325988 Construction of Human Respiratory Platform for Lung Organ In Vitro Study
Development of in vitro human organbio-assessment platform is essential to replace expensive and time-consuming animal testing models for biomedical research and drug discovery. However, a major challenge in the development of a human lung organ construct is to reconstitute physiologically realistic microenvironments that are capable of maintaining cell differentiation and tissue-specific function. For instance, in tissue culture experiments, primary human bronchiolar and alveolar epithelial cells do not differentiate into respiratory epithelium when submerged in culture medium. Here, we report a new approach that overcomes these limitations by integrating air-liquid interface using biocompatible porous hollow fibers and elastic membrane to mimic the physiological complexity for the growth of the lung bronchiole and alveoli compartments. The two phase flow system can simulate a dynamic liquid layer in both luminal surface of hollow fiber and apical side of membrane by alternatively changing air and liquid flow rate. This capability can be harnessed to develop well-differentiated human bronchiolar and alveolar lung tissue in the platform.
In this study, we have developed a specially-designed manifold enabling two-phase flow system to create a stable and reproducible air-liquid two phase culture for long term lung tissue culture. The flow direction of liquid phase was designed to be parallel to the porous polyethersulfone (PES) hollow fiber (HF) and poly-L-lactide (PLLA) membrane. In this way, the flow direction can prevent liquid shear stress directly acting on the adherent cells and minimize the impact of liquid shear force on cell differentiation. The air and liquid flow rates can also be directly controlled using a peristaltic pump in apical and basolateral sides, respectively. We systematically investigated the parameters associated with each process to quantitatively establish their effect on pressure and shear stress within the platform to identify optimal conditions for cell seeding and tissue culture. Finally, the results of coupling the HFs with co-cultured primary normal human bronchial epithelial (NHBE) cells and human lung microvascular endothelial (HLMVE) with the PLLA membrane supporting human alveolar cells and HLMVEs in the same universal blood circuit demonstrates the possibility to perform long term tissue culture in one platform. Consequently, the ability to create a functional air-liquid interface containing a mucin layer on the luminal side of airways and surfactant in the alveolar compartment in a system simulating the pulmonary architecture would represent a significant step forward toward realizing the goal of manufacturing in vitro lung organ constructs.