The Shay lab tissue engineering group seeds life-extended or immortalized human cells into a variety of three-dimensional (3-D) substrates in order to promote differentiation. The long-term goal is not only to produce functional organs (lung, heart, kidney) in vitro, but also to be able to have the cells express molecules that produce immune tolerance so that tissue-matching would no longer be required. Our most advanced model system uses bronchial lung epithelial cells seeded onto a decellularized lung matrix. This project has two broad aims: 1) develop novel cell reagents for lung tissue engineering and lung disease (cancer, lung fibrosis) modeling applications; 2) create 3-D tissue constructs using mutant adult lung progenitor cells for disease modeling (such as cystic fibrosis, pulmonary fibrosis and cancer) and normal progenitor cells for regenerative medicine applications. Primary cells isolated from adult patient lungs can normally only be passaged a few times before losing the ability to differentiate, making it difficult to isolate a sufficient number of cells for disease studies or tissue engineering. To address this, we have developed normal human bronchial epithelial cells (HBECs) which have been either immortalized by ectopic expression of CDK4 and hTERT/telomerase (HBEC3KTs) or conditionally life-extended using Rho-Kinase inhibitors while grown on an irradiated fibroblast feeder layer (or conditioned medium from irradiated feeder layers). These immortalized or conditionally life-extended cells are multipotent and form different lineages based on cues from the extracellular microenvironment. HBEC3KTs can form a well differentiated epithelium composed of basal, goblet, and ciliated cells when placed at the air-liquid interface (Figure 1A, B). HBEC3KTs can also form bronchial organoids/cyst-like structures when grown inside Matrigel® or highly branched structures when seeded on top of Matrigel® with a lung fibroblast feeder layer below the matrix (Figure 1C, D). In addition to primary bronchial cells from normal lung donors, we have isolated bronchial cells from patients with cystic fibrosis and lung cancer. We have thus created cellular reagents for the study of normal and diseased lung development that can be maintained in culture for a significantly extended period of time compared to traditional studies with primary bronchial cells. Figure 1. Differentiation of HBEC3KTs in different extracellular conditions. (A) Paraffin cross-section of well differentiated air-liquid interface (ALI) culture with immuno-histochemistry stain for MUC5AC+ goblet cells interspersed between ciliated cells. (B) Scanning electron micrograph also shows well differentiated goblet and ciliated cells at ALI. Scale = 10 µm (C) Phase contrast picture of 5 day bronchial organoids 50 µm in diameter embedded in Matrigel® with a fibroblast feeder layer. (D) Phase contrast picture of 10 day highly branched organotypic structure on top of Matrigel® with a fibroblast feeder layer below. The decellularized matrix of complex tissues preserves the tensional, extracellular molecular, and ultrastructure cues of the original organ. Cells seeded onto these matrices rapidly differentiate and form functional tissues. One current model decellularizes a mouse lung by perfusion with detergent, seeds life-extended bronchial basal cells into the tracheal cavity, and cultures the seeded lung in a pressurized bioreactor with perfusion of media through the vasculature. We are using this system to model normal and diseased lung development in an ex-vivo environment and to pursue tissue engineering of functional lungs for regenerative medicine applications.