Name: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells (Video) | JoVE Journal
Uploaded: 2012-01-03T12:00:00.000+00:00
Duration: 10 min 9 s
Description: Learn to select Plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells using an in vitro binding assay. This method enables study of cerebral malaria sequestration mechanisms.

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We thank Francisco Candal, CDC Technology Transfer Office, Atlanta Georgia for HBEC-5i cells. This work was funded by the Wellcome Trust (4 year PhD studentship to AC and Senior Fellowship in Basic Biomedical Science to JAR, grant number 084226).

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
<ol>
 <li>Prepare the necessary reagents.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>
 Medium to prepare</td>
 <td>
 Reagents</td>
 <td>
 Quantity</td>
 </tr>
 <tr>
 <td>"DMEM incomplete"</td>
 <td>DMEM-F12 Ham</td>
 <td>500ml</td>
 </tr>
 <tr>
 <td>  </td>
 <td>L-glutamine 200mM</td>
 <td>5ml</td>
 </tr>
 <tr>
 <td>  </td>
 <td>Penicillin/streptomycin 100X</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>  </td>
 <td>NaOH 1M</td>
 <td>1.3 ml (adjust pH to 7.4)</td>
 </tr>
 <tr>
 <td>"DMEM complete"</td>
 <td>"DMEM incomplete"</td>
 <td>450ml</td>
 </tr>
 <tr>
 <td>  </td>
 <td>Foetal Bovine Serum heat-inactivated</td>
 <td>50ml</td>
 </tr>
 <tr>
 <td>  </td>
 <td>Endothelial cell growth supplement</td>
 <td>5 ml</td>
 </tr>
 </tbody>
</table>
 
<ol start="2">
 <li>Culture HBEC-5i in a vented 25cm2 flask with 10ml DMEM complete medium in a 37°C incubator with 5% CO2.</li>
 <li>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.</li>
 <li>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.</li>
 <li>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).</li>
 <li>Discard the supernatant and resuspend the pellet with 10ml of DMEM complete medium. Pipette the solution up and down to thoroughly resuspend the cells.</li>
 <li>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.</li>
</ol>
2. Preparing endothelial cells for a selection
<ol>
 <li>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.</li>
 <li>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.</li>
 <li>Place the seeded Petri dish in the incubator. Note: Ideally, the confluency will be around 90% at the time of selection two days later.</li>
 <li>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.</li>
</ol>
3. Plasmodium falciparum routine culture
<ol>
 <li>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.</li>
</ol>
<table border="1">
<tbody>
 <tr>
 <td>Medium 			to prepare</td>
 <td>Reagents</td>
 <td>Quantity</td>
 </tr>
 <tr>
 <td>"RPMI 			incomplete"</td>
 <td>RPMI 1640 (with 			bicarbonate)</td>
 <td>500ml</td>
 </tr>
 <tr>
 <td> </td>
 <td>Hepes 1M</td>
 <td>12.5ml</td>
 </tr>
 <tr>
 <td> </td>
 <td>Glucose 20%</td>
 <td>5ml</td>
 </tr>
 <tr>
 <td> </td>
 <td>L-glutamine 			200mM</td>
 <td>5ml</td>
 </tr>
 <tr>
 <td> </td>
 <td>Gentamycin 			50mg/ml</td>
 <td>250μl</td>
 </tr>
 <tr>
 <td> </td>
 <td>NaOH 1M</td>
 <td>0.7 ml (adjust 			pH to 7.2)</td>
 </tr>
 <tr>
 <td>"RPMI 			complete"</td>
 <td>"RPMI 			incomplete"</td>
 <td>450ml</td>
 </tr>
 <tr>
 <td> </td>
 <td>Pooled human 			(non-immune) serum</td>
 <td>50ml</td>
 </tr>
 </tbody>
</table>
 
<ol start="2">
 <li>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).</li>
 <li>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%).</li>
</ol>
4. Selection of P. falciparum for cytoadhesion to endothelial cells
<ol>
 <li>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.</li>
 <li>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.</li>
 <li>Wash the HBEC-5i coated Petri dish twice aspirating medium and adding 3ml of incomplete DMEM.</li>
 <li>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.</li>
 <li>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.</li>
 <li>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.</li>
 <li>Remove medium from the dish and add 3ml of warm RPMI complete medium with 40μl packed cell volume of fresh uRBC.</li>
 <li>Place the dish in an airtight incubating chamber, gas it for 3 min and place the chamber in an incubator at 37°C overnight.</li>
 <li>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.</li>
 <li>Pellet the parasites, discard the supernatant, resuspend in 5ml RPMI complete medium and place the mixture in a flask for normal culturing.</li>
</ol>
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).
<img alt="Parasite selection diagram: HBEC-5i incubation, washing, culture process, stage-specific timing." src="/files/ftp_upload/3122/3122fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Overview of the selection process.
<img alt="Blood cell infection stages; microscopy images of ring stage iRBCs and pigmented trophozoite iRBCs." src="/files/ftp_upload/3122/3122fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Development stages of P. falciparum infected red blood cells. Giemsa smear visualized under microscope at 1000X magnification.
<img alt="Parasite binding assay: microscopy images, bar chart; HB3 cell comparison, experimental results." src="/files/ftp_upload/3122/3122fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
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.
<img alt="Selection table for HBEC-5i shows rounds needed for HB3, IT/FCR3, 3D7 (Yes) and Dd2 (No)." src="/files/ftp_upload/3122/3122table1.jpg" data-altupdatedat="1788235205000" data-alt="Table 1"> 
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.
<img alt="Cell culture microscopy of HDMEC and HPMEC showing HB3-Unselected and HB3-HBEC cells, stained nuclei." src="/files/ftp_upload/3122/3122fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
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 border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM-F12 Ham </td>
 <td>Sigma </td>
 <td>D6421 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>L-glutamine 200mM </td>
 <td>GIBCO </td>
 <td>25030 </td>
 <td>For DMEM and RPMI complete medium</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin 100X (10000 units/ml and 10mg/ml) </td>
 <td>ScienCell </td>
 <td>0503 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>Foetal Bovine Serum heat-inactivated </td>
 <td>ScienCell </td>
 <td>0025 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA (0.025% Trypsin,  0.5mM EDTA) </td>
 <td>ScienCell </td>
 <td>0103 </td>
 <td> </td>
 </tr>
 <tr>
 <td>Endothelial cell growth supplement </td>
 <td>ScienCell </td>
 <td>1052 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>Tissue culture treated 60 mm X 15 mm Petri dishes </td>
 <td>BD </td>
 <td>353002 </td>
 <td> </td>
 </tr>
 <tr>
 <td>Human Fibronectin </td>
 <td>Millipore </td>
 <td>FC010 </td>
 <td>Use at 2 μg/cm2</td>
 </tr>
 <tr>
 <td>TNF </td>
 <td>R&amp;D Systems </td>
 <td>210-TA </td>
 <td>optional, use at 50 μg/ml</td>
 </tr>
 <tr>
 <td>RPMI 1640 </td>
 <td>Lonza </td>
 <td>BE12-167F </td>
 <td>For RPMI complete medium</td>
 </tr>
 <tr>
 <td>Gentamycin 50mg/ml </td>
 <td>Lonza </td>
 <td>17-518Z </td>
 <td>For RPMI complete medium</td>
 </tr>
 <tr>
 <td>Hepes 1M </td>
 <td>Lonza </td>
 <td>BE17-737E </td>
 <td>For RPMI complete medium</td>
 </tr>
 <tr>
 <td>HDMEC </td>
 <td>ScienCell </td>
 <td>2000 </td>
 <td>Primary cell line</td>
 </tr>
 <tr>
 <td>HPMEC</td>
 <td>ScienCell</td>
 <td>3000</td>
 <td>Primary cell line</td>
 </tr>
 <tr>
 <td>HBEC-5i</td>
 <td> </td>
 <td> </td>
 <td>Obtained from Francisco Candal (fcandal@cdc.gov)</td>
 </tr>
 </tbody>
</table>
Table 2. Materials

<ol><li>Scherf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigenic+variation+in+malaria%3A+in+situ+switching%2C+relaxed+and+mutually+exclusive+transcription+of+var+genes+during+intra-erythrocytic+development+in+Plasmodium+falciparum%5BTitle%5D)+AND+%22Embo.+J%22%5BJournal%5D)">Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.</a> Embo. J. 17, 5418-5426 (1998).</li>
<li>MacPherson, G. G., Warrell, M. J., White, N. J., Looareesuwan, S., Warrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+cerebral+malaria.+A+quantitative+ultrastructural+analysis+of+parasitized+erythrocyte+sequestration%5BTitle%5D)+AND+%22Am.+J.+Pathol%22%5BJournal%5D)">Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration.</a> Am. J. Pathol. 119, 385-401 (1985).</li>
<li>Seydel, K. B., Milner, D. A., Kamiza, S. B., Molyneux, M. E., Taylor, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+distribution+and+intensity+of+parasite+sequestration+in+comatose+Malawian+children%5BTitle%5D)+AND+%22J.+Infect.+Dis%22%5BJournal%5D)">The distribution and intensity of parasite sequestration in comatose Malawian children.</a> J. Infect. Dis. 194, 208-208 (2006).</li>
<li>Taylor, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiating+the+pathologies+of+cerebral+malaria+by+postmortem+parasite+counts%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">Differentiating the pathologies of cerebral malaria by postmortem parasite counts.</a> Nat. Med. 10, 143-145 (2004).</li>
<li>van der Heyde, H. C., Nolan, J., Combes, V., Gramaglia, I., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unified+hypothesis+for+the+genesis+of+cerebral+malaria%3A+sequestration%2C+inflammation+and+hemostasis+leading+to+microcirculatory+dysfunction%5BTitle%5D)+AND+%22Trends.+Parasitol%22%5BJournal%5D)">A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction.</a> Trends. Parasitol. 22, 503-508 (2006).</li>
<li>Dorovini-Zis, K., Prameya, R., Bowman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+and+characterization+of+microvascular+endothelial+cells+derived+from+human+brain%5BTitle%5D)+AND+%22Lab.+Invest%22%5BJournal%5D)">Culture and characterization of microvascular endothelial cells derived from human brain.</a> Lab. Invest. 64, 425-436 (1991).</li>
<li>Rowe, J. A., Claessens, A., Corrigan, R. A., Arman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adhesion+of+Plasmodium+falciparum-infected+erythrocytes+to+human+cells%3A+molecular+mechanisms+and+therapeutic+implications%5BTitle%5D)+AND+%22Expert+reviews+in+molecular+medicine%22%5BJournal%5D)">Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications.</a> Expert reviews in molecular medicine. 11, e16-e16 (2009).</li>
<li>Xiao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasmodium+falciparum%3A+involvement+of+additional+receptors+in+the+cytoadherence+of+infected+erythrocytes+to+microvascular+endothelial+cells%5BTitle%5D)+AND+%22Exp.+Parasitol%22%5BJournal%5D)">Plasmodium falciparum: involvement of additional receptors in the cytoadherence of infected erythrocytes to microvascular endothelial cells.</a> Exp. Parasitol. 84, 42-55 (1996).</li>
<li>Corrigan, R. A., Rowe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strain+variation+in+early+innate+cytokine+induction+by+Plasmodium+falciparum%5BTitle%5D)+AND+%22Parasite.+Immunol%22%5BJournal%5D)">Strain variation in early innate cytokine induction by Plasmodium falciparum.</a> Parasite. Immunol. 32, 512-527 (2010).</li>
<li>Rowe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implications+of+mycoplasma+contamination+in+Plasmodium+falciparum+cultures+and+methods+for+its+detection+and+eradication%5BTitle%5D)+AND+%22Mol.+Biochem.+Parasitol%22%5BJournal%5D)">Implications of mycoplasma contamination in Plasmodium falciparum cultures and methods for its detection and eradication.</a> Mol. Biochem. Parasitol. 92, 177-180 (1998).</li>
<li>Moll, K., Ljungström, I., Perlmann, H., Scherf, A., Wahlgren, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMoll%2C+K+%22Methods+in+Malaria+Research%22">Methods in Malaria Research</a>. , MR4/ATCC. (2009).</li>
<li>Lambros, C., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture%5BTitle%5D)+AND+%22J.+Parasitol%22%5BJournal%5D)">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> J. Parasitol. 65, 418-420 (1979).</li>
<li>Herricks, T., Antia, M., Rathod, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deformability+limits+of+Plasmodium+falciparum-infected+red+blood+cells%5BTitle%5D)+AND+%22Cell.+Microbiol%22%5BJournal%5D)">Deformability limits of Plasmodium falciparum-infected red blood cells.</a> Cell. Microbiol. , (2009).</li>
<li>Nakamura, K., Hasler, T., Morehead, K., Howard, R. J., Aikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasmodium+falciparum-infected+erythrocyte+receptor%28s%29+for+CD36+and+thrombospondin+are+restricted+to+knobs+on+the+erythrocyte+surface%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.</a> J. Histochem. Cytochem. 40, 1419-1422 (1992).</li>
<li>Wassmer, S. C., Cianciolo, G. J., Combes, V., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LMP-420%2C+a+new+therapeutic+approach+for+cerebral+malaria%5BTitle%5D)+AND+%22Med.+Sci.+%28Paris%29%22%5BJournal%5D)">LMP-420, a new therapeutic approach for cerebral malaria.</a> Med. Sci. (Paris). 22, 343-345 (2006).</li>
<li>Wassmer, S. C., Combes, V., Candal, F. J., Juhan-Vague, I., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelets+potentiate+brain+endothelial+alterations+induced+by+Plasmodium+falciparum%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum.</a> Infect. Immun. 74, 645-653 (2006).</li>
</ol>

We have nothing to disclose.

The hallmark of cerebral malaria is sequestration of P. falciparum iRBC within the brain microvasculature2, 3. However, in vitro cultures of P. falciparum only poorly cytoadhere to HBEC-5i, a model for human brain microvascular endothelium. Here we developed a straightforward assay to enrich a population for binding to HBEC-5i, a more "in-vivo like" phenotype. Three out of 4 P. falciparum strains were successfully selected using this method. Furthermore, HB3 was also selected on HDMEC and HPMEC, indicating that this protocol can be used for various parasite and endothelial cell types.
Different hypotheses may explain the lack of binding with the Dd2 strain. The most likely being the fact that this parasite line is knobless, which deeply impedes the cytoadherence13, 14.
We recommend culturing unselected parasites alongside the selection process to provide a control for comparison. This will allow, for example, comparing the transcriptome of binding and non-binding parasites, with the hope of discovering parasite ligand candidates.
TNF is a cytokine found at high level in cerebral malaria patients and has been show to induce the expression of many surface proteins of HBEC-5i (Claessens et al, in preparation and 8, 15, 16). In this case, the amount of bound iRBC was similar in normal HBEC-5i compared to activated HBEC-5i.
This "sequestration model" can also be used to study the molecular interaction between the iRBC and the endothelial cell, as well as the effect of putative anti-cytoadherence drugs. In this case, we recommend plating HBEC-5i in smaller wells, such as "8-well chamber slides" (BD 354628) or "CultureWell" (Sigma C7735-20EA).

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

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

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

The overall goal of this procedure is to select plasmodium, fpar, and parasites for binding to human brain endothelial cells. This is accomplished by first incubating a culture of plasmodium, fpar, and parasites, which contain a mixture of infected and uninfected red blood cells with a layer of human brain endothelial cells, the next step is to wash off uninfected and unbound red blood cells by washing several times with culture. Medium, fresh red blood cells are then added to allow further growth of the parasite population.The final step a few weeks later is to repeat the overall selection as soon as a sufficient paras is reached. Ultimately, microscopy is used to show a high amount of parasites bound to HBE five i. This method can help answer key questions in the cerebral malaria field, such as the identification of parasite ligands or host receptor molecules involved in the cyto endurance process.Prepare human red blood cells from group O positive whole blood by separating the red blood cells from the white blood cells by passage through a leukocyte depletion filter. Centrifuge the cells at 1000 G for 10 minutes and Reese's spend the pellet with all PMI. Incomplete, medium.Repeat this wash step once more, then re suspend the pellet. In our PMI incomplete medium at 50%hematocrit, red blood cells are stored at four degrees Celsius for a week. Culture Odium Valspar infected red blood cells.With RPMI complete medium at 2%hematocrit and incubate at 37 degrees Celsius with 3%carbon dioxide, 1%oxygen, and 96%nitrogen every day. Make a gem smear to assess the developmental stage of the parasites. Change the medium daily by spinning the culture at 500 G for five minutes.Aspirating out the supernatant and adding fresh RPMI complete medium about once a week. Synchronize the parasites by treatment with aqueous 5%D sorbitol. The selection assay may be performed the day after a ring stage culture with a minimum of 5%to 10%Parsit is observed.In addition. Enough parasite culture to form a 30 microliter P cell volume is needed per 60 millimeter dish of hbe five eye cells. Prepare a 60 millimeter tissue culture dish to receive the human brain microvascular endothelial cell line or H back fivei by adding two micrograms per centimeter squared of fibronectin and sterile saline to the dish.Incubate the dish at 37 degrees Celsius for five to 20 minutes, then aspirate the fibronectin solution. Then harvest the HBE five I cells from a 25 centimeter square tissue flask by ionization and centrifugation Reese, suspend the cell pellet in 10 milliliters of DMEM, complete medium. Then add 1.5 milliliters of this cell suspension to the fibronectin treated tissue culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide for two to three days until the culture reaches 50 to 100%co fluency and the parasite cultures are ready for cyto aian on the day of the assay.The HBE five I culture should be at 50 to 100%co fluency. While the parasite culture should be at the pigmented trophozoites stage with at least five to 10%paras and 2%hematocrit. First pipette 1.5 milliliters of parasite culture from the culture flask and transfer to a 15 milliliter centrifuge tube.Centrifuge the culture at 500 G for five minutes and re bend in 10 milliliters. A freshly made warm DMEM incomplete, medium. Repeat this wash step a second time after the final centrifugation is complete.Asberry the DMEM incomplete, medium, and re spend the 30 microliter pellet in 1.5 milliliters of DMEM. Incomplete medium, supplemented with 1%BSA. Next, retrieve the 60 millimeter dish of hvac.Five.Eye cells aspirate the culture media and wash twice with three milliliters of DMEM. Incomplete medium. Add the solution of parasites, the dish of HPE five eye cells and incubate in the tissue culture incubator for 75 minutes in total.Twice during the incubation resus, suspend the parasites by gently rocking and swirling the dish following the incubation. Wash the dish five times by aspirating the medium using a plastic pasta pipette to add three milliliters of warm DMEM incomplete medium, and then gently rocking the plate after the final wash, examine the dish under an inverted microscope. If many uninfected red blood cells are still visible, repeat the wash procedure as described above to remove them.After aspirating the media from the dish Resus suspend 40 microliters packed cell volume of fresh red blood cells In warmed RPMI complete medium and add the cell suspension to the dish. Place the dish in an air sight incubating chamber. Then gas the dish with 3%carbon dioxide, 1%oxygen and 96%nitrogen for three minutes.And place the chamber in an incubator at 37 degrees Celsius overnight. During this incubation, schiz on stage parasites burst and maites reinve the red blood cells. The next day, harvest the ring stage parasites by washing.With RPMI incomplete medium in a similar manner as before, but with more vigorous pipetting of media and rocking of the plate. Keep all of the media used, which will contain resuspended red blood cells in a 15 milliliter conical tube. Check the plate under an inverted microscope to ensure that all red blood cells have been removed from the dish centrifuge.The tube containing the media from the washed plate to pellet the parasites discard the supernatant Resus suspending five milliliters. RPMI complete medium and place the mixture in a 25 centimeter square flask for culturing. The parasites are cultured for two to four weeks until enough material is obtained and a new round of selection can be performed.Unselected HB three parasites show a low level of binding to hbe five eye cells. This image shows hbe five eye cells fixed with 2%glutaraldehyde and stained with giemsa visualized under the microscope at 1000 times magnification peaches were taken after uninfected red blood cells and unbound infected red blood cells were washed away. After each round of selection, more and more parasites bind into the endothelial cells and the selections can be repeated at shorter time intervals after five rounds of selection.High binding parasite populations are obtained as shown here with HB three HBE parasites. This table shows a summary of the plasmodium alspar strains that were successfully selected for binding to HE five. I note that HB three was also selected on TNF activated HE five I after five rounds of selection.The DD two strain showed no increase in binding to HBE five I compared to unselected DD two. This 400 times magnification image shows binding of plasmodium alspar HB three parasites to HD MEK dermal endothelial cells before selection. Here, the binding of plasmodium alspar HB three parasites to the same dermal endothelial cell line is shown after four rounds of selection.This figure shows binding of plasmodium spar HB three parasites to HP ME pulmonary endothelial cells before selection. Now the binding of plasmodium spar HB three parasites to the H HP ME cells is increased after four rounds of selection. Although the culture medium for hd e, C and H HP EC slightly differs, the protocol used for the selection was identical as with hbe five I.Following this procedure of the methods like R-T-P-C-R or micro analysis can be performed to answer additional questions like characterizing the transcription of variant surface antigen in unselected and selected parasites.

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Research

JoVE Journal

Immunology and Infection

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

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