(568e) Dynamic Culture of Organoid Models of Disease Progression: Transitional Fatty Liver

Organoids are tissue-like constructs that contain some of the structural, cellular, and functional abilities of in vivo tissue. As such, they are a valuable tool for in vitro development of disease models. Current in vitro organoid disease models rely on animal-derived matrices (e.g. Matrigel/Cultrex), which do not capture the high stiffness regimes present in many disease tissues (1 – 15 kPa). Additionally, the mechanical properties are inherent to the matrix and cannot be tuned. This presents a challenge for modeling the environment of tissue during disease, which can transition from soft and healthy (<1 kPa) to stiff and diseased (> 5 kPa). To address this, we developed a small molecule approach to transiently disrupt hydrogel crosslinking to on-demand tune the mechanics of an engineered, recombinant matrix during organoid culture.

A key issue in developing therapeutics for non-alcoholic fatty liver disease (NAFLD) is the transition in liver mechanics from a soft (healthy) to a stiff (diseased) liver. NAFLD affects ~30% of the global population and is the leading cause of end-stage liver disorders. Despite directly impacting a third of the world's population, there are currently no FDA-approved treatments for NAFLD. This is partly due to a critical gap in understanding the onset and progression of NAFLD, which presents both metabolic and mechanical changes in the liver. Identification of a mechanistic pathway has been hindered by the concurrence of multiple NAFLD symptoms: (1) lipid accumulation due to elevated levels of circulating free fatty acids (FFA), (2) metabolic dysregulation and production of oxidative species, and (3) altered mechanosignaling due to increased extracellular matrix deposition and remodeling (fibrosis).

Using hyaluronan (HA) and an engineered elastin-like protein (ELP), we assembled HA-ELP (HELP) hydrogels, crosslinked via dynamic covalent hydrazone chemistry, to model NAFLD progression in patient-derived, induced pluripotent stem cell (iPSC) hepatic organoids (HO). By altering the concentration and crosslinking of HELP, we produced static and dynamic hydrogels that match the stiffness of healthy (~0.8 kPa), early diseased (3 kPa), and advanced diseased (6 kPa) human liver. Importantly, using our small molecule approach, we can on-demand tune the stiffness of each environment during HO culture and investigate transitional cues in driving NAFLD.

Exposure to oleic acid, an FFA elevated in NAFLD, drove intrahepatic lipid accumulation in all conditions; however, high-stiffness hydrogels produced significantly greater volumes of lipid accumulation. Immunocytochemistry imaging of fatty liver HO reveals altered lipid droplet signatures after exposure to oleic acid, including an increased number and size. Using Coherent Anti-Raman Spectroscopy, a non-linear microscopy technique that probes vibration frequencies, reveals environment-induced differences in lipid droplet structures, including phase and fluidity of lipids. Gene expression, in combination with lipidomic-style approaches, reveals a link between mechanosignaling and altered metabolic activity. Critically, we see that HO cultured in a dynamic environment that matches the transitional phase between healthy and diseased stiffness more readily adopt an exacerbated fatty liver phenotype. This highlights the growing need to study the dynamic and transitional phases between NAFLD stages.