Abstract
Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.
Highlights
Formation of a functional epithelial barrier was evaluated by measuring Transepithelial Electrical Resistance (TEER) from epithelial monocultures and co-cultures on Transwell® inserts and collagen and hyaluronic acid bilayered (CHyA-B) scaffold (Figure S1)
Fibroblast addition to Collagen and Hyaluronic Acid (CHyA)-B scaffolds led to an increase in TEER values from 830 ± 20.83 Ω cm2 in epithelial monocultures to 912 ± 75.94 Ω cm2 in epithelial co-cultures but this was not statistically significant (Figure 1B and Figure S1B)
A polarised and functional epithelial barrier was formed in both Transwell® inserts and CHyA-B scaffolds with similar values to those previously reported and the presence of fibroblasts in co-cultures appeared to improve epithelial barrier integrity [13]
Summary
Several factors hamper the progression of novel respiratory treatments into clinical applications including poor understanding of disease pathophysiology and the lack of proper in vitro models to assess drug efficacy and safety early in the development process. The majority of pre-clinical in vitro models for drug assessment fail to recapitulate the complex physiology of the upper respiratory tract by not including relevant airway cell types as well as by not mimicking the three-dimensional (3D) environment in which respiratory tissues are located. There is an urgent need to develop more accurate and complex 3D models of the airway to be used as tools during drug discovery to augment clinical translation of novel therapies for respiratory medicine
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