(I-343) In situ differentiation and self-assembly of macroscopic intestinal tubes

Introduction:: Organoids and organs-on-chips are in vitro miniature organ-mimicking systems. They have become essential tools for understanding development and hold great promise in translational medicine. Currently, most organoid systems lack tissue-relevant complexity, in terms of size and geometry, which is one of the main hurdles for their applications in tissue engineering. Here, we introduce a series of methods to fabricate macroscopic collagen-based cell-laden patterns, including disks, strips, and three-dimensional free-form constructs. We demonstrate that induced pluripotent stem cells (iPSCs) can rapidly self-assemble on the macroscopic strips while maintaining pluripotency. Under the endoderm and intestine inductions, the pluripotent strip-like tissue undergoes dramatic cell organization and differentiates into intestine-like tubes. Furthermore, we show that strips containing high-density primary mouse intestinal crypts also self-assemble into intestinal tubes with the proper spatial organization. This study provides new insights into the possibility of large-scale organ printing.


Materials and Methods:: Unmodified collagen bioink with tissue-relevant cell density (iPSCs or mouse intestinal organoids, density: ~ 4×10⁷cells/mL) was fabricated into various geometries using rapid collagen gelation technique or 3D bioprinting. To generate the macroscopic tissue strips, the bioink was rapidly gelled in a multi-channel device with a 400μm channel diameter. The iPSC strips were then embedded in Matrigel, followed by definitive endoderm (DE) induction, mid-hind gut induction, and intestinal expansion¹. The strips were expanded for up to 5 weeks. For the mouse intestinal strips, they were allowed to grow and spontaneously assemble for 6 days in IntestiCult medium (STEMCELL Technologies). Immunofluorescent staining and confocal imaging were used to examine the cellular organization and markers for cell lineage commitment.

Results, Conclusions, and Discussions:: Our rapid molding technique enables the scalable and instantaneous synthesis of macroscopic cell-laden strips with a uniform cell distribution. We utilize this to create pluripotent tissues. These strips can maintain pluripotency for days under standard stem cell maintenance media. We successfully differentiate the strips into intestinal cells in situ. Over time, the pluripotent strips evolve into self-organized tubular tissues with numerous crypt-like domains surrounding a central lumen - mimicking physiological intestinal tissue. Moreover, primary mouse intestinal organoids-laden strips can self-assemble into macroscopic intestinal tubes within just 6 days. Both types of organoids demonstrate the potential to build patient-derived organoids for improved preclinical drug screening and even organ repair.

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Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).