Amniotic Membrane‐Derived Extracellular Matrix for Developing a Cost‐Effective Xenofree Hepatocellular Carcinoma Organoid Model

Current limitations in the treatment of hepatocellular carcinoma (HCC) include tumor recurrence, chemoresistance, and severe side effects, all of which call for novel cancer models that better represent the tumor microenvironment (TME). 3D organoids hold promise due to their increased relevance to the TME hallmarks. Herein, we aim to establish an HCC organoid model that mimics the HCC microenvironment and its metabolic interactome. The organoid comprises a decellularized human amniotic membrane (dAM) as a biomimetic matrix, Huh-7 cell line, bone marrow mesenchymal stromal cells (BM-MSC), and human umbilical vein endothelial cell-conditioned medium (HUVEC-CM). The structure integrity of the HCC organoid was monitored using H&E staining at 7, 14, and 21 days and transmission electron microscopy (TEM) and scanning electron microscopy (SEM) at 21 days. The established organoid model maintained its viability over 21 days as tested by propidium iodide (PI) fluorescence staining, MTT, upregulated expression of proliferating cell nuclear antigen (PCNA), and alpha-fetoprotein (AFP). The expression of vascular endothelial growth factor (VEGF) in the HCC organoid induced a neo-angiogenic response in ovo. Metabolic reprogramming in the HCC organoid showed a shift toward glycolysis as indicated by promoted glucose consumption, upregulated lactate production, and reduced cellular pyruvate concentration. Oxidative phosphorylation was suppressed as indicated by reduced reactive oxygen species (ROS), and hydrogen peroxide (H₂O₂), and halted urea cycle progression. The dataset shows that the dAM may hold a promise for its use as extracellular matrix (ECM) source for HCC organoid models, by replicating the HCC microenvironment and metabolic signature, thus holding a promise for developing targeted therapeutic strategies.