Development of an in vitro media perfusion model of Leishmania major macrophage infection

Abstract

Background In vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites. Methods Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360μl/min, L . major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured. Results Mean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10 −9 m/s and 1.23 x 10 −7 m/s for cells at the base of the chamber and cells on an insert, respectively. L . major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran. Conclusions Perfusion of culture medium in an in vitro L . major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds.

Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs

“…The activities of amphotericin B ( Figure 1 a), miltefosine ( Figure 1 b), paromomycin sulphate ( Figure 1 c) and sodium stibogluconate ( Figure 1 d) in the PEM– L. major amastigote model were determined at three different culture media flow rates, (i) static (0 m/s), (ii) low flow (1.45 × 10 −9 m/s; cells placed at bottom of well) and (iii) high flow (1.23 × 10 −7 m/s; cells placed on top of inserts) [ 42 ]. EC 50 and all measurable EC 90 values were altered by varying amounts as the speed of media perfusion was increased ( Table 2 ).…”

“…The influence of perfusion of the culture medium on the infection levels of Leishmania was investigated using the QV900 media perfusion system (Kirkstall LTD, York, UK) developed and detailed in elsewhere [ 42 ]. The method for the static infection of PEMs seeded on 12 mm glass cover slips and their transfer to the QV900 is also described in O’Keeffe et al [ 42 ].…”

“…The influence of perfusion of the culture medium on the infection levels of Leishmania was investigated using the QV900 media perfusion system (Kirkstall LTD, York, UK) developed and detailed in elsewhere [ 42 ]. The method for the static infection of PEMs seeded on 12 mm glass cover slips and their transfer to the QV900 is also described in O’Keeffe et al [ 42 ]. Briefly, PEMS are seeded onto glass coverslips and infected with promastigotes in static conditions for 24 h. Subsequently two thirds of the glass coverslips containing infected macrophages were transferred to the media perfusion system and maintained under flow conditions either at the bottom of the well or on top of an insert, at a flow speed of 360 μL/min for 72 h in a 34 °C, 5% CO 2 incubator.…”

“…The remaining coverslips were used for the static control, with macrophages maintained in the same culture medium without flow. This creates three different culture media flow rates, (i) static (0 m/s), (ii) low flow (1.45 × 10 −9 m/s; cells placed at bottom of well) and (iii) high flow (1.23 × 10 −7 m/s; cells placed on top of inserts) [ 42 ]. After the initial 24 h of static infection, the media used for the activity of drug studies was supplemented with drug (miltefosine at 20 μM, 5 μM or 1.25 μM, amphotericin B (AmB) at 200 nM, 50 nM or 12.5 nM, sodium stibogluconate at 600 μg, 200 μg or 60 μg of Sb V /mL or paromomycin sulphate salt at 300 μM, 100 μM or 30 μM).…”

Activity of Chitosan and Its Derivatives against Leishmania major and Leishmania mexicana In Vitro

“…After each time point, the slides were examined as described above for the assay of chitosan activity against intracellular amastigotes of L. major and L. mexicana. The inhibition activity of the uptake (phagocytosis or pinocytosis) of the two inhibitors was evaluated on a fluorescence plate reader using fluorescent latex beads and pHrodo red dextran (72). We showed that cytochalasin caused 94 and 84% inhibition of phagocytosis of fluorescent latex beads (Sigma-Aldrich, UK) after 4 h and 24 h, respectively, and dynasore caused 95 and 90% inhibition of pinocytosis of pHrodo red dextran (M w ϭ 10,000; Thermo Fisher, UK) after 4 h and 24 h, respectively ( Table S6 in the supplemental material).…”