Three-dimensional (3D) bioprinted co-culture models: a new paradigm for reproducing the tumor microenvironment and precision therapy

Multicellular three-dimensional (3D) bioprinting technology, a pivotal in vitro approach in tissue engineering and disease modeling, enables the co-culture of multiple cell populations within 3D architectures while preserving physiological interactions (1). This strategy facilitates the precise simulation of human histogenesis and pathogenesis through four principal modalities: organoid-based systems that recapitulate tissue self-organization, air-liquid interface (ALI) platforms facilitating epithelial-mesenchymal crosstalk analysis, 3D microfluidic devices for spatiotemporal control of biomolecular gradients, and bioprinting techniques achieving micron-level spatial patterning of heterogeneous cells (2). Each modality addresses distinct experimental demands in reconstructing multicellular microenvironments, collectively enhancing the fidelity of drug screening and advancing mechanobiological research.

Recent advancements in organ-on-a-chip technology have substantially improved the biomimetic fidelity of microenvironmental simulations, offering innovative tools to elucidate multicellular interaction mechanisms in complex biological systems. As a transformative biofabrication technique, 3D bioprinting leverages computer-assisted precision deposition to spatially arrange multiple cell types and biomaterials into hierarchical tissue constructs. Comparative analyses of conventional two-dimensional (2D) monolayers, 3D monocultures, and bioprinted co-culture systems have demonstrated the latter’s superior capability in recapitulating native tissue complexity, as evidenced by studies using primary human intrahepatic cholangiocarcinoma cells. These studies revealed that 3D-bioprinted tumor models retained key malignant phenotypes—including elevated tumor markers [carbohydrate antigen 199 (CA199), carcinoembryonic antigen (CEA)], stemness markers [CD133, epithelial cell adhesion molecule (EpCAM)], secretory protein expression, and invasive/metastatic potential—all of which were markedly diminished under 2D culture conditions (3). The dual utility of 3D bioprinting lies in its ability to reconstruct tumor microenvironments (TMEs) for mechanistic investigations of cellular crosstalk and drug sensitivity profiling, while also enabling longitudinal tracking of functionally distinct cellular subpopulations to decode their dynamic transitions during disease progression. Together, these features establish a robust platform for both oncological research and pathophysiological mechanism exploration.

The TME constitutes a complex niche in which neoplastic cells reside, comprising both cellular components—such as vascular cells, immune cells, and fibroblasts—and acellular elements, including the extracellular matrix (ECM). The bidirectional interplay between tumor cells and their surrounding microenvironment plays a pivotal role in modulating oncogenesis and malignant progression. Traditional 2D culture systems fundamentally fail to replicate the spatial heterogeneity and architectural complexity of native tumor tissues. In contrast, 3D bioprinted co-culture models address this limitation by structurally integrating stromal components within biomimetic matrices, thereby enabling systematic investigation of paracrine and mechanical influences exerted by specific cellular subsets while recapitulating tissue-level histomorphological features. Pioneering work by Sgarminato et al. exemplified this approach through the use of gelatin methacryloyl (GelMA)-based hydrogels embedded with human fibroblasts, successfully replicating pancreatic exocrine tubular–acinar structures with enhanced proliferative capacity and sustained cell viability (4). Such engineered platforms not only reproduce the topobiological constraints of the native TME but also serve as high-fidelity systems for dissecting intercellular signaling networks and evaluating therapeutic vulnerabilities under physiologically relevant conditions.

Xu et al. pioneered a co-bioprinted prostate cancer model comprising a five-layered grid hydrogel core encapsulating tumor cells, concentrically surrounded by circular strata containing fibroblasts. This architecturally engineered system demonstrated that hyaluronic acid exerts a stronger pro-proliferative effect than fibroblast-derived signals within the TME. Mechanistic investigations using this platform revealed that 3D-bioprinted co-cultures exhibited maximal expression of epithelial-mesenchymal transition (EMT) markers and cancer stem cell (CSC) signatures. Additionally, the study elucidated hyaluronic acid’s dual regulatory role in enhancing cytokine secretion and orchestrating the expression of pro-angiogenic factors (5). These findings establish a causal link between spatially defined ECM biochemistry and malignant phenotypic evolution, highlighting how precisely reconstructed TME topologies can faithfully recapitulate tumor-stroma coevolution dynamics. Such physiomimetic models not only uncover microenvironmental determinants of therapeutic resistance but also offer a predictive framework for patient-specific drug response profiling, ultimately bridging the gap between reductionist in vitro systems and the complexity of clinical pathophysiology.

3D-bioprinted co-culture systems exhibit superior pathophysiological fidelity compared to conventional 2D models, particularly in replicating clinically relevant gene expression profiles and drug response phenotypes of malignant cells (6). Growing evidence continues to validate this technological advantage. For instance, a tri-culture system incorporating hepatocellular carcinoma (HCC)—HepG2, cholangiocarcinoma (CCA)—RBE, and human umbilical vein endothelial cells (HUVECs) successfully mimicked bile duct stricture formation driven by cholangiocellular hyperplasia. This architecture-dependent variation in drug sensitivity is especially evident in intrahepatic cholangiocarcinoma models, where 3D constructs display pharmacological responses that cannot be captured in monolayer cultures (7). Notably, Wang et al. demonstrated that colorectal cancer cells cultured in multicellular configurations—incorporating THP-1 cells and HUVEC cells as stromal components—exhibited markedly increased chemoresistance compared to monoculture controls, implicating stromal-mediated cytoprotective mechanisms under pharmacological stress (8). These findings underscore the critical importance of faithfully recapitulating the TME. Engineered systems that integrate neoplastic, stromal, and immune components within ECM-mimetic matrices are transforming personalized oncology by enabling predictive modeling of tumor-stroma-immune interactions and mapping patient-specific therapeutic vulnerabilities.

3D bioprinted co-culture models have demonstrated the ability to replicate the metastatic and invasive characteristics inherent to native tumor tissues. Fan et al. developed a microchannel-integrated 3D-printed platform incorporating glioblastoma organoids (GB), dorsal forebrain organoids (DO), and ventral forebrain organoids (VO). Through multicellular 3D cultivation, their results revealed that DO exerted a more pronounced regulatory influence on GB gene expression profiles compared to VO, manifesting as enhanced invasive potential of GB cells. This differential effect was attributed to region-specific intercellular crosstalk, potentially mediated by spatially organized paracrine signaling (9). The integration of cellular interaction networks with the spatial architecture of 3D cultures provides a robust experimental foundation for mechanistic studies of tumor metastasis and invasion, particularly in recapitulating microenvironment-driven phenotypic plasticity.

3D bioprinting technology not only enables the precise fabrication of high-throughput in vitro models for tumor drug sensitivity testing but also facilitates the construction of disease models that replicate specific pathological processes ex vivo. These models offer new perspectives for elucidating disease mechanisms and developing therapeutic strategies. Previous studies have demonstrated significant progress in applying 3D bioprinted co-culture systems to model vascular and neurological diseases.

In vascular modeling, 3D bioprinting offers high resolution and structural stability in replicating the coaxial architecture of multilayered arteries, thereby enabling the investigation of the pathophysiology of atherosclerosis and facilitating drug screening. Gao et al. developed an arterial structure using in-bath coaxial cell printing to study the pathophysiology of an atherosclerosis model. They employed 3% vascular tissue-derived decellularized extracellular matrix (dECM) as the bath material and encapsulated human dermal fibroblasts to assess their support for resident cell growth. A triple-coaxial nozzle was used to fabricate the three-layered structure (10). Similarly, Zhang et al. developed a thrombosis-on-a-chip model using GelMA hydrogel to recapitulate the processes of thrombogenesis, thrombolysis, and fibrosis (11).

In addition, researchers have proposed a neuroimmune co-culture system that recapitulates key pathological features of Parkinson’s disease (PD), aiming to investigate multicellular responses to complex PD-mimicking microenvironments. An extrusion-based 3D bioprinter was used to construct a midbrain dopaminergic (DA) neuron model relevant to PD pathology, simulating the interdependence between cells and their surrounding microenvironment. The bioink, composed of ECM-derived hydrogels incorporated with conductive nanostructures, facilitated the differentiation of midbrain neural progenitor cells (NPCs) into DA neurons (12).

3D-bioprinted co-culture systems leverage spatially heterogeneous cellular architectures to recapitulate multicellular interactions and pathophysiological processes with unprecedented fidelity, offering transformative potential for modeling tumor metastasis and invasion by emulating the spatial dynamics and intercellular crosstalk inherent to native tissues (13). These systems provide a robust platform for dissecting tumor-stroma-immune interplay, where gene expression profiles, metabolic reprogramming patterns, and drug response data exhibit enhanced clinical correlation compared to conventional models (14). Beyond oncology, 3D bioprinting enables the assembly of physiomimetic tissue constructs that decode disease progression mechanisms at cellular, molecular, and regulatory levels, exemplified by its utility in reconstructing neurovascular interfaces and multi-organoid systems.

However, several technical bottlenecks persist in current 3D bioprinted co-culture systems. First, standardization of model construction parameters—such as cell ratios, scaffold topology, and culture conditions—remains a significant challenge. Second, issues including scaffold structural instability during long-term culture and hypoxia in the central regions of constructs require urgent resolution (15). Most critically, existing systems are typically limited to modeling the pathological processes of a single organ. Given that the human body functions as an integrated, multi-organ system, disease mechanisms often involve complex inter-organ interactions. In this context, the development of co-culture platforms that integrate multiple organ systems may represent a pivotal direction for future advancement in this field.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, HepatoBiliary Surgery and Nutrition. The article did not undergo external peer review.

Funding: This work was supported by the National Natural Science Foundation of China (No. 32271470).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://hbsn.amegroups.com/article/view/10.21037/hbsn-2025-297/coif). H.Y. serves as an unpaid editorial board member of HepatoBiliary Surgery and Nutrition. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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