Microstructured hydrogels to guide self-assembly and function of lung alveolospheres

Epithelial cell organoids have increased opportunities to probe questions on tissue development and disease in vitro and for therapeutic cell transplantation. Despite their potential, current protocols to grow these organoids almost exclusively depend on culture within three-dimensional (3D) Matrigel, which limits defined culture conditions, introduces animal components, and results in heterogenous organoids (i.e., shape, size, composition). Here, we describe a method that relies on polymeric hydrogel substrates for the generation and expansion of lung alveolar organoids (alveolospheres). Using synthetic hydrogels with defined chemical and physical properties, human induced pluripotent stem cell (iPSC)-derived alveolar type 2 cells (iAT2s) self-assemble into alveolospheres and propagate in Matrigel-free conditions. By engineering pre-defined microcavities within these hydrogels, the heterogeneity of alveolosphere size and structure was reduced when compared to 3D culture while maintaining alveolar type 2 cell fate of human iAT2 and primary mouse tissue-derived progenitor cells. This hydrogel system is a facile and accessible culture system for the culture of primary and iPSC-derived lung progenitors and the method could be expanded to the culture of other epithelial progenitor and stem cell aggregates.