Figure 1: MPCCs Can Be infected With Clinical P. vivax Isolates.

(A) Schematic of the MPCC system where primary human hepatocyte (PHH) islands are patterned in 96-well plates. Sporozoites are overlaid onto cultures 1 day post seeding and incubated for 3 hours, followed by addition of mouse fibroblasts.

(B) Size distribution histograms of P. vivax EEFs in the MPCC system are shown after infection with 2 clinical isolates, performed in two separate experiments. VK247 experiment: day 5, 310 parasites; day 8, 199 parasites; day 18, 49 parasites. VK210 experiment: day 5, 207 parasites; day 8, 179 parasites; day 18, 41 parasites. At least 3 wells per time point pooled. See also Figure S1B.

(C) Representative images of small and large parasites on days 5 and 8, stained with an anti-MSP1 antibody. Scale bar, 5μm.

(D) Representative images of merozoite release in live P. vivax (VK210) cultures on day 12 (observed in five out of five wells). Released merosomes (white arrows) and merozoite-releasing merosome are shown (black arrow). Inset shows close up view of two merosomes on the same day. Scale bars, 50μm. See also Figure S1A and Supplementary Video 1–3.

(E) Representative images of P. vivax infected reticulocytes. Reticulocyte-enriched red blood cells were overlaid onto P. vivax-infected MPCCs, 10 days post-infection. Giemsa staining of collected blood cells revealed ring-stage parasites, one day later. Experiment performed 3 times by adding blood cells to at least 6 infected wells, all of which became positive for blood stage infection as assessed by blood smear. (6/1018, 10/1070 and 4/995 infected red blood cells counted) Scale bars, 5μm.

(F) P. vivax EEFs from MPCC cultures were fixed on days 5 and 8. Antibodies against UIS4, BIP, HSP60 and H3K9Ac were used to visualize parasite structures. Day 8 structures were further characterized with antibodies against HSP70, MIF and ACP. A representative small and large form is shown for all day 8 proteins. Scale bars, 5μm. See also Figures S1C and S2.