(137c) Tissue-Mimetic Organoids in 4D Biomaterials to Study Regenerative and Pathological Processes

Three-dimensional (3D) organoids have emerged as powerful models for studying development, disease progression, and generating functional tissues for drug screening. While physiologically relevant biochemical cues regulate certain functional aspects of organoids, recent studies have demonstrated that physical properties of the extracellular environment—such as matrix viscoelasticity and extracellular fluid viscosity—also influence cell behavior and function. However, current organoid culture systems lack mechanical tunability of the extracellular environment. Organoids are typically cultured in naturally derived extracellular matrices (ECMs) such as Matrigel, which hampers reproducibility and lacks the ability to fine-tune ECM properties on demand. Furthermore, physiological ECM properties are dynamic, exhibiting temporal variations during development and pathological progression. Thus, there is a critical need for biomaterial platforms with tunable properties that allow precise spatial and temporal control, enabling the development of more physiologically relevant organoid models.

We employ a poly(ethylene glycol) (PEG)-based “blank-slate” hydrogel platform as a synthetic ECM for organoid studies. First, we demonstrate the use of photopatternable hydrogels to replicate the crypt-villi architecture of the intestinal epithelium and investigate the temporal sequence of mechanotransduction events in intestinal stem cell (ISC)-derived organoids. By selectively cleaving crosslinks in the hydrogel matrix using 405 nm laser light, we spatially and temporally control softening in targeted regions, thereby directing the initiation of crypt-like budding from organoids. Our analysis revealed nuclear morphology differences between differentiated Paneth cells and non-differentiated cells, which were consistent with in vivo findings. Temporal control of crypt growth enabled synchronized bud formation, allowing us to characterize the dynamic sequence of nuclear shape changes. Additionally, we found that increasing nuclear mechanotransduction—either by matrix photopatterning or pharmacological intervention—promoted crypt formation with differentiated cells, even in media conditions typically favoring stem cell maintenance. This highlights that mechanical cues act alongside biochemical signals to regulate stem cell fate.

Next, we leverage our photopatternable hydrogel platform to model in vitro the process of crypt fission. Crypt fission, the primary mechanism of crypt regeneration following injury, has been studied exclusively in vivo or in Matrigel-based organoid cultures, which lack systematic control over the process. Without an engineered system, the influence of crypt shape or ECM composition on crypt fission remains unclear. Here, we present the first in vitro system that dictates the precise location and timing of crypt fission events. Our system recapitulates the presence of differentiated Paneth cells both before and after crypt division, demonstrating the derivation of functional crypts post-fission. Temporal control over fission allows us to isolate early cellular changes occurring during this process. By tracking β-catenin localization at different time points, we uncover the dynamic role of Wnt signaling in initiating and driving daughter crypt formation and growth.

Finally, we explore how synthetic hydrogels can be used to mimic pathological abnormalities, such as tumor formation, and gain novel mechanistic insights. Pancreatic ductal adenocarcinoma (PDA), which accounts for ~90% of all pancreatic cancers, is characterized by a dense stromal microenvironment that constitutes a major portion of the tumor mass. Using organoids derived from pancreatic cancer patients, we demonstrate that ECM composition and stiffness critically regulate tumor cell plasticity and proliferation. Specifically, PDA organoids grown from single cells in PEG hydrogels functionalized with fibronectin-mimetic adhesive peptides (RGD) initially formed proliferative colonies, but their morphology gradually deviated from Matrigel-grown organoids. Over time, the organoid lumen collapsed, giving rise to solid cribriform structures indicative of polarity loss and a phenotypic switch. Interestingly, incorporating full-length laminin into the hydrogel restored lumen formation and organoid morphology to match Matrigel-grown controls, underscoring the importance of ECM composition in regulating organoid fate. Furthermore, by using confocal light-guided microscopic softening around organoids, we selectively enriched proliferative cells in specific regions. Our hydrogel system also supports organoid-stromal cell co-culture, making it a versatile platform for engineering complex tissue-mimetic models with precise control over matrix properties.

This presentation will also outline the research and training objectives of my future independent lab, which aims to address healthcare challenges through a multidisciplinary approach that integrates principles of chemical engineering, biomaterials, microfluidics, advanced microscopy, molecular biology, electronics, and both mathematical and in vivo models.

Acknowledgments: This research was supported by the National Science Foundation grant RECODE-2033723, the National Institutes of Health grants R01-DK120921, 1S10OD034320 and the Helen Hay Whitney Foundation award F-1339.