Engineering High-Quality Cartilage Microtissues Using Hydrocortisone Functionalized Microwells

Engineering clinically relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large-scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies which support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this article, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications. Impact statement This study addresses a key challenge facing emerging modular biofabrication strategies that use microtissues as biological building blocks. Namely, achieving the necessary robust and consistent differentiation of clinically relevant cell sources, for example, mesenchymal stem/stromal cells (MSCs), and the accumulation of sufficient tissue-specific extracellular matrix (ECM) to engineer tissue of scale. We achieved this by establishing hydrocortisone as a simple and potent method for improving MSC chondrogenesis, resulting in the biofabrication of high-quality (ECM rich) cartilage microtissues. These findings could enable the generation of more scalable engineered cartilage by ensuring the formation of high-quality microtissue building blocks generated using heterogeneous MSC populations.