Table 1.
Information on the 19 norovirus strains used in the study.
Figure 1.
Formation of a uniform monolayer around the collagen beads coated with Int-407 cells.
Cells were removed from an STLV after 7 days of culture in GTSF-2 medium at 37°C and the 3D structures visualized by light microscopy (1∶40 magnification).
Figure 2.
Electron micrographs of microvilli development in Caco-2 cells during a 5-week growth period.
Caco-2 cells were cultured in GTSF-2 medium for up to 5 weeks at 37°C. At 2, 3 and 5 weeks of culture, under either 2D, or 3D conditions in a rotating wall vessel, cells were removed and processed for visualization under electron microscope. Top Panel [A (1200x), B (440x), C (1400x)]: 3D structures Lower Panel [D (690x), E (890x), F (690x)]: 2D structures.
Figure 3.
Immunohistochemical imaging of differentiation markers in Int-407 and Caco-2 cells.
Image A. β-catenin localization in three-dimensional structures of Caco-2 cells after 3 weeks in culture in STLV (1∶40). Image B. Localization of ZO-1 in 3D of Int-407 cells after 4 weeks of culture in STLV and HARV (1∶40). Image C. Distribution of claudin in 3D of Caco-2 cells after 3 weeks in culture in STLV (cell nuclei were stained with DAPI, 1∶40 magnification).
Figure 4.
Morphology of 3D Int-407 cells after inoculation with norovirus.
Images A and B: control Int-407 cells (left) and (right) Int-407 cells inoculated with GII.4 norovirus at 48 h post inoculation (light microscopy, 1∶20 magnification).
Figure 5.
No evidence of Norovirus replication in 3D Caco-2 cells.
No significant increase of RNA copy number was detected in supernatants of Caco-2 cells harvested at 0, 24 and 48 hours post inoculation. Different norovirus strains and different virus concentrations were evaluated. Viral RNA was detected by quantitative reverse transcription-realtime RT-PCR. Inoculation of 3 different concentrations (MOI 1, 10, 100) of: A) GII.4 (#19 in table 1) on 3D Caco-2 cultures (Lab B) and B) GI.3 strain (#18 in table 1) on 3D Caco-2 cultures (Lab B). C) Comparison of 2D and 3D Caco-2 cells by GI.3 (#7 in table 1) and GII.4 (#6 in table 1) (Lab A).
Table 2.
No evidence of Norovirus replication in 3D Int-407 cells.