt RBCs can be stored under these conditions for 5 d.

thawing the glycerol-frozen parasites ● tIMInG 1 h9| Take a cryovial containing the malaria parasites from the liquid nitrogen and thaw for 2–3 min in a 37 °C water bath or by warming in the hand.

10| Spray the cryovial with 70% ethanol before introducing under the laminar flow hood.

11| Transfer the contents of the cryovial to a 50-ml tube using a 1-ml serological pipette. Note the total volume transferred (V). crItIcal step The cultures are stored diluted 1:1 in freezing solution.

12| Slowly add 0.1 × V of solution A dropwise while swirling the tube. For example, if V = 600 µl (300 µl infected RBC + 300 µl freezing solution), then solution A is 0.1 × 600 = 60 µl.

13| Let the tube stand for 5 min at room temperature. crItIcal step Thawing must be gradual.

14| Slowly add 10 × V of solution B dropwise at first while swirling the tube. For example, if V = 600 µl (300 µl infected RBC + 300 µl freezing solution), then solution B is 10 × 600 = 6,000 µl.

15| Centrifuge at 250g for 5 min at 37 °C. crItIcal Never use centrifugation speeds above 300g for infected RBC unless performing Percoll synchronization.

16| Discard the supernatant. Resuspend the pellet in 10 ml washing medium and transfer to a 15-ml sterile tube.

17| Centrifuge at 250g for 5 min at 37 °C and remove the supernatant.

18| Estimate the pellet volume and add an equal amount of fresh RBC and mix well. This step is required to provide the parasites with fresh RBC for invasion, although it reduces the parasitemia by half.

19| Resuspend the mixture in complete medium to keep the hematocrit at 1–1.5%. For example, for a 1% hematocrit: 600 µl washed RBC + 60 ml complete medium in a 75-cm2 tissue culture flask.

20| Maintain the flask in 5% CO2 at 37 °C for 48 h.

assessing parasitemia levels and viability of cultures by preparing and staining smears ● tIMInG 15–45 min21| After incubation for 48 h as described in Step 20, assess the viability of cultures by counting ring-stage parasites in smears. To prepare smears from the settled culture, first take 0.5–1 ml of the culture and centrifuge it at 250g for 3 min at


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room temperature. Discard the supernatant and pipette 2–3 µl onto a clean glass slide. Using another slide positioned at an angle of 30–45° to the first, slowly drag this slide backward through the drop to produce a blood smear on the underside of the top slide. Finally, smear this blood forward across the bottom slide and allow it to dry before staining. crItIcal step Keep the culture flask under the laminar flow hood for 20 min while preparing the smears.

22| To stain smears, use either Wright’s eosin methylene blue following option A or Giemsa, following option B.(a) Wright’s azur eosin methylene blue staining ● tIMInG 15 min (i) Completely cover the smear with Wright’s solution for 4 min.

! cautIon Do not exceed this time. This incubation time should be adjusted according to the weather; e.g., when working in temperate climates such as an environmental temperature of 36 °C at low humidity, 1 min is sufficient.

(ii) Add distilled or PBS (pH 7.2) until the slide is immersed. (iii) Incubate for 9 min. (iv) Clean both sides of the slide with water. A tissue paper soaked in methanol can be used to remove excess stain from

the underside. ? trouBlesHootInG

(v) Leave to dry before microscopic investigation. To assess the parasitemia level and growth stage, 1,000–1,200 cells are counted using PlasmoScore 1.3 software46 in different image fields. Parasites are observed on a red cell background. Viability is confirmed by visualization of the characteristic P. falciparum morphology along its intraerythrocytic cycle, which is shown in Box 2. The invasion window can also be estimated from the smears as described in Box 2.

(B) Giemsa’s azur eosin methylene blue staining ● tIMInG 45 min (i) Fix each thin blood film with methanol for a few seconds. (ii) Completely cover the smear with Giemsa 3% (vol/vol) solution for 30–45 min (or for a rapid method, use Giemsa

10% (vol/vol) solution for 5–8 min; prepare this stain at two concentrations according to standard methods43). (iii) Clean both sides of the slide with distilled water. (iv) Leave to dry before microscopic observation. The parasitemia level is estimated and parasite viability is confirmed

as described in Step 22A(v). When Giemsa 3% (vol/vol) is used, a pale pinkish background is observed. In contrast, using the Giemsa 10% method, the preparation gives rise to a greyish background. The invasion window can also be estimated from the smears as described in Box 2.

change of culture medium ● tIMInG 30 min23| After 48 h incubation (Step 20), the parasites should be at the ring stage at around 10% parasitemia. Change the medium of cultures and transfer the parasites in fresh complete medium to a 150-cm2 flask following the instructions indicated in Box 3. Keep the culture flask in 5% CO2 at 37 °C until a new medium change would be required (~18 to 24 h later, when parasites reach the late trophozoite stage). crItIcal step The addition of red cells is not required. Remember to add RBCs only when the culture reaches the late trophozoite stage.? trouBlesHootInG

assessment and maintenance of cultures before sorbitol synchronization ● tIMInG 1 h24| After a further ~24 h incubation (Step 23), check the culture again as described in Steps 21 and 22 to determine parasite stage (see Box 2), change the medium and restore the 1–1.5% hematocrit using a fresh RBC suspension (see Box 3). Keep the culture flask in 5% CO2 at 37 °C until sorbitol synchronization (~18 to 24 h later).? trouBlesHootInG

25| Count the ring-stage parasites 8 h after the estimated invasion time (as calculated in Step 22; see Box 2), following the method described in Steps 21 and 22. If the ring-stage parasitemia is >10% (see Box 2), the culture is ready for sorbitol synchronization. crItIcal step The level of parasitemia depends on the viability and number of rings in the inoculum population. The use of inocula containing at least 10% rings is recommended to achieve 20–30% parasitemia at this step (~4 d after thawing).? trouBlesHootInG

sorbitol synchronization ● tIMInG 1 h26| Warm a 10-ml aliquot of 5% (wt/vol) sorbitol for 5 min at 37 °C.

27| Remove the medium from the culture flasks from Step 24 using a pump leaving ~15 ml.

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crItIcal step The rings must be very young, not older than 10 h. Do not synchronize more than three flasks of 150 cm2 at once and do not undertake the synchronization procedure more than twice during a week of continuous culture. Repeated sorbitol treatments over a short period of time reduce parasite viability.

28| Mix with care and transfer the flask’s contents to 15-ml tubes.

29| Centrifuge the parasite culture in a swinging bucket at 250g for 5 min at 37 °C.

30| Discard the supernatant, estimate the pellet volume and add 9–10 volumes of 5% (wt/vol) sorbitol (e.g., 1 ml pellet + 9 ml 5% sorbitol solution).

31| Vortex vigorously for 30 s to rupture old RBC and mature parasite forms.

32| Incubate for 8 min at 37 °C, preferably under shaking at 240 rpm.

33| Vortex for 15 s. If incubation was performed under shaking in Step 32, this step is omitted.

34| Centrifuge at 250g for 5 min at 37 °C and remove the red supernatant. The red color of the supernatant indicates hemolysis of RBC containing mature forms of the parasite.

35| Cleaning step. Add 10 ml washing medium and centrifuge at 250g for 5 min at 37 °C.

36| Discard the supernatant and repeat Step 35.

37| Prepare a smear using the pellet from Step 36 to assess ring enrichment and absence of mature forms as described in Steps 21 and 22.

38| Transfer the pellet from Step 36 to a clean 150-cm2 culture flask containing complete medium (see Box 3). This must be performed carefully, by adding a 1-ml aliquot of complete culture medium to the bottom of the 15-ml tube from Step 36. Then, using a plastic pipette, gently resuspend the pellet by aspirating and releasing up and down and transfer to the clean culture flask containing complete medium prepared earlier in this step. Repeat at least twice to harvest all parasites. crItIcal step During this procedure, avoid dragging the cell debris stuck to the 15-ml tube walls.

39| Return to the incubator at 37 °C in a 5% CO2 atmosphere and culture under these conditions for 12–24 h.

assessment and maintenance of cultures following sorbitol synchronization ● tIMInG 1 h40| At 12–24 h after sorbitol synchronization, take a smear as described in Steps 21 and 22 to estimate the peak invasion time (see Box 2).? trouBlesHootInG

41| Change the medium and add freshly prepared RBCs to give a 1–1.5% hematocrit (see Box 3). Place the incubator back at 37 °C in a 5% CO2 atmosphere and culture under these conditions for ~12 to 24 h until parasitemia is checked. crItIcal step Cultures at high parasitemia require frequent medium changes.? trouBlesHootInG

42| Check parasitemia and culture growth in smears as described in Steps 21 and 22. At this point, parasitemia will be around 50%.

43| This step can be performed in two alternative ways depending on the experimental use of the culture and the extent of synchronization required for future experiments. Option A should be used when high levels of synchronization are not required. Option B, which includes a further synchronization by the Percoll 70% method, should be used when high synchronization is needed, e.g., to extract parasite mRNA. For both options, subculturing will enhance parasitemia, increasing the biomass and reducing any interference of hemoglobin or uninfected erythrocyte proteins in the extraction procedure.(a) Maintenance of synchronous cultures at high parasitemia ● tIMInG 2 h (i) Remove medium from cultures in Step 41 and divide the culture into three 150-cm2 flasks. Add fresh complete medium.

Do not add RBCs. This allows for prolonging the medium change time up to 24 h (see Box 3). Keep the culture in the incubator at 37 °C in a 5% CO2 atmosphere for 24 h. ? trouBlesHootInG


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(ii) Take a smear as described in Steps 21 and 22 to estimate parasitemia and window invasion time (Box 2). (iii) Change the medium of each flask with fresh complete medium. Add RBCs to dilute the parasitemia down to 17–20%

(see Box 3). Return to the incubator at 37 °C in a 5% CO2 atmosphere. Keep the culture under these conditions until 8 h after the estimated invasion window time. ? trouBlesHootInG

(iv) Take a smear as described in Steps 21 and 22 to estimate parasitemia and parasite stage (see Box 2). Expectedly, parasites will be in the ring form at a parasitemia level around 60%.

(v) If these ring forms are required, harvest two out of the three flasks as indicated in Steps 51–56. If trophozoite or schizont forms are required, maintain the culture to reach these stages (20–30 h later). In this case, the medium should be changed in the three flasks and then every 12 h (see Box 3). Once the required parasite stage is reached, harvest two out of the three flasks (Steps 51–56) and keep the other to establish a steady-state culture at high parasitemia to resume further harvesting cycles. crItIcal step After the last addition of RBC to a synchronic culture (Step 43A(v) or Step 43B(xviii)) and just before harvesting, the medium change should be undertaken without a pump to minimize parasite losses. crItIcal step To finish the culture, the three flasks can be harvested at once in this step.

(vi) Subculture the remaining flask from Step 43A(v) by repeating Steps 43A(i–iii). Hereafter, culture yield increases up to 5 ml of infected RBC per week with around 60% parasitemia. To maintain the synchronization window along the harvesting cycles, follow Steps 43B(v–xiv) or Steps 26–39, once a week.

(B) Maintenance of cultures and further short window synchronization ● tIMInG 3 h 45 min (i) Remove the medium from cultures in Step 41 and divide the culture into two 150-cm2 flasks containing fresh complete

medium (Box 3). ? trouBlesHootInG

(ii) Return to the incubator at 37 °C in a 5% CO2 atmosphere. Keep the culture at these conditions for 12 h. (iii) Take a smear as described in Steps 21 and 22 to estimate window invasion time (Box 2). If mature trophozoites

and/or schizonts (i.e., 38–42 h after invasion) are seen in the smears, go to Step 43B(v); if not, go to Step 43B(iv). (iv) Change the medium by fresh complete medium (see Box 3). Keep the culture at 37 °C in a 5% CO2 atmosphere until

the time that mature trophozoites and/or schizonts are expected (i.e., 38–42 h post invasion). ? trouBlesHootInG

(v) Percoll synchronization. Warm the 70% (vol/vol) Percoll solution to 37 °C; mix well; and warm both the complete and washing media at 37 °C.

(vi) Remove the medium from cultures in Step 43B(iii or iv) using a pump leaving ~15 ml of culture including infected RBCs. Transfer the contents to 15-ml centrifuge tubes. ? trouBlesHootInG

(vii) Centrifuge the tubes at 250g for 5 min at 37 °C. (viii) During centrifugation, prepare two new 15-ml tubes by adding 3–4 ml 70% (vol/vol) Percoll with a 5-ml plastic pipette. (ix) Discard most of the culture medium from the tubes in Step 43B(vii) using a 5-ml plastic pipette. Save the infected

red cell pellets. If a thin upper layer is left in the tubes, it is easier to remove the pellets using a 1-ml plastic pipette. To the top of both of the Percoll tubes from Step 43B(viii), add 0.5–1.0 ml of the pellet by quickly—but carefully—pouring down the side of the tube to avoid RBC and Percoll mixing.

(x) Centrifuge at 800g for 10 min at 37 °C, without the brake. crItIcal step Brake must not be used, otherwise the cushion is lost.

(xi) Using a sterile Pasteur pipette, carefully remove the top layer from both tubes. This contains the RBC parasitized with schizonts and late trophozoites. Transfer each top layer to a sterile 15-ml tube containing 10 ml washing medium. Mix carefully by inversion. crItIcal step During this procedure, avoid taking Percoll solution dragged to the parasite layer.

(xii) Centrifuge at 300g for 7 min at 37 °C and discard the supernatant. Save the pellets. crItIcal step This step removes any Percoll solution dragged in Step 43B(xi). Note that for every 1 ml that is synchronized, only 100 µl of mature forms are recovered.

(xiii) Prepare a smear of the washed pellets as described in Steps 21 and 22. The mature forms of parasites will be close to 90%. ? trouBlesHootInG

(xiv) Transfer the washed pellets to two sterile flasks and add fresh RBC to dilute down to 10% parasitemia and complete with culture medium to obtain a 1% hematocrit (greater parasitemias do not improve next invasion). Incubate at 37 °C in 5% CO2 for 12–20 h.

(xv) Take a smear as described in Steps 21 and 22 to estimate parasitemia and the invasion window (see Box 2). Expect-edly, parasites will be in the ring form. Change culture medium to both flasks (see Box 3). Keep the culture at 37 °C in a 5% CO2 atmosphere until 6–8 h before expected invasion time. ? trouBlesHootInG

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(xvi) Take a smear as described in Steps 21 and 22 to estimate parasitemia. Subculture each flask into two 150-cm2 flasks and add fresh RBCs to achieve 17–20% parasitemia with 1–1.5% hematocrit. Keep the culture at 37 °C in a 5% CO2 atmosphere during ~12 to 20 h. crItIcal step High synchronization persists over three subsequent invasions. ? trouBlesHootInG

(xvii) Take a smear as described in Steps 21 and 22 to estimate parasitemia and parasite stage (see Box 2). Expectedly, parasites will be in the ring form at a parasitemia level around 60%.

(xviii) If these ring forms are wanted, three out of the four flasks are harvested as indicated in Steps 51–56. If trophozoite or schizont forms are required, maintain the culture to reach these stages (20–30 h later). In this case, the medium should be changed in the four flasks and then every 12 h (see Box 3). Once the required parasite stage is reached, harvest three out of the four flasks (Steps 51–56) and keep the other to establish a steady-state culture at high parasitemia to resume further harvesting cycles. To finish the culture, the four flasks can be harvested at once in this step. crItIcal step After the last addition of RBC to a synchronic culture (Step 43A(v) or 43B(xviii)) and just before harvesting, the medium change should be undertaken without a pump to minimize parasite losses. ? trouBlesHootInG

(xix) The remaining flask is subcultured following Step 43A(i–iii). Hereafter, culture yield increases up to 5 ml of infected RBC per week with around 60% parasitemia. To maintain the synchronization window along the harvesting cycles, follow Step 43B(v–xv) or Steps 26–39, once a week. ? trouBlesHootInG

Freeze-storing infected rBc in liquid nitrogen ● tIMInG 45 min44| Take flask from the incubator and aspirate the medium with a pump. crItIcal step For freezing, synchronized parasite cultures should contain a minimum of 10% young rings ( < 10 h preferably from synchronized cultures; see Box 2). Check it in a smear as described in Steps 21 and 22.? trouBlesHootInG

45| Resuspend the infected RBCs in 3–4 ml of washing medium and transfer to a 15-ml tube.

46| Centrifuge at 250g for 5 min at 37 °C.

47| Discard the supernatant.

48| Estimate the volume of the RBC pellet and add an equal amount of freezing solution dropwise while swirling the tube. Mix using a plastic pipette.

49| Aliquot the mixture into 1-ml sterile cryovials. The aliquots in each vial must be 500 to 600 µl.

50| Store immediately in liquid nitrogen.

parasite harvesting ● tIMInG 30–45 min51| Divide the contents of each 150-cm2 flask (from Steps 43A(v) or 43B(xviii)) into three 50-ml tubes. Centrifuge the 50-ml tubes at 300g for 7 min at 37 °C to remove all medium.

52| Wash each infected red cell pellet thoroughly with 20 ml of washing medium.

53| Centrifuge the 50-ml tubes again at 300g for 7 min at 37 °C. Discard the supernatant. crItIcal step The supernatant must be clear, indicating the absence of parasites. Always check the supernatant in subsequent centrifugations.

54| Resuspend each pellet in 2 ml of washing medium and combine them in a new 15-ml tube. Wash the tubes with additional 2 ml of washing medium and bring them to the new 15-ml tube containing the combined pellets.

a b c

Figure 3 | State of the culture 48 h after thawing. Using 300 µl of a synchronous inoculum containing 10–20% rings, up to 600 µl at 10% parasitemia can be obtained after 48 h (Step 23). Note that not all frozen parasites survive thawing, resulting in a parasitemia below the initial level. However, the smears stained with Giemsa 3% (a) or 10% (b) and Wright’s (c) show the synchronization window remains after first invasion. Panels show a culture 48 h after thawing, in which only the surviving parasites have multiplied. After 48 h, the parasite appears as young rings, which within the red cells may vary in size from small to medium. Ring and comma forms are common shapes, chromatin does not yet appear condensed (no dots) and the parasite cytoplasm is regular, fine to fleshy. For further details on parasite stage morphology and synchronization window, see Box 2. Scale bar = 10 µm.


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55| Centrifuge at 300g for 7 min at 37 °C.

56| Discard the supernatant and store the parasite pellet at –80 °C for further analysis (for RNA expression analysis, the immediate addition of an RNA stabilization reagent (e.g., RNALater, Qiagen) is recommended).

● tIMInGSteps 1–8, preparation of materials and RBCs: 5 hSteps 9–20, thawing the glycerol-frozen parasites: 1 hSteps 21 and 22, assessing parasitemia levels and viability of cultures by preparing and staining smears: 15–45 minStep 23, change of culture medium: 30 minSteps 24 and 25, assessment and maintenance of cultures before sorbitol synchronization: 1 hSteps 26–39, sorbitol synchronization: 1 hSteps 40–42, assessment and maintenance of cultures following sorbitol synchronization: 1 hStep 43A, maintenance of synchronous cultures at high parasitemia: 2 hStep 43B, maintenance of cultures and further short window synchronization: 3 h 45 minSteps 44–50, freeze-storing infected RBC in liquid nitrogen: 45 minSteps 51–56, parasite harvesting: 30–45 min

? trouBlesHootInGTroubleshooting advice can be found in table 1.

taBle 1 | Troubleshooting table.

step problem possible reason solution

22A(iv) The smears are too dark Excess incubation time with the dye Excess dye evaporation in dry, hot weather

Wash smears with methanol 100% and allow drying before microscope observation Reduce incubation time by 1–4 min

23 At 48 h after launch, the para-sitemia is below 1%

The inoculum had too many trophozoites and shizonts. These are less resistant to thawing

Ring parasitemia of the inoculum was too low

Wait for another invasion cycle and check the culture’s progress. If parasitemia rises above threefold, the culture can safely be continued. If the parasitemia increase is insufficient ( < 3-fold), we recommend discarding the culture and launching a new one. Be careful during freezing. Only use cultures rich in young rings ( < 10 h postinvasion)

23, 24, 41, 43A(i and iii), 43B(i, iv, vi, xvi, xvi and xviii) and 44

Loss of parasites when changing the culture medium

Insufficient time allowed for the culture flask to settle in the laminar flow cabinet before starting the change Culture shaken before the change

Increase the settling time

Be careful when the culture does not mix during transfer from the CO2 incubator to the laminar flow cabinet

25 The ring-stage parasitemia is < 10%

Ring parasitemia of the inoculum was too low The hematocrit is higher than 2%

Wait for another invasion cycle to achieve higher parasitemia Change the medium 24 h later (6–8 h before invasion), divide the flask into two new flasks and adjust hematocrit to 1%

40 After sorbitol treatment, the synchronization window is too high (above 10 h up to 18 h)

Wrong estimation of invasion time Possible mistakes when executing Steps 31–34 of the sorbitol synchronization procedure

Try Percoll synchronization if one requires synchronic culture as soon as possible Wait for a cycle without adding sorbitol and repeat the synchronization step. Carefully follow the instructions for the sorbitol synchronization procedure

(continued)


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antIcIpateD resultsGrowth from frozen parasitesAfter 48 h of thawing a synchronous inoculum containing >10% rings to set up the culture, new ring forms should be clearly observed when they are stained (Fig. 3).

sorbitol synchronization. Sorbitol treatment lyses trophozoites and schizonts. For best results, it is preferable to carry out this synchronization before all the schizonts have disappeared to achieve the narrowest window. At the time when there are still mature schizonts in the culture, young rings start to become visible but, despite their presence, very young ring forms might not be observable under the microscope (Fig. 4a). However, if the culture is checked 24 h after the sorbitol procedure, only mature forms will be seen (Fig. 4b). When sorbitol synchronization is incorrectly timed, the window increases to 18 h (Fig. 4c).

taBle 1 | Troubleshooting table (continued).

step problem possible reason solution

43B(xiii) Many trophozoites of intermediate age (~34 to 38 h postinvasion) appear together with the mature forms after Percoll synchronization

Wrong estimation of invasion time

RBC and Percoll layers have mixed during Step 43B(xi) of the Percoll procedure

For highly synchronized cultures (Step 43B), try sorbitol synchronization within 10 h of Percoll treatment

43B(xv) Reduced parasitemia after Percoll

Percoll synchronization is incorrectly timed

Wait until parasitemia increases before attempting further synchronization

Synchronization window is too wide after Percoll

43B(xix) Parasitemia level falls more than expected after a normal growth rate is achieved

Blood is too old Replace with fresh blood (whole blood < 15 days after extraction or washed RBC younger than 5 days)

Figure 4 | Synchronization of cultures using sorbitol. When the cultures reach a high parasitemia level (more than 30%), the synchronization window can be maintained or shortened by the sorbitol or Percoll methods. Scale bar = 10 µm. (a) The best situation for sorbitol synchronization is when very young rings and very mature schizonts are seen in the same smear (Step 25), meaning that most forms will be rings (including as yet invisible young rings: < 4 hours) at the time of sorbitol treatment and that parasitemia loss will therefore be minimal. At the schizont stage, the malaria parasite starts to reproduce. Very mature schizonts quickly release merozoites and are usually found in the same smear associated with many young ring forms. Mature schizonts have 18–24 merozoites in a compact cluster containing malaria pigment as a single refringent dark brown mass. (b) After 26 h of treatment with sorbitol (Steps 40–41), fresh red blood cells are added to achieve 17–20% parasitemia at 1.0–1.5% hematocrit. Note the synchronization window is around 10 h. That is, the time between the early and mature schizonts seen in the picture (for further details on parasite stage morphology and synchronization window, see Box 2). (c) The synchronization window increases to 18 h when sorbitol treatment is slightly delayed, since this is the time between the schizonts and the late rings observed in the same smear, which is equivalent to an 18-h synchronization window (see Box 2). Columns 1, 2 and 3 in all items were stained with Giemsa 3%, Giemsa 10% and Wright, respectively.

1 2 3

a

b

c


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percoll synchronizationFor this synchronization protocol, parasites must be predominant in the schizont stage (Fig. 5a) to obtain optimal results (Figs. 5b,c). Avoid using this procedure when there are still a large number of trophozoites (>15% relative parasitemia) in the culture. If Percoll synchronization is incorrectly timed, parasitemia is dramatically reduced in the following invasion (Fig. 5d) or the synchrony window widens.

parasite harvestingAfter both synchronization methods, 60% parasitemia cultures can be collected at any stage within a 10- or 4-h synchrony window, following optional Steps 43A or 43B respectively. Three 150-cm2 culture flasks will allow to isolate 1.17 × 1010 parasites at any intraerythrocytic stage from a starting inoculum of 2.34 × 108 parasites. From this

1 32

a

b

c

d

e f

Figure 6 | High-density synchronized cultures of Plasmodium falciparum. Starting from 300 µl of thawed inoculum, a synchronous culture of 50–60% parasitemia is achieved by Step 43A (a and b). Optionally, cultures with a short synchrony window and above 60% parasitemia are achieved by Step 43B (c and d). Hereafter, 3–5 ml of synchronous culture showing close to 60% parasitemia can be harvested every week. Parasitemia was determined by counting at least 1,200 cells in the Wright’s or Giemsa procedures. Scale bar = 10 µm (a) Cultures at the late ring stage (~88% rings of relative parasitemia, synchronization window 14–22 h). (b) Cultures at the early trophozoite stage. Note the synchrony of early trophozoite forms remains above 85% with a synchronization window of 22–30 h. (c) Cultures at the trophozoite stage (more than 90% trophozoites of relative parasitemia, synchronization window 30–34 h). (d) Cultures at schizont stage (synchronization window 38–42 h). Note the high parasitemia and synchrony persisting across two invasion cycles after double synchronization with sorbitol and Percoll 70%. Columns 1, 2 and 3 in a–d were stained with Giemsa 3%, Giemsa 10% and Wright’s, respectively. The pictures in a–d show P. falciparum strain Dd2, but the protocol has also been successfully applied to 3D7 and to a clinical isolate. (e) Rings of the 3D7 clone at 45% parasitemia stained with Wright’s. (f) Trophozoites in an isolate from a patient from Equatorial Guinea at 53% parasitemia stained with Giemsa 10%.

a

b

c

d

1 2 3Figure 5 | Synchronization of cultures using Percoll 70%. By monitoring the culture process preparing periodically smears, the best time to enrich the culture in mature parasite forms using Percoll 70% can be easily determined. Scale bar = 10 µm. (a) The optimal time is when only very late trophozoites and schizonts together with a few young rings are seen in high parasitemia cultures (Step 43B(iii/iv)). These forms are separated in two layers of the Percoll cushion. Late trophozoites are seen as large round or elliptical forms occupying most of the red cell that have completely loss their ring-like shape; they contain the malaria pigment as a single refringent dark brown mass and the cytoplasm is homogeneous with no segmentation. Schizonts, which develop from trophozoites, undergo cellular segmentation and differentiation to form the merozoite cells within the erythrocyte and also contain a mass of malaria pigment (for further details on parasite stage morphology and the synchronization window, see Box 2). (b) The upper layer after Percoll treatment contains close to 90% parasitemia (Step 43B(xiii)) before it is diluted with fresh red blood cells to 10% and a 1–1.5% hematocrit. (c) In this panel, a picture of a smear taken 20 h after Percoll treatment indicates 30% parasitemia (Step 43B(xv)). Note the short synchronization window of 4 h. The detection of young rings in the smear indicates a 4-h period as described in Box 2. Young and intermediate rings are distinguished by the absence of chromatin condensation; this may be seen as 1–2 dots in mature rings (for further details on parasite stage morphology and the synchronization window, see Box 2). (d) A reduced parasitemia (around 15% or less) and more than 4-h synchrony window can be obtained after Percoll 70% treatment of an incorrectly timed culture. This photograph shows a culture 40 h after Percoll treatment. Columns 1, 2 and 3 in all items were stained with Giemsa 3%, Giemsa 10% and Wright, respectively.


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100-fold enriched culture, for each ml of harvested infected RBCs at 50% parasitemia in early trophozoites (around 24 h post-invasion), one can expect to obtain 1.2 ± 0.3 mg (n = 6) of total parasite protein. This means up to 7.5 mg of total parasite protein can be obtained weekly. Figure 6 shows different harvesting times for intraerythrocyte stage cultures of the Dd2 and 3D7 clones and the clinical isolate.

acknoWleDGMents 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.

autHor 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.

Published online at http://www.natureprotocols.com/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.

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