Growth and organotypic branching of lung-specific microvascular cells on 2D and in 3D lung-derived matrices

Abstract

Tissue-specific endothelial cells have vital roles in maintenance and functioning of native tissues with constant reciprocal crosstalk with resident cells. Three-dimensional (3D) physio-mimetic in vitro models which incorporate lung-specific microvasculature are needed to model lung-related diseases which involve modulation of endothelial cell behavior like cancer. In this study, we investigated the growth kinetics, morphological changes and responses to biological cues of lung microvasculature on two-dimensional (2D) and in lung matrix-derived 3D hydrogels. HUVEC and HULEC-5a cells were cultured on 2D and compared for their growth, morphologies, and responses to varying growth medium formulations. Brightfield and immunofluorescence imaging was performed to assess differences in morphology. For 3D cultures, native bovine lungs were decellularized, lyophilized, solubilized, and reconstituted into hydrogel form in which endothelial cells were embedded. Cell growth and organotypic branching was monitored in 3D hydrogels in the presence of varying biological cues including lung cancer cell secretome. HUVEC and HULEC-5a cells demonstrated comparable growth and morphology on 2D. However, in 3D lung-derived ECM hydrogels, tissue-specific HULEC-5a cells exhibited much better adaptation to their microenvironment, characterized by enhanced organotypic branching and longer branches. HULEC-5a growth was responsive to lung cancer cell-conditioned medium in both 2D and 3D conditions. In 3D, the concentration of ECM ligand significantly affected cell growth in long-term culture where molecular crowding had an inhibitory role. Our data reveals that HULEC-5a cells offer a reliable alternative to frequently pursued HUVECs with comparable growth and morphology. Due to their intrinsic program for cellular crosstalk with resident cells, the use of tissue-specific endothelium constitutes a vital aspect for modeling physiological and pathological processes. Furthermore, our study is the first demonstration of the synergy between lung-specific microvasculature with lung-specific ECM within a 3D in vitro model.