Fig. 3

Characterizing Plasmodium developmental phenotypes in the primary human hepatocyte (PHH) culture system. a A total of ten cryopreserved PHH donor lots were screened for infectability and liver stage (LS) development by cryopreserved P. vivax and P. falciparum sporozoites in comparison to donors PDC and differentiated HepaRG line. Four donors (lot H, J, P, and Q) were significantly higher in percent of LS parasites relative to PDC while two donors (lot F and N) showed no LS parasite growth. Alternatively, specific donors appear to only be susceptible to a single Plasmodium species (lot G and P to P. vivax, lot H and I to P. falciparum). b Plasmodium sporozoites were added to PHHs at 5000 sporozoites per well with P. vivax field isolates showing highest LS development rates between 2 and 8.3%, P. falciparum NF54-WT between 0.6 and 2%, and P. falciparum NF54-GFP between 0.04 and 0.4%. c On day 6 of LS development, P. falciparum has a relatively synchronized growth with a large parasite population at a mean size of >100 µm². d, e P. vivax LS parasites have a larger distribution of size from day 6 to day 8 (d) with a consistent hypnozoite (~60%) to developing schizont (~40%) ratio in the total population (e). f, g Representation of a single 384-well view showing day 6 P. falciparum LS schizonts stained with anti-GAPDH (left) and day 8 P. vivax developing LS schizonts and dormant hypnozoites stained with anti-rUIS4 (right) imaged on the Operetta HCI system, Perkin Elmer. Graph bars represent means with s.d. from an independent experimental replicate (n = 3, a) or for biological replicates (n = 3) with experimental replicates (n = 3, b). Statistical significance was determined using a two-way ANOVA followed by Dunnett’s multiple comparisons to PDC (a, b) where significance is represented by P < 0.05 (*) and P < 0.0001 (****). Plasmodium spp. mean parasite size distribution (c–e) was calculated from biological replicates (n = 3) with experimental replicates (n = 26, P. vivax) or (n = 8, P. falciparum). Gray scale bars represent 10 µm