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3R-Project 118-10

Engineering of an in vitro hepatocyte tissue system for malaria liver infection research

Dalu Mancama and Claire Rossouw
Systems Biology, Biosciences Unit, CSIR, Pretoria 0184, South Africa
dmancama@csir.co.za,, crossouw@csir.co.za

Keywords: human; hepatocyte; parasites; infectious diseases; malaria; reduction; replacement; infectiosity

Duration: 1 year Project Completion: 2011

Background and Aim
Malaria continues to be an important global infectious disease for which mortality levels remain unacceptably high. The illness is caused by the Plasmodium parasite, most often Plasmodium falciparum, which is transmitted to humans in sporozoite form by infected Anopheles mosquitoes. Drug therapy represents an important intervention in controlling the disease. However the parasite has become increasingly resistant to treatment with the current line of available drugs. In certain regions tolerance is now exhibited even to artemisinin based therapy (Wongsrichanalai et al., 2008), which is widely considered to be the antimalarial of last defence. There is therefore an increasing urgency to develop new approaches and technologies that speed up the creation of effective new drugs and, ultimately, vaccines against the disease. Despite advances in our understanding of P. falciparum and its intra-erythrocytic development, significantly less is known about the parasite’s development in hepatocytes. A major reason underlying this shortfall has been the unavailability of suitable in vitro models through which to study parasite-host hepatocyte development on a large-scale. Consequently, surrogate animal models based on related parasite species are widely employed for this purpose. Through joint collaboration between the CSIR Biosciences and Material Sciences and Manufacturing units, work has been initiated to engineer a novel 3D polymer-based hepatocyte tissue culture system. This development is driven by a need to create new in vitro systems that correlate more closely with the in vivo liver state, providing a more reliable predictive model for research, in particular for investigating parasite-host interaction. Through the successful establishment of such a system (3), we envisage the possibility of perfomring detailed molecular investigations of P. falciparum hepatocyte development, leading to new opportunities in drug and vaccine discovery.

Method and Results
Through the use of conventional non-woven polymers and smart polymer technology (3), it has been possible to develop a proprietary three-dimensional scaffold system that is capable of supporting the growth of hepatocyte (and other anchorage dependent) cells in an enhanced manner. In particular, the system preserves proteins and glycans expressed on the extracellular matrix that are critical, together with other factors, to facilitating malaria sporozoite invasion and development. Primary hepatocyte cell lines (e.g. hNHEPS and HC04 (Sattabongkot et al., 2006)) will be employed to facilitate infection by Plasmodium falciparum sporozoites that have been isolated from Anopheles mosquito hosts. Conditions will be optimized throughout to achieve maximum rates of infection and development, culminating in the assessment of merozoite viability by exposing mature merozoites from the new system to conventional red blood cell cultures. Successful merozoite invasion of red blood cells, and subsequent intra-erythtrocytic development in these cells, will confirm the validity of the new in vitro model.

Conclusions and Relevance for 3R
This project aims to develop a 3D-based hepatocyte culture system that facilitates large-scale molecular analysis of the human form of malaria, P. falciparum, in this tissue. To date, this research has largely relied on the use of surrogate animal models, that utilize related species of this parasite. New opportunities in malaria drug and vaccine discovery research are anticipated from the new system.

References
1. Wongsrichanalai C and Meshnick SR. Declining Artesunate-Mefloquine Efficacy against Falciparum Malaria on the Cambodia–Thailand Border (2008) Emerging Infectious Diseases 14(5): 716-719.

2. Sattabongkot J, Yimamnuaychoke N, Leelaudomlipi, S, Rasameesoraj, M, Jenwithisuk, R, Coleman, Russell, Udomsangpetch, R, Cui, L, Brewer, T. Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax (2006) Am J Trop Med Hyg 74: 708-715.

3. Claire L. Rossouw , Avashnee Chetty, Francis Sean Moolman, Lyn-Marie Birkholtz, Heinrich Hoppe, Dalu T. Mancama, Thermo-responsive non-woven scaffolds for “smart” 3D cell culture (2012). Biotechnology and Bioengineering, Vol. 109/8, 2147–2158, doi:10.1002/bit.24469.

Figures

Figure 1
Figure 1: Electron micrograph of differentiated HepG2 hepatocytes grown on the 3D scaffold surrounded by extracellular matrix.

Figure 2
Fluorescence confocal image at 400X magnification illustrating differentiated HepG2 hepatocyte cell viability grown on the 3D scaffold following fluorescein diacetate staining.

Figure 2
Figure 2



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