SUMMARY The Plasmodium liver stage is an attractive target for the development of anti-malarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable and reproducible in vitro liver stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum including the release of infected merozoites and infection of overlaid erythrocytes, and also the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput anti-malarial drug screening and vaccine development.

Studying human hepatotropic pathogens such as hepatitis B and C viruses and malaria will be necessary for understanding host-pathogen interactions, and developing therapy and prophylaxis. Unfortunately, existing in vitro liver models typically employ either cell lines that exhibit aberrant physiology, or primary human hepatocytes in culture configurations wherein they rapidly lose their hepatic functional phenotype. Stable, robust, and reliable in vitro primary human hepatocyte models are needed as platforms for infectious disease applications. For this purpose, we describe the application of micropatterned co-cultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive cells. Using this system, we demonstrate how to recapitulate in vitro liver infection by the hepatitis B and C viruses and Plasmodium pathogens. In turn, the MPCC platform can be used to uncover aspects of host-pathogen interactions, and has the potential to be used for medium-throughput drug screening and vaccine development.

SummaryMalaria eradication is a major goal in public health but is challenged by relapsing malaria species, expanding drug resistance, and the influence of host genetics on antimalarial drug efficacy. To overcome these hurdles, it is imperative to establish in vitro assays of liver-stage malaria for drug testing. Induced pluripotent stem cells (iPSC) potentially allow the assessment of donor-specific drug responses, and iPSC-derived hepatocyte-like cells (iHLCs) can facilitate the study of host genetics on host-pathogen interactions and the discovery of novel targets for antimalarial drug development. We establish in vitro liver-stage malaria infections in iHLCs using P. berghei, P. yoelii, P. falciparum, and P. vivax and show that differentiating cells acquire permissiveness to malaria infection at the hepatoblast stage. We also characterize antimalarial drug metabolism capabilities of iHLCs using prototypical antimalarial drugs and demonstrate that chemical maturation of iHLCs can improve their potential for antimalarial drug testing applications.

The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.

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