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?vivax is underexplored. Based on our ability to establish the liver stage of P.?falciparum in MPCCs and maintain the hepatocyte phenotype for 4�C6?weeks and the observation that some strains of P.?vivax can reactivate over a similar timescale, we next explored check details the feasibility of establishing the liver stages of P.?vivax in MPCCs over time. As for the P.?falciparum experiments, the viability of the cryopreserved P.?vivax sporozoites was evaluated by assessing their gliding motility prior to each experiment ( Figure?6A). We explored several strains of P.?vivax including Chesson, a strain known to efficiently form dormant forms and reactivate at shorter timescales ( Hollingdale et?al., 1986?and?Krotoski et?al., 1986). Cultures were infected and then fixed at various time points and stained for circumsporozoite protein (CSP). Using immunofluorescence microscopy, we analyzed the size distribution and localization of the P.?vivax forms over a 3-week period. We readily observed P.?vivax liver forms in MPCC, including mature, liver-stage schizonts larger than 20?��m found at day 6 postinfection ( Figures 6C, 6B, S4A, and S4B). In addition, a population of smaller P.?vivax CSP (PvCSP)-positive forms (RecBCD S4A�CS4C). The infection efficiency, based on the percentage of hepatocytes containing large and small PvCSP-positive forms at day 6 postinfection, was 0.013% and 2%, respectively ( Figure?S4D). Similar numbers of small forms were observed up to day 21 (data not shown). Importantly, 85% of the small Selleck Antidiabetic Compound Library forms observed were intracellular, based on immunostaining performed before and after permeabilization ( Figure?S4C). In contrast, cultures infected with P.?falciparum contained very few small forms after 15?days in culture, and those were predominantly extracellular ( Figure?S4E). These P.?vivax small forms may represent the dormant hypnozoite stage of the parasite life cycle, which is responsible for clinical relapses in P.?vivax malaria patients; however, further characterization will be required to substantiate this hypothesis. Both small and large forms were observed when other strains of fresh and frozen P.?vivax sporozoites were examined ( Figure?S4A and data not shown). Finally, we demonstrated that the MPCC system can support maturation of hepatic P.?vivax schizonts, based on the detection of the late-stage antigen PvMSP-1 at day 12 postinfection ( Figure?S4B). In this report, we describe an in?vitro cell-based platform that recapitulates the human liver stage of P.?falciparum and P.?vivax infection. Although some attempts to infect cryopreserved human primary hepatocytes have been described in the past ( Meis et?al., 1985?and?Silvie et?al., 2004), this source of hepatocytes has not been routinely adopted by the field to date.