3D bioprinting of patient-derived cholangiocarcinoma organoids in a decellularized liver matrix-based bioink for drug testing

Abstract

Cholangiocarcinoma (CCA) shows marked interpatient heterogeneity in chemotherapy response, highlighting the need for physiologically relevant, standardized, and reproducible in vitro models for individualized drug screening. However, Matrigel-based organoid models lack hepatobiliary extracellular matrix cues, and many bioprinting strategies rely on dissociated or fragmented organoids, disrupting native architecture and spatial organization. To address these limitations, we developed a composite bioink for direct bioprinting of patient-derived CCA organoid (CCAO) fragments that retained multicellular organization, enabling standardized culture, imaging, and drug-response evaluation. Although decellularized liver matrix (DLM) was used to reconstruct the tissue-specific microenvironment, its weak gelation, poor mechanical properties, and limited printability restricted its application. Tris(2,2’-bipyridyl)ruthenium(II)/sodium persulfate (Ru/SPS) and gelatin methacryloyl (GelMA) were introduced to improve photocrosslinking, mechanical support, and printability. DLM retained key components of the hepatic extracellular matrix, including collagen, fibronectin, laminin, and glycosaminoglycans. With GelMA and Ru/SPS, the bioink achieved rapid visible-light crosslinking (405 nm, 50 seconds), tunable mechanical properties, and good extrusion-printing compatibility. Among the tested formulations, the DLM-Ru/SPS:GelMA (4:1) group showed the best balance of printability, transparency, and organoid-compatible microstructure. Using this optimized bioink, organoid fragments were patterned and reassembled into viable spheroids that retained key CCA features and maintained high viability (>80%). The platform also enabled reproducible assessment of drug response, demonstrating dose-dependent cisplatin sensitivity and enhanced cytotoxicity with cisplatin–gemcitabine treatment. In summary, this study established a DLM-based direct bioprinting platform for CCAOs and a standardized system for individualized therapeutic evaluation.

Keywords

Three-dimensional bioprinting / Bioink / Cholangiocarcinoma / Decellularized liver matrix / Patient-derived organoids / Drug screening

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