Abstract
BackgroundIn 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.MethodsLeishmania 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.ResultsMean 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.ConclusionsPerfusion 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.
Highlights
Traditional cell culture methods typically rely on either immortalized cell lines or primary isolated cells grown in designed nutritious media on non-physiological substrates, such as functionalized plastic and glass
Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo Quasi Vivo 900 media perfusion system (QV900) macro-perfusion system
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
Summary
Traditional cell culture methods typically rely on either immortalized cell lines or primary isolated cells grown in designed nutritious media on non-physiological substrates, such as functionalized plastic and glass. These methods have been at the core of in vitro studies on many basic biological processes, they provide a limited platform owing to both their inadequate representation of key physiological characteristics and their relevance to disease models [1]. One area that is often overlooked in cell culture models is the transport and movement of nutrients around cells, which occurs through fluid flow in the body This could impact on the growth and survival of pathogens in intracellular models as infection is reliant on nutrients provided by the host cell and cell-cell interactions. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites
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