In vitro model alveoli from photodegradable microsphere templates

Abstract

Recreating the 3D cyst-like architecture of the alveolar epithelium in vitro has been challenging to achieve in a controlled fashion with primary lung epithelial cells. Here, we demonstrate model alveoli formed within a tunable synthetic biomaterial platform using photodegradable microspheres as templates to create physiologically relevant, cyst structures. Poly(ethylene glycol) (PEG)-based hydrogels were polymerized in suspension to form microspheres on the order of 120 μm in diameter. The gel chemistry was designed to allow erosion of the microspheres with cytocompatible light doses (≤15 min exposure to 10 mW cm−2 of 365 nm light) via cleavage of a photolabile nitrobenzyl ether crosslinker. Epithelial cells were incubated with intact microspheres, modified with adhesive peptide sequences to facilitate cellular attachment to and proliferation on the surface. A tumor-derived alveolar epithelial cell line, A549, completely covered the microspheres after only 24 hours, whereas primary mouse alveolar epithelial type II (ATII) cells took ~3 days. The cell-laden microsphere structures were embedded within a second hydrogel formulation at user defined densities; the microsphere templates were subsequently removed with light to render hollow epithelial cysts that were cultured for an additional 6 days. The resulting primary cysts stained positive for cell–cell junction proteins (β-catenin and ZO-1), indicating the formation of a functional epithelial layer. Typically, primary ATII cells differentiated in culture to the alveolar epithelial type I (ATI) phenotype; however, each cyst contained ~1–5 cells that stained positive for an ATII marker (surfactant protein C), which is consistent with ATII cell numbers in native mouse alveoli. This biomaterial-templated alveoli culture system should be useful for future experiments to study lung development and disease progression, and is ideally suited for co-culture experiments where pulmonary fibroblasts or endothelial cells could be presented in the hydrogel surrounding the epithelial cysts.

Designing Microgels for Cell Culture and Controlled Assembly of Tissue Microenvironments

“…[98] Microgel carriers have also been used to model alveolar cysts in culture, where degradable particles are used to template hollow epithelial spheres representative of their native morphology in the lung. [100] Thus, by modifying the composition and structure of microgel carriers, users can isolate specific cell populations, control their proliferation and fate, and create physiologically relevant structures in culture. Further development of these types of systems could help bridge the gap between cell isolation and transplantation, creating a single cell culture device capable of isolation, expansion, differentiation, and transplantation.…”

Looking ahead: where to next for animal models of bronchopulmonary dysplasia?

“…An increasing number of reports are appearing in the literature detailing such ex vivo studies. These approaches include the use of co-cultures (Greer et al 2014 ; Lewis et al 2015 ; Pieretti et al 2014 ) to address cell-cell interactions and cell phenotypic transformation during alveolarization, plus in-vitro-generated lung organoids that appear to undergo processes akin to alveolarization (Dye et al 2015 ; Quantius et al 2016 ). Evidence has also been presented that lung sections harvested from developing mouse lungs and maintained in culture continue to undergo secondary septation (Pieretti et al 2014 ).…”

Recent advances in the mechanisms of lung alveolarization and the pathogenesis of bronchopulmonary dysplasia

“…As an alternative to animal models, several recent reports have detailed the used of ex vivo lung culture (277), epithelialmesenchymal cocultures (122), in vitro-generated human pluripotent stem cell-derived lung organoids (90), 3D multicell microtissue culture models of airway smooth muscle (363), and a biomaterial-templated alveoli culture system (189) for the study of alveolarization and alveolar biology.…”