doi: 10.3967/bes2018.016
Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections
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Abstract:
Objective Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. Methods A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV. -
Key words:
- 3D cell culture /
- Human airway epithelium (HAE) /
- Human rhinovirus C /
- Human bocavirus /
- Propagation
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Figure 3. Signature protein expressions of organotypic tissue in 3D cultured HAE cells. The 2D and 3D cultured cells were immunostained with anti-cytokeratin 5 (A), anti-ZO-1 (B), anti-pan-cytokeratin (C) and anti-β-tublin (D), and the nuclei were counterstained with DAPI. The representative confocal images are shown (×10).
Figure 6. Cytokine secretion measured in the 2D and 3D cultured HAE cells after infection with HRV-C. TNF-α (A), IL-6 (B), IL-8 (C), and IP-10 (D) levels in the media of HAE cultures at the indicated day after infection were measured by ELISA assay. Each data point represents the mean of three independent experiments at least.
Figure 7. Cytokine secretion measured in the 2D and 3D cultured HAE cells after infection with HBoV. TNF-α (A), IL-6 (B), IL-8 (C), and IP-10 (D) levels in the media of HAE cultures at the indicated day after infection were measured by ELISA assay. Each data point represents the mean of three independent experiments at least.
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