Figure 3: Establishment of liver stage malaria infection by two rodent Plasmodium species in p-HEALs in vitro.

(A) Hepatocyte‐specific infection of p‐HEALs with Pb‐GFP-luc in vitro. PEG cryogels seeded either with HEP + FIB (p-HEAL) or FIB only (control) were exposed to Pb‐GFP-luc spz and imaged by bioluminescence imaging (BLI) at 48 h post-infection (3 wells per condition are shown). Each red circle demarcates the region of interest (ROI) that is defined for the quantification of the Pb-GFP-luc-derived BLI signal, as determined by the physical extent of a PEG cryogel. (B) BLI quantification of Pb‐GFP-luc infection in p-HEALs over time. n = 3. (C) Representative confocal images of Pb-GFP-luc (green) EEFs in p‐HEALs at 48 h post-infection. DAPI nuclear staining (blue) is shown either alone (right) or overlayed with GFP (left). A GFP-positive EEF (white dotted line) is observed adjacent to a HEP nucleus (white arrow). Arrowheads denote FIB nuclei. (D) Coculture of HEPs with FIBs maintains hepatocyte infectibility with Pb‐GFP-luc over time better than monoculture. PEG cryogels were seeded with HEP + FIB cocultures or HEP only monocultures and exposed to Pb-GFP-luc spz at 2 or 7 days after cell seeding. BLI quantification of Pb‐GFP-luc infection at 48 h post-infection is shown here. n = 3. (E) Dose response of Pb-GFP-luc-infected p-HEALs to antimalarial primaquine at 48 h post-infection. Primaquine treatment started at 3 h post-infection. **p < 0.01, ***p < 0.001, One way ANOVA (p = 0.0009) with Tukey’s multiple comparison test. n = 3. (F) Representative BLI images of Pb-GFP-luc-infected p-HEALs treated with various primaquine doses at 48 h post-infection (only 1 well per primaquine dose is shown here). Red circles demarcate the ROI that was quantified in (E). (G) Contribution of FIB density-driven heterotypic HEP/FIB interactions on infectibility of p-HEALs with an alternate Plasmodium species. At 2 days post-seeding, PEG cryogels seeded with varying HEP:FIB ratios were exposed to Py-luc spz, which is more restrictive in its host cell targeting, and imaged by BLI at 48 h post-infection. *p < 0.05, One way ANOVA (p = 0.0098) with Tukey’s multiple comparison test. n = 3. Error bars represent SEM. Scale bars: 10 μm.