On the survival of 48 h Plasmodium vivax Aotus monkey-derived ex vivo cultures: the role of leucocytes filtration and chemically defined lipid concentrate media supplementation

The Aotus-adapted P. vivax Salvador I (SAL-I) strain originally obtained from William C. Collins (CDC, Atlanta, USA) [44], and further adapted by serial passage to spleen intact Panamanian Aotus lemurinus lemurinus monkeys [45] was used for inoculation of the donor monkeys of this study.

Twenty-two (22) laboratory-bred, spleen-intact, male and female Aotus l. lemurinus (Panamanian Owl monkey), karyotype VIII and IX [46], weighing between 758 and 829 g were used as P. vivax-infected blood donors. The animals were housed at the Gorgas Memorial Institute of Health Studies (GMIHS) monkey colony in Panama City, Panama, and cared and maintained as described [47].

Briefly, monkeys were immobilized with ketamine administered intramuscularly in the thigh muscle at 10 mg/kg and inoculated intravenously (iv) in the saphenous vein using a 25-g butterfly needle catheter attached to a 3-ml syringe containing P. vivax-infected blood from a donor monkey. Previously, 1 ml of venous blood had been obtained from the femoral vein of the donor animal with a 21-g butterfly needle catheter attached to a 3-ml syringe containing Sodium Citrate Solution (Sigma-Aldrich, St. Louis, MO, USA). Blood was then centrifuged at low speed (1200 rpm) for 5 min and plasma removed and further washed three times with phosphate buffer saline (PBS). The iRBCs pellet was then re-suspended in 1 ml of RPMI incomplete media to contain 5 × 10⁶ iRBCs x ml. The animals were followed up daily for parasitaemia determination from day 5 post-inoculation (PI) until peak parasitaemia that occurred approximately between days 12 and 21 (PI) [45], with daily thick and thin blood smears obtained from a prick in the marginal ear vein using a lancet and stained with Giemsa as described [48]. At peak parasitaemia, 3 ml of blood were obtained from the femoral vein of fasted animals for ex vivo cultures as described above [45, 49], at this point the animals received radical treatment with mefloquine at 20 mg/kg by gastric intubation as described [45].

Briefly, anticoagulated whole blood was centrifuged at 1200 rpm for 5 min, followed by removal of the buffy-coat and plasma with the help of a serological pipette. The remaining iRBCs column was further re-suspended and washed twice by centrifugation as above with incomplete McCoy’s5A medium (Sigma-Aldrich, St. Louis, MO, USA), re-suspended in incomplete McCoy’s5A medium and either used directly (washed) or passed through a Plasmodipur^® filter (EuroProxima, Amhem, The Netherlands) to remove remaining WBCs. Filter-retained iRBCs were back flushed from the Plasmodipur^® filter previously filled with 3 ml of incomplete McCoy’s media, applying steady suction with the help of a 10-ml syringe, and further washed three times as described above before plating (Fig. 1).

Plasmodipur^®-filtered, filter-retained or washed-only (unfiltered) iRBCs, were re-suspended to 5% haematocrit (HCT) in complete McCoy’s5A Modified medium (Sigma-Aldrich, St. Louis, MO, USA) containing 25 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 2 g/mL sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 2 g/L d-glucose (Sigma-Aldrich, St. Louis, MO, USA) and 40 μg/mL gentamycin (Sigma-Aldrich, St. Louis, MO, USA) [25], supplemented with 25% heat inactivated human AB + serum alone or 20% in combination with 5% chemically defined lipid concentrate (CDLC) (Cat No. 11905031, GIBCO, Grand Island, NY, USA) alone or in combination with 200 nM GlutaMax™-I 100X (GIBCO, Life Technologies, CA, USA) (a stabilized form of l-glutamine, L-alanyl-l-glutamine, that prevents degradation and ammonium build-up during long-term cultures); IMDM™ 1x + GlutaMAX™ (Iscove’s modified Dulbecco’s containing + 25 mM HEPES + 3.024 g/L Sodium Bicarbonate) (GIBCO, Life Technologies, CA, USA) culture medium supplemented with 10% heat inactivated human AB + serum as described [32]; and AIM-V 1x (+ l-Glutamine + 50 ug/ml Streptomycin Sulfate + 10 ug/mL Gentamicin Sulfate) (GIBCO, Life Technologies, CA, USA) culture medium supplemented with 10% heat inactivated human AB + serum as described [27]. In general, 25 μL of packed iRBCs (filtered, filter-retained or washed) were re-suspended in a 1.8 ml sterile Eppendorf™ tube containing 475 μL of complete culture media supplemented with either 25% serum alone or 20% in combination with 5% CDLC, previously brought to 37 °C in an incubator to obtain a 5% HCT final cell suspension. Each iRBC cell suspension tube was then seeded into one well of a 4-well round Nunc culture plate (Thermo Scientific, USA) (Fig. 1). For the co-cultivation experiment Aotus P. vivax iRBCs were mixed in a 50:50 proportion with malaria-naïve Aotus RBCs or hRBCs and then brought to 5% HTC concentration in McCoy’s5A medium supplemented with 25% sera as described above. Plates were incubated at 37 °C in a gas chamber under a 5% O₂ + 5% CO₂ and 90% N₂ gas atmosphere in a Thermo^® incubator in static conditions. Culture media was replaced at 24 h.

Parasitaemias were followed up through one cycle of maturation. Thin smears were prepared at 0, 24 and 48 h and in three donor animals up to 72 h of incubation, stained with Giemsa for the determination of  % parasitaemia and parasite stages differential proportions (% parasitaemia = number of iRBCs in 5000 RBCs × 100%). At the end of the 48–72 h of incubation, cultures were harvested, and samples were preserved in Trizol and IFA slides prepared for a parallel experiment.

Survival of ex vivo cultures was defined as cultures with any microscopic detectable parasitaemia through one cycle of maturation (48–72 h of incubation).

In order to determine the optimal serum concentration for the survival of P. vivax ex vivo culture in the reference group (McCoy’s5A complete + serum), washed iRBCs obtained from Aotus donor 22033 were cultured with McCoy’s5A media supplemented with either 25 or 50% serum alone in triplicate as described above, determining its parasitaemias at 0, 24 and 48 h of incubation.

Plotting and parametric and non-parametric statistical analysis were performed using the Prism 6 for Mac OS x (Graph Pad^® Software, San Diego, CA, USA).

The observation that Plasmodipur^® filter-retained iRBCS (retained) ex vivo cultures, survived longer than filtered iRBCs during attempts to grow Aotus-derived P. vivax parasites in vitro, prompted the design of an experiment to assess the effect of filtration on the survival of filtered versus retained P. vivax SAL-1 Aotus-derived iRBCs ex vivo cultures, maintained with McCoy’s5A culture medium supplemented with either 25% serum alone or 20% in combination with 5% CDLC, determining their endpoint parasitaemias at 48 or 72 h of incubation (Fig. 1). Ten Aotus P. vivax-infected blood donors, bled at peak parasitaemia (Mean = 101.46 × 10³ × µL), were used in the experiment (Additional file 1: Table S1).

First, to determine the optimal serum concentration for culturing P. vivax Aotus derived iRBCs, washed iRBCs,obtained from donor Aotus monkey MN22033 were maintained with McCoy’s5A medium supplemented with either 25 or 50% serum in three technical replicates for 48 h of incubation as described above. As shown on Additional file 2: Fig. S1, cultures maintained with 25% serum remained viable up to 48 h of incubation while those cultures maintained with media supplemented with 50% serum did not. Hence, 25% serum concentration was chosen for the reference group.

Analysis of the survival proportions of Plasmodipur^® filtered and filter-retained ex vivo cultures at 48 h of incubation (Figs. 2, 3), detected a statistically significant difference in survival proportions between the filtered lipid (5/10) and the retained lipid (9/10) ex vivo cultures (p = 0.0255; one-tailed Chi square) (Fig. 3b), and a borderline significant difference between the filtered lipid (5/10) and retained serum (8/10) cultures (p = 0.0798; one-tailed Chi square), and filtered serum (6/10) and retained lipid (9/10) (p = 0.0607; one-tailed Chi square), suggesting that filtration was detrimental for the survival of ex vivo cultures independently of their media supplementation status. Similarly, no significant differences in the proportion of surviving cultures was detected between the retained serum (8/10) and retained lipid (9/10) ex vivo cultures (p = 0.2556; one-tailed Chi square) (Figs. 2 and 3b), indicating that filter-retained ex vivo cultures survived in higher proportions independently of their media supplementation status (Table 1). Taking together, this experiment demonstrates that filtration of leukocytes is not essential but appears deleterious for the survival of P. vivax Aotus-derived ex vivo cultures.

To further characterize these findings, an analysis of the parasite maturation cycle of the ex vivo cultures was carried out, determining their ring ratios or proportion of rings to mature stages (ring ratio = rings/(trophozoites, schizonts and gametocytes) for each of the 10 P. vivax Aotus monkeys derived ex vivo cultures (Figs. 2, 3 and Table 1). This time, after 48 h of incubation, a statistically significant difference in the survival proportion of ex vivo cultures was observed in the retained lipid cultures compared to the filtered lipid (Fig. 3b) (p = 0.0255; one-tailed Chi square test), while there was no statistically significant difference between the retained serum and filtered serum cultures (p = 0.1646; one-tailed Chi square test). When the ring ratios and  % parasitaemia of the different treatment groups was analysed at time 0, 24 and 48 h of incubation, as shown in Fig. 3c, d, the parasites in the retained serum and retained lipid ex vivo cultures appeared cycling as evidenced by a higher relative proportion of rings (35–40%) (ring ratios: 0.67 and 0.54, respectively) observed in these two groups; this compared to only 13% in the filtered serum and 28% in the filtered lipid ex vivo cultures (ring ratios: 0.15 and 0.39, respectively) (p = 0.0004; one-tailed Chi square test) (Fig. 3b, c and Table 1), indicating that cycling in the retained cultures were maintained in a steady state as indicated by their constant rings ratios, which suffered little variation from baseline, compared to the filtered cultures that had a substantial drop by 48 h of incubation. Likewise, the mean parasitaemias in the retained lipid ex vivo cultures were significantly different compared to the filtered sera group at 24 h of incubation (p = 0.0218; unpaired two-tailed t test), but not at 48 h (Fig. 3d); suggesting that the parasites in the filtered sera cultures either had stalled (stop cycling) or failed at re-invading, as exemplified by the parasite bizarre forms compatible with failed re-invasions, observed attached but growing outside of RBCs at 48 h of incubation in washed only ex vivo cultures supplemented with 25% serum alone (Fig. 4, panels o-p), or were affected as shown in partially ruptured schizonts (Fig. 4, panels g, h, j). Strikingly, as shown in Fig. 5, by 72 h of incubation both the filtered sera and filtered lipid cultures of Aotus donors 25036 and 30016 were dead, but the retained serum and retained lipid cultures continued cycling, as evidenced by the higher proportion of schizonts observed in these groups (Fig. 5). In contrast, the parasites in the washed ex vivo cultures derived from Aotus 28002 supplemented with 25% serum alone (Fig. 5), were all dead by 72 h of incubation. While, those supplemented with sera plus GlutaMax™, serum plus CDLC and serum plus CDLC and GlutaMax appeared cycling up to 72 h of incubation, suggesting a deleterious effect of filtration in Aotus donors 25036 and 30016 ex vivo cultures, but a protective effect of the CDLC supplement, even in the presence of low levels of WBCs. Nevertheless, it cannot be ruled out that the lower starting parasitaemias at time 0 h (< 0.2%) in the filtered cultures derived from Aotus 30016, composed of a higher proportion of rings (ring ratios: 1.17 and 1.08), when compared to the retained cultures (ring ratios: 0.64 and 0.67) (p = 0.0292; one-tailed Chi square test) (Fig. 3c), might have played a role in the complete disappearance of the parasites by 72 h of incubation. Interesting to note is the low frequency of gametocytes present in Aotus blood at time of bleeding, when only 2/10 monkeys were positive for gametocytes (Additional file 1: Table S1), suggesting that the high passage P. vivax SAL-1 Aotus-adapted strain used in this study is a poor producer of gametocytes, as has been reported for squirrel monkeys (Saimiri boliviensis) infected with the Thai 561 strain of P. vivax [50].

Further analysis using parasitaemia fold change as an indicator of survival and cycling as shown in Fig. 3e, revealed that the retained lipid cultures maintained with McCoy’s5A medium supplemented with 20% serum plus CDLC, had the lowest mean negative fold parasitaemia change at 48 h of incubation (Mean ± SEM = -91 ± 79), compared to the filtered serum (Mean ± SEM = − 154 ± 71), filtered lipid (Mean ± SEM = − 161 ± 76), or retained serum cultures (Mean ± SEM = − 157 ± 96) (Additional file 1: Table S2), supporting a protective effect of CDLC supplementation.

It is known that Plasmodium is incapable of de novo biosynthesis of fatty acids and cholesterol and must obtain it from the host [33], and that malaria parasites have a high demand for lipids to support their replication and enlarge their parasitophorous vacuole membrane (PVM) [38]. Hence, an experiment was design to test the effect of McCoy’s5A medium supplemented with 20% serum alone or in combination with 5% CDLC or GlutaMAX™ (a stabilized form of l-glutamine that prevents degradation and ammonia building in culture) or both, benchmarked against IMDM™ and AIM-V™ media; both that had been reported to be superior to McCoy’s5A [21, 28, 32] over parasitaemia levels of P. vivax Aotus-washed ex vivo cultures.

For this purpose, washed P. vivax Aotus iRBCs ex vivo cultures were maintained with McCoy’s5A modified, IMDM™ or AIM-V™ culture media as delineated in Fig. 1, determining their mean parasitaemia densities at 0, 24 and 48 h of incubation. After normalization of starting parasitaemias at time 0 h, by 24 h of incubation parasitaemia was significantly higher in the IMDM™ media group (p < 0.003; Mann–Whitney, two-tailed t-test), compared to McCoy’s5A medium supplemented with serum alone (reference group) or in combination with GlutaMAX™ (p < 0.004; Mann–Whitney, two-tailed t-test), but not with McCoy’s5A medium supplemented with serum plus CDLC alone or in combination with GlutaMAX™ (p = 0.1528; Mann–Whitney, two-tailed t-test), nor with the AIM-V™ culture medium group.

By 48 h of incubation, parasitaemia was still significantly higher in the IMDM™ group, compared to McCoy’s5A supplemented with serum (reference group) (p = 0.006; Mann–Whitney, two-tailed t-test) or sera plus GlutaMAX™ (p = 0.02; Mann–Whitney, two-tailed t-test), but not with McCoy’s5A medium supplemented with serum plus CDLC and GlutaMAX™ (p = 0.3387; Mann–Whitney, two-tailed t-test), nor with the AIM-V™ medium (Fig. 6).

In general, an additive effect pattern of McCoy’s5A medium supplementation emerged as follows: serum < serum + GlutaMAX™ < serum + CDLC < serum + CDLC + GlutaMAX™ (Fig. 6), indicating the superiority of McCoy’s5A medium supplemented with serum in combination with CDLC and GlutaMAX™ over serum alone, and no significant difference in survival of parasites compared to IMDM™ and AIM-V™ media [27, 32], although substantial variation in parasitaemia densities was observed in both the IMDM™ and AIM-V™ media ex vivo culture groups.

Next, to test the effect of co-cultivation with hRBCs on the survival of Aotus-derived P. vivax ex vivo cultures, Aotus iRBCs were co-cultivated with malaria-naïve Aotus or hRBCs in a 50:50 proportion at 5% HTC concentration, and maintained with McCoy’s5A medium supplemented with 25% sera. This time, by 24 h of incubation, parasitaemia densities in the hRBCs co-cultivated ex vivo cultures were significantly higher than in the Aotus blood-only supplemented cultures (p < 0.0295; Mann–Whitney U non-parametric one-tail test) (Fig. 7), with re-invasions observed in both cultures at 48 h of incubation (Fig. 8), though there was only a borderline statistically significant difference in parasitaemia densities between groups (p = 0.0530; Mann–Whitney U non-parametric one-tail test), suggesting that co-cultivation with hRBCs improved the survival of P. vivax Aotus-derived iRBCs ex vivo cultures.