Influenza A virus (IAV) claims ∼250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (two-dimensional [2D] cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineered Lung Model (3D-HTLM), we describe the 3D culture of primary human small airway epithelial cells (HSAEpCs) and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2. The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.

The pulmonary epithelium is divided into upper, lower, and alveolar (or small) airway epithelia and acts as the mechanical and immunological barrier between the external environment and the underlying submucosa. Of these, the small airway epithelium is the principal area of gas exchange and has high immunological activity, making it a major area of cell biology, immunology, and pharmaceutical research. As animal models do not faithfully represent the human pulmonary system and ex vivo human lung samples have reliability and availability issues, cell lines, and primary cells are widely used as small airway epithelial models. In vitro, these cells are mostly cultured as monolayers (2-dimensional cultures), either media submerged or at air-liquid interface. However, these 2-dimensional cultures lack a three dimension-a scaffolding extracellular matrix, which establishes the intercellular network in the in vivo airway epithelium. Therefore, 3-dimensional cell culture is currently a major area of development, where cells are cultured in a matrix or are cultured in a manner that they develop ECM-like scaffolds between them, thus mimicking the in vivo phenotype more faithfully. This review focuses on the commonly used small airway epithelial cells, their 2-dimensional and 3-dimensional culture techniques, and their comparative phenotype when cultured under these systems.

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Introduction Monocyte chemoattractant protein-1 (MCP-1) is a bioactive molecule that is expressed in significant amounts in all stages of atherosclerosis. The role of MCP-1 in this disease is to recruit monocytes across the endothelium and into the arterial tissue. Eventually, the monocytes differentiate into cholesterol-engorged macrophages called “foam cells” that result in atherosclerotic plaque formation. The mechanism that MCP-1 uses to mediate monocyte transendothelial migration is believed to be via its concentration gradient. However, the formation of the MCP-1 concentration gradient in the extracellular matrix is still poorly understood. Methods A 3D in vitro vascular tissue model has been developed to study the cellular mechanisms involved in the early stages of atherosclerosis. In the present study, a mathematical model is used to determine the gradient of MCP-1 in the collagen matrix of the 3D in vitro vascular tissue model. Experiments were performed to investigate the stability of MCP-1 and the interaction between MCP-1 and the collagen matrix. Results and Conclusions MCP-1 is stable for at least 24 hours under experimental conditions and MCP-1 interacts with the collagen matrix. The diffusion coefficient for the transport of MCP-1 in the collagen matrix and the rate constant for the binding of MCP-1 to collagen were determined to be 0.108 mm2 hr−1 and 0.858 hr−1, respectively. Numerical results from the model indicate that the concentration gradients of both soluble and matrix-bound (or static) MCP-1 are formed inside the collagen matrix.