Reagents and in vitro culture conditions
RPMI culture medium: 0.5 gr of hypoxanthine (Sigma, art nr. H-9377-25G) was added to 2 liter Milli-Q and placed on a magnetic stirring plate at room temperature (RT) to dissolve. Subsequently 59.4 gr HEPES (BDH Prolabo, 441487M or alternative 441476L) and 1 pot RPMI1640 powder (Life Technology Invitrogen, 518 − 00035) was added. The volume was topped up with Milli-Q to 4800 ml and left to dissolve by magnetic stirring. Media was transferred to a pressure vessel and an additional 4800 ml Milli-Q was added, giving a total volume of 9600 ml. The pressure vessel inlet was attached to a N2 gas cylinder and the outlet to silicon tubing with a 0.2 µm hollow fiber medium filter (Mediakap-10 Spectrum, me2m-10b-12s). Under 1 bar of pressure, the media was filter sterilized and collected in a customized sterile 3000 ml Erlenmeyer with tap placed inside a sterile biological safety cabinet. Media was filled out in 80 ml aliquots in sterilized glass bottles that were closed with aluminum caps with rubber inlay and frozen at -20°C. Medium was thawed in a water bath at 37°C before use. For so-called incomplete medium, 3.7 ml of autoclaved 5% sodium hydrogen bicarbonate (Merck, 1.06329.1000) in Milli-Q was added. For complete medium, another 8 ml pooled human serum was supplemented. Thawed medium was stored in the fridge at 4°C and used for a maximum of 7 days.
Serum: Serum was obtained in 200 ml IV bags by the national blood bank (Sanquin; Nijmegen, The Netherlands). Bags of at least 10 malaria naïve donors (group A or AB, Rh + and Rh- mixed) were pooled and collected in a customized 3000 ml Erlenmeyer with tap. Aliquots of 100 were filled out in sterilized glass bottles and closed with aluminum caps with rubber inlay, frozen at -20°C and thawed in a water bath at 37°C before use. Thawed serum was stored in the fridge at 4°C and used for a maximum of 7 days.
Erythrocytes: Blood from malaria naïve donors (group O, Rh + and Rh-blood mixed) was drawn twice weekly in 10 ml lithium heparinized tubes (BD, Vacutainer) after skin disinfection with 70% Isopropyl Alcohol (FA Alcohol Swab-S), by the national blood bank (Sanquin;Nijmegen, The Netherlands). Tubes were centrifuged at 750 xg for 5 minutes in a benchtop centrifuge at RT. After removal of the plasma and buffy-coat layer, blood from at least 6 donors was pooled and transferred to 15 ml tubes (Corning, 430766). Blood was washed 2x by adding 10 ml incomplete medium to each tube and centrifuged as above. After removal of the supernatant the volume of erythrocytes was diluted with an equivalent volume of complete medium to obtain a 50% hematocrit solution. Tubes were stored in the fridge at 4°C and used for a maximum of 7 days for parasite culture or 9 days for SMFA blood meal preparation. Unprocessed heparin blood was used for routine mosquito colony maintenance.
Culture conditions: Parasites were cultured at 37°C and continually supplied with a pre-mixed gas mixture of 3% O2, 4% CO2 and 93% N2. Isolates were adapted in an semi-automated shaker system [16], a modified version of the 1981 Butcher model [17], and cultured for gametocyte production in a semi-automated tipper system [18].
Plasmodium falciparum collection and culture adaptation
Peripheral blood was collected in EDTA Vacutainers (BD). Tubes were spun down for 5 minutes at 750 xg at RT in a tabletop centrifuge without break. The plasma and buffy-coat layer was gently removed before erythrocytes were transferred to a 15 ml tube (Corning, 430766). Subsequently, blood was washed 2x by topping up the volume with incomplete RPMI medium. Tubes were gently mixed by inverting and spun down as above. After the final wash the supernatant was removed and a Giemsa stained blood smear was made from the pellet to assess parasites morphologically and determine the percentage of infected erythrocytes. 0.2 ml of patients’ blood was added to 0.1 ml of 50% pre-washed erythrocytes (as described above) and topped up to 10 ml with complete RPMI medium, supplemented with AB serum. Cultures were transferred to the semi-automated shaker system [16]. After one week adaptation A serum was used for culture. Every 2–3 days parasitemia was evaluated by microscopy after Giemsa staining and 0.5-1.0% erythrocytes were added until parasites were adapted and 5% hematocrit was reached.
Cryopreservation and retrieval of parasites
For cryopreservation, a modified methodology of Diggs et al was used [19]. Briefly, cultures were transferred to a 15 ml tube (Corning, 430766) and spun down at 750 xg for 5 minutes in a benchtop centrifuge at RT. The supernatant was removed and the residual volume of the erythrocyte pellet was determined. 1x the volume of complete culture medium was added to obtain 50% hematocrit. Subsequently 2x the initial volume of 30% glycerol in PBS (Gibco 10010-015) was added dropwise and the volume was gently mixed with a pipette. A minimum of 0.5 ml of suspension was distributed per cryovial (Nunc, 368632). Vials were transferred to a ‘Mr frosty’ freezing container (Nalgene, 5100-0001) and frozen at -80ºC overnight before they were placed in liquid nitrogen at -196ºC for long term storage.
To retrieve isolates from liquid nitrogen a modified methodology of Diggs et al. was used [19]. Briefly, parasites were retrieved from liquid nitrogen by rapidly thawing of the suspension at 37°C. After the vial was directly placed on ice. The volume was determined and transferred to a 15 ml tube (Corning, 430766). Two volumes of 27% sorbitol (Merck, 1.07758) in 1x PBS (Gibco 10010-015) was gently added via the side of the inclined tube. The tube was gently mixed by rotating in a nearly horizontal position before it was placed on ice for 12 minutes. Thereafter, two volumes of cold 5% sorbitol in 1x PBS was added and the tube was again gently mixed before incubated on ice for 10 minutes. The tube was centrifuged at 300 xg for 5 minutes in a benchtop centrifuge set at 4°C. The supernatant was removed and the pellet was suspended in 2 volumes of 5% sorbitol and incubated on ice for 8 minutes. After centrifugation at 750 xg for 5 minutes the supernatant was removed and the pellet was washed 2x with 10 ml incomplete culture medium. After the final wash, 10 ml complete medium and 2.5% fresh erythrocytes were added. Every 2–3 days, growth was assessed after Giemsa staining and ~ 1% of erythrocytes was added, based on parasitemia, until 5% hematocrit was reached.
Plasmodium falciparum routine asexual- and sexual parasite culture
To maintain asexual parasite growth, cultures were Giemsa stained two (tipper system [18]) or three times (shaker system [16]) a week, irrespective of the parasitemia, to assess the percentage of infected erythrocytes by microscopy at 1000x magnification. Cultures were diluted back to 0.5 or 1.0% parasitemia, for respectively shaker or tipper system, and 5% hematocrit per 10 ml by adding erythrocytes. Successful culture adaptation of isolates was determined as continued asexual growth with increasing parasitemia after cyclic replication.
For gametocyte production, 1% parasitemia and 5% hematocrit was seeded at day zero in the tipper system [18]. Successful gametocyte culture was evaluated by assessing induction, maturation and transmission. Gametocyte induction and maturation were assessed microscopically eight days after seeding by the presence of stage II gametocytes (D-shaped), and fourteen days after seeding by the presence of mature stage V male and female gametocytes, using the nomenclature classification of Hawking et al [20] as demonstrated in Ponnudurai et al [16]. Special attention was paid to; (a) thickness of the gametocyte, (b) pace of development, and (c) localization of the pigment. Mature male gametocytes were activated to test for their ability to exflagellate. To assess exflagellation, evaluated 14 days post seeding, 200 µl of culture material was spun down with a benchtop centrifuge at 17700 xg for 20 seconds. After removal of the supernatant, 3 µl of pelleted erythrocytes was mixed on a microscope slide with 10 µl of Fetal Bovine Serum (FBS, Invitrogen, art no 10270106) and incubated for 10 min at room temperature in a humidified box. A coverslip with Vaseline coated edges was applied on top and exflagellation was assessed by microscopy at 400x magnification and determined as the presence or absence of exflagellation centers without further quantification.
Exflagellating gametocyte cultures were fed to mosquitoes to test their ability to establish infection [21]. An infective blood meal was prepared as previously described [21]. In brief at 37°C, 300 µl ml of culture material was added to 180 µl pellet erythrocytes and spun down in a benchtop centrifuge as described above. Supernatant was removed, without disturbing the pellets surface, and 150 µl serum was added. Blood meal was gently vortexed and fed by midi-feeders to a total of 50 mosquitoes.
Cloning of isolates
For cloning, a modified methodology of Thaithong was used [22]. Briefly, a 100x dilution of culture material was prepared to count the number of erythrocytes with a hemocytometer. The number of infected erythrocytes per µl was calculated. Three dilutions were made in complete medium with 1% fresh erythrocytes; 100, 10 and 0.3 parasites per 100 µl. For each dilution 100 µl was transferred to respectively 10, 10 and 40 wells of a 96 well flat-bottom microplate (Nunc, 167008), outer wells were filled with 150 µl sterile MilliQ to diminish evaporation. Plates were maintained in humidified modular chamber (Billups and Rothenberg) at 37°C and an atmosphere of 3% O2, 4% CO2, and 93% N2. Medium was every other day, by placing the plate on a 45 degrees angle stand for 30 minutes before removing the medium carefully without disturbing the settled erythrocytes at the bottom of the wells. Every other day of medium change 0.5% fresh erythrocytes were added. After 10 days, thick smears were prepared of the control wells, that were seeded with 10 and 100 parasites. Without fixation they were Giemsa stained to confirm parasite growth. After two to three weeks 10 µl of each experimental well was analyzed for growth by microscopy after Giemsa staining. For positive wells, usually maximal 10 per plate, the remaining culture volume was transferred to a shaker flask and 1% fresh erythrocytes were added. Every 2–3 days growth was assessed by microscopy and ~ 0.5% of fresh erythrocytes was added, based on parasitemia, until 5% hematocrit was reached.
Clonality was confirmed by assessment of the merozoite surface protein 1 and 2 (MSP1 and MSP2) and Glutamate-Rich Protein (GLURP) polymorphic region by PCR, as described before [14], a methodology adapted from Snounou et al [23]. DNA was extracted from pelleted cultures using the QIAamp DNA Blood Kit spin protocol for DNA purification from blood or body fluids (Qiagen, 51104). GoTaq G2 Flexi DNA Polymerase kit (Promega, M7801) was used for DNA amplification. Only if single bands were observed for each of the three target genes, clonality was assumed.