Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

General recommendations

Human brain microvascular endothelial cell (HBEC-5i) culture has been previously described in ^{6, 8}. P. falciparum parasites were cultured as in ⁹. Both HBEC-5i and P. falciparum parasite cultures should be kept in sterile conditions at all times. All reagents should be pre-warmed at 37°C. We recommend to regularly check for mycoplasma contamination¹⁰ by PCR (Minevera Biolabs, following manufacturer’s instructions). The protocol is summarized in Figure 1.

1. Endothelial cell routine culture

1. Prepare the necessary reagents.

2. Culture HBEC-5i in a vented 25cm² flask with 10ml DMEM complete medium in a 37°C incubator with 5% CO₂.
3. Passage cells when they become confluent. Remove the old medium by suction and wash twice using DMEM incomplete medium or tissue culture grade PBS (Ca²⁺ and Mg²⁺ free) pre-warmed to 37°C.
4. Add 1ml of pre-warmed Trypsin-EDTA (0.025% Trypsin, 0.5mM EDTA), swirl to cover the entirety of the flask and incubate for ˜2 min at 37°C.
5. Check under inverted microscope that at least 90% of cells have been detached. If necessary, gently knock the bottom of the flask to dislodge adherent cells. Add 10ml of DMEM complete medium to block the trypsin and transfer cells into a 15ml conical tube. Centrifuge for 4 min at 300 g at room temperature (RT).
6. Discard the supernatant and resuspend the pellet with 10ml of DMEM complete medium. Pipette the solution up and down to thoroughly resuspend the cells.
7. Add 1 or 2 ml of the cell suspension into a new culture flask and add 8ml of fresh medium to maintain the culture. Assess culture growth every day under an inverted microscope and change medium every 2 or 3 days before it turns yellow.

2. Preparing endothelial cells for a selection

1. Two days prior to the day of the selection, add fibronectin diluted in sterile PBS (2 μg/cm²) in one (or more) 60 mm Petri dish. Incubate the dish for 5 to 20 min at 37°C, then remove the fibronectin solution, which can be stored at 4°C for a month and re-used once.
2. Passage cells as described in sections 1.3. to 1.5. Assuming 100% confluent cells were detached and resuspended in 10ml DMEM complete medium (section 1.6., resuspend with an equivalent volume of medium if the confluency is lower, e.g. resuspend with 8ml if the confluency was 80%), add 1.5ml of the suspended cells to the fibronectin coated Petri dish and another 1.5ml of DMEM complete medium.
3. Place the seeded Petri dish in the incubator. Note: Ideally, the confluency will be around 90% at the time of selection two days later.
4. Optional. To activate HBEC-5i, add the TNF (Tumour Necrosis Factor) cytokine at a final concentration of 50μg/ml 24 hours prior to the selection.

3. Plasmodium falciparum routine culture

1. Prepare the necessary reagents (see table here under). Prepare human red blood cells (RBC) by separating whole blood (group O+) by passage through a leukocyte depletion filter (see “Methods in Malaria Research” publication¹¹ for general malaria parasites culturing). Wash RBC twice by centrifuging at 400 g for 5 min and resuspending them with 10 ml RPMI incomplete. Keep washed RBC at 4°C in incomplete medium at 50% haematocrit.

2. Culture P. falciparum infected RBC with RPMI complete medium at 2% haematocrit and incubate at 37°C with 3% CO₂, 1% O₂, and 96% N₂. Make Giemsa smear¹¹ daily to assess the stage of development of parasites (Figure 2).
3. Regularly (approximately once a week) synchronize culture by sorbitol treatment¹². The day prior the selection assay, aim for a ring-stage culture at 5% parasitaemia or more (ideally at least 10%).

4. Selection of P. falciparum for cytoadhesion to endothelial cells

1. On the day of the assay, the parasite culture should be at pigmented trophozoite stage (Figure 2)(ideally 10% parasitaemia) while HBEC-5i culture should be at 50 to 100% confluency (ideally 90%). 30μl packed cell volume of parasite culture is needed per HBEC-5i Petri dish.
2. Wash parasites twice by centrifuging (500g for 5 min) 1.5ml of parasite culture. Discard supernatant and resuspend with 10ml of freshly made, warmed, DMEM incomplete medium. Repeat the wash a second time. Resuspend the 30μl packed cell volume with 1.5ml of DMEM incomplete with 1% BSA.
3. Wash the HBEC-5i coated Petri dish twice aspirating medium and adding 3ml of incomplete DMEM.
4. Add the solution of parasites to the HBEC-5i dish and incubate at 37°C for 75min. Resuspend parasites twice (after 30 and 60 min) during the incubation by gently rocking the dish in the four directions, as well as clockwise and anti-clockwise.
5. After incubation, wash the dish 5 times by aspirating medium, using a plastic Pasteur pipette to add 3 ml of warm DMEM incomplete medium, and gentle rocking. If many dishes are being used, keep them on a warm surface, such as a large flask filled with water at 37°C.
6. Check the dish under an inverted microscope. If many uninfected RBC are still visible, do more washes as described above. Handle the Petri dish very carefully to avoid any risk of contamination.
7. Remove medium from the dish and add 3ml of warm RPMI complete medium with 40μl packed cell volume of fresh uRBC.
8. Place the dish in an airtight incubating chamber, gas it for 3 min and place the chamber in an incubator at 37°C overnight.
9. The day after, harvest the parasites by washing with RPMI incomplete medium, in a similar way as described in section 4.4. but more vigorously. Keep all medium used (containing resuspended RBC) in a 15ml conical tube. Check under inverted microscope that all RBC have been removed from the dish.
10. Pellet the parasites, discard the supernatant, resuspend in 5ml RPMI complete medium and place the mixture in a flask for normal culturing.

5. Representative Results

Unselected parasites show a low level of binding to HBEC-5i (Figure 3A). Thus, after the first round of selection, very few parasites will be harvested and it may take up to a month of culturing to reach a parasitaemia sufficient for the second round of selection. After each round of selection, more and more parasites bind to the endothelial cells and the selections can be repeated at shorter time intervals. After 4-5 rounds of selection, high-binding parasite populations are obtained (Figure 3).

In our hands, the binding of HB3-HBEC to HBEC-5i or TNF activated HBEC-5i was of similar level (data not shown).

The protocol described here was tested with 4 P. falciparum strains: HB3, IT/FCR3, 3D7 and Dd2 (Table 1). Only the latter proved not able to bind to HBEC-5i, even after 5 rounds of selection.

HB3 parasites were also selected for cytoadherence to Human Dermal and Pulmonary Microvascular Endothelial Cells (HDMEC and HPMEC). After 4 rounds of selection, using the method described here, a high-binding population was obtained on both HDMEC and HPMEC (Figure 4).

Figure 1. Overview of the selection process.

Figure 2. Development stages of P. falciparum infected red blood cells. Giemsa smear visualized under microscope at 1000X magnification.

Figure 3. Typical example of parasites binding to HBEC-5i. (A) The blue layer is HBEC-5i fixed with glutaraldehyde and stained with Giemsa, visualized under microscope at 1000X magnification. Pictures were taken after uRBC and unbound iRBC were washed away. The left panel shows a single P. falciparum HB3 (unselected) parasite bound to HBEC-5i. In the right panel the HB3-HBEC parasites had been selected for 5 rounds. (B) Data represents the average of two independent experiments, each performed in duplicate. The number of parasites bound per endothelial cell was counted.

Table 1. Summary of Plasmodium falciparum strains that were successfully selected for binding to HBEC-5i. Note that HB3 was also selected on TNF-activated HBEC-5i. After 5 rounds of selection, the Dd2 strain showed no increase in binding to HBEC-5i compared to unselected-Dd2.

Figure 4. Binding of P. falciparum HB3 parasites to dermal (HDMEC) and pulmonary (HPMEC) endothelial cells, before and after 4 rounds of selection. Although the culture medium for HDMEC and HPMEC slightly differs (see supplier’s instructions), the protocol used for the selection was identical as with HBEC-5i. Pictures taken at 400X magnification.

Table 2. Materials