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.

We describe the development of a well-based cell culture platform that enables experimenters to control the geometry and connectivity of cellular microenvironments spatiotemporally. The base material is a hydrogel comprised of photolabile and enzyme-labile crosslinks and pendant cell adhesion sequences, enabling spatially-specific, in situ patterning with light and cell-dictated microenvironment remodeling through enzyme secretion. Arrays of culture wells of varying shape and size were patterned into the hydrogel surface using photolithography, where well depth was correlated with irradiation dose. The geometry of these devices can be subsequently modified through sequential patterning, while simultaneously monitoring changes in cell geometry and connectivity. Towards establishing the utility of these devices for dynamic evaluation of the influence of physical cues on tissue morphogenesis, the effect of well shape on lung epithelial cell differentiation (i.e., primary mouse alveolar type II cells, ATII cells) was assessed. Shapes inspired by alveoli were degraded into hydrogel surfaces. ATII cells were seeded within the well-based arrays and encapsulated by the addition of a top hydrogel layer. Cell differentiation in response to these geometries was characterized over 7 days of culture with immunocytochemistry (surfactant protein C, ATII; T1α protein, alveolar type I (ATI) differentiated epithelial cells) and confocal image analysis. Individual cell clusters were further connected by eroding channels between wells during culture via controlled two-photon irradiation. Collectively, these studies demonstrate the development and utility of responsive hydrogel culture devices to study how a range of microenvironment geometries of evolving shape and connectivity might influence or direct cell function.

Poly(ethylene glycol) (PEG) hydrogels represent a versatile material scaffold for culturing cells in two or three dimensions with the advantages of limited protein fouling and cytocompatible polymerization to enable cell encapsulation. By using light-based chemistries for gelation and for incorporating biomolecules into the network, dynamic niches can be created that facilitate the study of how cells respond to user-dictated or cell-dictated changes in environmental signals. Specifically, we demonstrate integration of a photo-cleavable molecule into network cross-links and into pendant functional groups to construct gels with biophysical and biochemical properties that are spatiotemporally tunable with light. Complementary to this approach, an enzymatically cleavable peptide sequence can be introduced within hydrogel networks, in this case through photoinitiated addition reactions between thiol-containing biomacromolecules and ene-containing synthetic polymers, to enable cellular remodeling of their surrounding hydrogel microenvironment. With such tunable material platforms, researchers can employ a systematic approach for 3D cell culture experiments, spatially and temporally modulating physical properties (e.g., stiffness) as well as biological signals (e.g., adhesive ligands) to study cell behavior in response to environmental stimuli. Collectively, these material systems suggest routes for new experimentation to study and manipulate cellular functions in four dimensions.