Development of novel in vitro platforms to model the lung
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Chronic respiratory diseases are the leading cause of death and disability. Estimates suggest a ~40% increase in population with chronic respiratory diseases and ~18% increase in respiratory diseases-related deaths between 1990 and 2017 with ~545 million affected and ~3.9 million deaths in 2017. The leading risk factor for men was found to be smoking irrespective of the region while the leading cause of respiratory illness for women varied depending on the region – household air pollution from cooking fuels in part of Asia and Africa, inhalation of particulate matters in parts of Asia and Oceania, and smoking in other regions. With such a large population affected, it is imperative to understand the disease etiologies and find therapies to prevent and manage respiratory illnesses. Animal models of pulmonary diseases serve as invaluable tools towards this; these models have immensely contributed to our understanding especially at a whole-organism level. However, species differences between the models and humans often result in failed translation of therapies and drugs. Additionally, animal models do not allow decoupling of factors contributing to diseases thus making the findings confounding. These bottlenecks can be overcome by utilizing in vitro models wherein human cells can be used and the effect of factors contributing to disorders can be studied systematically. Conventional in vitro models suffer from their own limitations such as being too simplistic and unrepresentative of in vivo conditions. Emergence of organ-on-a-chip and organoid technologies have tried to bridge this gap by recreating key organ-specific features and tissue microenvironment. However, challenges remain with incorporating complex and heterogeneous mechanical cues, long-term culture of primary cells in vitro and creation of multicellular models that accurately capture the architecture and functionalities of the organ.To address several of these challenges, we have developed platforms to capture the complexity of the lung within in vitro devices. Specifically, we developed a breathing alveolus-on-a-platform that utilizes a microfluidic-pneumatic mechanism to subject the cells to heterogeneous strain arising from out-of-plane stretching akin to expansion and contraction of an alveolus during breathing. Using the device, we showed that breathing induced changes in cells such as their alignment, surfactant production, and wound healing response. We used the device to model lung conditions associated with altered pulmonary biomechanics such as changes in lung compliance and ventilator-induced lung injury. We also demonstrated the ability of the device to be used a tool for screening toxicological effects of chemicals such as compounds used as flavors in electronic cigarettes. Finally, we demonstrated the ability of the device to support the culture of primary human alveolar type 2 (AT2) cells and iPSC-derived AT2 cells. One of the limitations of in vitro systems is the inability to culture primary cells, including AT2 cells, for a long time without loss in their phenotype and identity. Until recently, a feeder layer of supporting stromal cells was required to culture AT2 cells in vitro. While medium conditions have now been optimized and defined to avoid the use of feeder cells, Matrigel is still the current state-of-art platform to culture these cells. However, its undefined nature and limited tunability present a barrier to expanding the use and application of such in vitro tools. Through a screening of ECM hydrogels, we identified laminin-111 as a key ECM molecule that supports the culture of AT2 cells and formation of organoids known as alveolospheres. We further showed that laminin-111 can be used by itself or in combination with other ECM proteins such as fibrin or synthetic polymers to grow alveolospheres on par or better than Matrigel. Further, we optimized the laminin and fibrinogen concentrations that can support the formation of alveolospheres and demonstrated cell-mediated degradation of matrix as being critical for alveolosphere formation. We showed the versatility of our system by culturing alveolospheres grown from AT2 cells from various sources – murine, human, and iPSCs-derived. Identification of matrix properties that regulate cellular function such as self-renewal and lineage-specific differentiation will significantly advance the efforts towards organ-specific microphysiological systems. Lung is a common site for metastasis and lung cancer is the most lethal cancer. So, we next examined the applicability of using lung-on-chip platform to study metastasis of distant cancers into the lung. With culture conditions for alveolosphere optimized, we worked towards building more complex models of the lung by including other components such as lung fibroblasts and functional vasculature for their potential use as platforms to model lung metastasis of cancers such as undifferentiated pleomorphic sarcoma. Towards this, we first adapted a vasculature-on-a-chip model and showed that heterogeneous cells populations from undifferentiated pleomorphic sarcoma had different extravasation efficiencies in the vasculature-on-a-chip. We then integrated the vasculature with iAT2 alveolospheres and normal human lung fibroblasts to create multi-cellular models of the lung. To better support the phenotype of individual cells, we also developed novel compartmentalized devices that can house multiple cell types in their corresponding medium while allowing cross-talk with other cell types by means of soluble factors. Such multicellular platforms recapitulating the complexity of the lung using human cells can serve as important tools for gaining mechanistic insights into pulmonary functions as well as models for studying lung disorders, identifying therapeutic targets, and screening candidate drugs.
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