**3. Results**

#### **3.1 Morphological characteristics of feline intestinal cells** *in vitro*

The attachment of FIECs to the substrate was observed by phase-contrast microscopy, in situ. In 5 days, the cells aligned and polarized, with the nuclei located in the same plane as the organization of the columnar epithelium (**Figure 1A**). Ultrastructural analysis by scanning electron microscopy showed the absorptive characteristics of these cells, including the identification of plasma membrane projections that established focal adhesion points (**Figure 1B**). Long and thin finger-like projections were visible and often established cell-cell contacts and extensive cytoplasmic contacts, indicating the formation of specialized membrane areas, such as cellular junctions (**Figure 1B**). Transmission electron microscopy demonstrated that epithelial cells in culture retained a great number of cytological features typical of intestinal epithelial cells, such as large numbers of microvilli (i.e., a brush border at the apical pole) (**Figure 1C**–**D**). Lateral interdigitations are observed below the junctional complex between two adjacent epithelial cells (**Figure 1C**). The junctional areas presented tight junctions (zonula occludens) in which the outer leaflets of the plasma membranes were fused, intermediate junctions (zonula adherens), characterized by plasma membranes separated by a space, and desmosomes (macula adherens) (**Figure 1D**). All these characteristics confirmed the intestinal epithelial nature of FIECs as enterocytes that were maintained for up to six passages.

#### **3.2 Expression of intestinal markers in FIEC**

To confirm the epithelial nature of FIEC, we investigated the intermediate filaments by employing an anti-pan-cytokeratin antibody that recognizes a range of cytokeratins (1, 5, 6, 8, and 10). Confocal laser scanning microscopy showed that secondary cultures

#### **Figure 1.**

*Morphological characterization of cells isolated from felid small intestines. (A) Cells with 24 h of cultivation showed epithelial characteristics, such as alignment and polarization. The nuclei of the cells are located in the same plane, similar to the organization of the intestinal epithelium. (B) Scanning electron microscopy revealed thin and long cytoplasmic projections and focal points of adhesion to the substrate (thin arrows). Cell-cell contact areas form specialized contact points, such as intercellular junctions (thick arrows). (C-D) By transmission electron microscopy, many microvilli (mv) are observed at the apical pole. Junctional complex areas (ZO = zonula occludens; ZA = zonula adherens; D = desmosomes) are located at interdigitations. Bars: (A) 20 μm; (B) 5 μm; (C) 0.5 μm; (D) 0.2 μm.*

of FIECs preserved the morphological and functional characteristics of immature enterocytes. These cells sustained strong expression of cytokeratin concentrated around the nuclei after two weeks, indicating they were truly epithelial (**Figure 2A**–**D**). Double staining by phalloidin-FITC to identify actin filaments and the anti-cytokeratin antibody revealed little to no co-localization between these proteins (**Figure 2A**). The localization of actin filaments was mostly observed at focal adhesion points for the substrate at the cellular membrane (**Figure 2A**–**G**). The functional properties of FIECs were evaluated based on the expression of intestinal alkaline phosphatase, which is an enzyme secreted by the intestinal epithelium (**Figure 2B**–**D**). Intestinal alkaline phosphatase expression was initially detected after 5 days of culture (**Figure 2B**). The labeling showed a progressive increase in the enzyme concentration inside the cells, which occurred between 7 and 9 days post-cultivation (**Figure 2C**–**D**). The immunocytochemistry assays targeting vimentin and desmin failed in the FIECs until up to 15 days in secondary culture (data not shown), as expected for healthy intestinal cells.
