Using the EpiAlveolar Lung Model to Predict Multiple Carbon Nanotubes' Fibrosis Potential

EpiAlveolar is in vitro An organotypic model of human alveolar tissue consisting of human primary alveolar epithelial cells and lung fibroblasts grown on the apical surface of the Transwell tab and primary pulmonary endothelial cells growing on the basal surface of the tab. Fabrics were cultured in ALI to differentiate between differentiation and simulation in vivoSimilar to aerosol exposure.

The EpiAlveolar fabric model was evaluated for its ability to mimic the characteristics of the alveolar micro environment. in vivo. The alveoli are composed of an extremely thin epithelial layer surrounded by a network of capillaries to exchange gas. To assess whether the overall morphology of EpiAlveolar tissues is similar, the tissue cross is photographed with hematoxylin and eosin (H&E). Figure 1a shows that the EpiAlveolar tissues are about 2-4 cell layers on a thick apical surface, with a thin monolithic of endothelial cells on the basal surface. In addition, the tissue sections were stained with an epithelial marker, cytokeratin 19 (CK19), and a mesenchymal cell marker, Vimentin (VIM) to reveal a layer of fibroblast (apical side) and endothelial cell (stem) alveolar epithelial cells. Lateral) layers (Figure 1b). in vivo The alveolar epithelium consists of two cell types, type I (ATI) and type II (ATII). The presence of both cell types in EpiAlveolar tissues was positively correlated with cytokeratin 19 (CK19), ATI market, and cytokeratin 8 (CK8) with a positive atonic sign (Fig. 1c). The transmitter electron microscopic micrograph confirmed the presence of lamellar bodies (additional Figure 2a), membrane-bound structures containing membrane membranes on the surface membranes. (51) This result was further confirmed by the complete staining of tissues for the expression of prosurfactant C (pSP-c), a critical characteristic of type II alveolar epithelial cells (Figure 1d). In addition, the surface tension of EpiAlveolar tissues, which is directly related to surfactant production, was rated on day 2 (additional figure 2b, c), as previously described in the literature. (52-54) The method is based on the fact that the diameter (D) The deposited drop is reduced by reducing the surface tension and vice versa. EpiAlveolar tissues tested in three biological replicas (N. = 3), while A549 cells served as a test control and were measured only once (N. = 1). The graph (Fig. 2b) shows the relationship between the drop ratio and the surface tension. The surface tension of EpiAlveolar tissues is slightly higher than measured in A549 cells, (52), which may be attributed to the fact that the EpiAlveolar tissue consists of a mixture of alveolar epithelial type I and II cells. Although the measured surface tension is higher than it appears in vivo(54) It is sufficient to maintain a regular shape of the drop (additional figure 2c), which confirms the presence of surfactants on the cell surface. (52,53) A further quantitative indicator of the ratio of cell types, surface tension, and surfactant secretion continues. We have taken into account the existence of different types of alveolar cells and spatial organization, we want to determine whether EpiAlveolar tissues represent a barrier, which is an important function. in vivo Tissue and is not sufficiently developed when using alveolar cell lines, such as A549 cells. (55) The threshold function was assessed by visualization of close connections (visualization 1e) and measurement of transpithelial electrical resistance (TEER) over time. TEER value ≥300 Ω · cm2 It was considered to be based on previous experiments and recommendations of the manufacturer indicating an intact barrier. The mean TEER value of EpiAlveolar tissue was highest in the first week after the end of differentiation, decreased slightly, and then stabilized at approximately 1000 Ω · cm.2 (Figure 1f). The TEER culture remained high for about a month, indicating that the tissue barrier remained intact. This is important because it allows the integrity of the threshold to be assessed in long-term, repeated exposure experiments. Following the characterization of the EpiAlveolar model, the tissues were treated with various therapies, and the endpoints were evaluated in humans in relation to the path of pulmonary fibrosis, as described in the AOP scheme (additional Figure 1).

Figure 1

Figure 1. Characterization of the EpiAlveolar model. (A) Hematoxylin and eosin staining, showing tissue thickness, i.e., 2-3 layers of cell layers on the surface of the apical and thin monolithic cells of endothelial cells on the basal surface. (B) Tissue sections showing the location of the cell inside the tissue, i.e. the layer of alveolar epithelial cells on top of the fibroblast (apical lateral) and endothelial cell (base lateral) layer, the fibroblast (apical side) and endothelial cytokine (stem lateral margin) layers: e. 19 (CK19, red) mesenchymal cell marker vimentin (VIM, green) and DAPI (blue) cell sister nuclei. (C) Alveolar epithelial cell staining: ATI (CK19, red) and ATII cytokatin 8 (CK8, green). (D) prosurfactant C (pSP-C, red) whole wire staining, functional marker of ATII cells and DAPI (blue) dye of cell nuclei. (e) Immunofluorescence staining for the epithelial close connection marker ZO-1 (red) and cell nuclei (DAPI, blue). (F) transferential electrical resistance values ​​within 42 days of tissue development; The data are presented as mean ± standard deviation, N. = 3. Data obtained in the laboratory 1.