Geometric engineering of organoid culture for enhanced oganogenesis in a dish

Introduction:: Conventional organoid culture utilizes extracellular matrix (ECM) hydrogel drops to generate 3D self-organized multicellular tissue constructs that resemble structural and functional characteristics of living organs in the body. However, this approach has limited capacity to support sustained development and maturation of organoids necessary to accurately model the complexity of native organs. To address this issue, we developed a microengineered platform termed OCTOPUS (Organoid Culture-based Three-dimensional Organogenesis Platform with Unrestricted Supply of soluble signals) by redesigning the three-dimensional configuration of conventional organoid culture. We present a simple device that allows reconfiguration of the 3D culture environment of organoids to eliminate the problem of restricted supply of nutrients. Our method provides uninterrupted, long-term culture of stem cells to generate organoids with significantly increased structural and functional maturity. Moreover, using patient-derived cells, we present an organoid model of inflammatory bowel disease (IBD) and demonstrate its ability to reproduce key pathological features of IBD. Finally, we demonstrate the advanced capability of our platform to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD.


Materials and Methods:: Our approach can be conceptualized as i) removing the necrotic core from the hydrogel drop and ii) radially segmenting the outer layer and spreading it out to form a thin array of organoids (Fig. 1a). Using this idea, we created OCTOPUS which consists of 8 identical organoid culture chambers (Figs. 1b, 1c). Using human and mouse intestinal stem cells, we demonstrated long-term viability and sustained growth of intestinal organoids without the need for passaging (Fig. 2B). Furthermore, we cultured stem cell suspension isolated from the small intestine IBD patients in our microdevice for over 3 weeks to establish a patient-derived organoid model of IBD and examined key pathological features of IBD. We also performed single-cell RNA sequencing to examine cellular heterogeneity and expression of IBD-associated genes. Using this system, we engineered vascularized, perfusable human enteroids and studied innate immune cell recruitment in IBD. In addition to procedural simplicity and convenience, our organoid culture platform also enabled new capabilities over conventional organoid culture method by facilitating the handling, manipulation, and analysis of organoid models established in our device.


Results, Conclusions, and Discussions:: Mouse intestinal adult stem cells cultured in OCTOPUS and Matrigel drop underwent proliferation process and self-assembled into 3D intestinal organoids (Fig. 2a). However, when culture period was extended beyond 7 days, organoids in drops started to lose their viability while the ones in OCTOPUS continued to grow resulting in significant increase in their size (Fig. 2b). During culture for 14 days, intestinal organoids in OCTOPUS showed the formation of buds that resemble crypt and villus of in vivo intestinal epithelium (Fig. 2c). Importantly, our device allowed organoids to develop significantly elongated and increased number of buds compared to that measured in drop (Fig. 2c). In addition, the 3D culture environment in OCTOPUS led to epithelial differentiation and maturation during organoid development (Fig. 2d). Using our system, we also demonstrated a patient-derived organoid model of inflammatory bowel disease and its interrogation using single-cell RNA sequencing (Figs. 3a-3d). Finally, we developed vascularized and perfusable human enteroids in a microengineered device (Fig. 3e), which was used to model the recruitment of innate immune cells to the disease intestinal epithelium in IBD. We believe that our system enables uninterrupted culture of organoids for prolonged periods to significantly increase the lifespan and maturity of organoids for in vitro modeling of organogenesis and disease pathophysiology. Overall, these data suggest our advanced capabilities of i) engineering vascularized organoids and ii) using this model to study biological crosstalk between the vasculature and organoids during the development and disease progression.


Acknowledgements (Optional): : This work was supported by the National Institutes of Health (NIH) (1DP2HL127720-01), the National Science Foundation (CMMI: 15-48571), the Ministry of Trade, Industry & Energy of the Republic of Korea and the University of Pennsylvania.


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