Tumor Organoid-Immune Cell Co-Culture Models

In cancer research, co-culture systems have become essential tools for modeling interactions among multiple cell types within a controlled environment. Given the limited microenvironmental complexity of conventional tumor organoids, integrating immune and stromal components enables a more accurate recapitulation of tumor biology. Such systems enable the study of cellular crosstalk, signaling pathways, and tumor heterogeneity, and are therefore indispensable in modern cancer research^[1].

To capture cellular heterogeneity within the tumor microenvironment (TME) and heterotypic cell-cell interactions, three primary strategies have emerged: submerged immune cell-tumor organoid co-culture, 3D microfluidic culture, and ALI culture. Among these, submerged co-culture serves as a reconstructed TME model, while the latter two represent holistic TME models. The main characteristics and applications of different organoid models in simulating the TME are summarized in Figure 1.

Immune Cell-Tumor Organoid Submerged Co-culture

Physical co-culture of immune cells with tumor organoids mimics immune cell infiltration in vivo, and is widely employed to investigate cytotoxic T cell activity and macrophage-mediated phagocytosis.

Matrix-embedded method: In this approach, immune cells and organoids are co-embedded within Matrigel or collagen matrices, preserving 3D architecture while allowing cell migration. Direct co-culture of pancreatic cancer organoids with T cells in Matrigel successfully induced the activation of cancer-associated fibroblasts.

Suspension co-culture method: Suspension co-culture using ultra-low attachment plates is suitable for high-throughput drug screening. For instance, co-culture of colorectal cancer organoids with CD8⁺ T cells facilitates rapid assessment of T cell cytotoxicity enhanced by PD-1 inhibitors.

Three-Dimensional Microfluidic Culture

Microfluidic systems provide dynamic and biomimetic environments that enable precise control of biophysical parameters such as fluid shear stress and mechanical traction forces. In addition, these systems incorporate biochemical gradients to accurately emulate the physiological microenvironment of human organs. These systems overcome two fundamental limitations of conventional organoid models: the absence of physiological vascular perfusion and the lack of a functional immune system.

ALI Culture

ALI culture is a 3D cultivation paradigm that simulates the luminal environment of hollow visceral organs. In this system, cells are seeded onto the permeable membrane of a collagen-coated insert; the basal surface remains submerged in the culture medium, while the apical surface is directly exposed to air, thereby mimicking in vivo epithelial architecture. Primarily applied to epidermal and respiratory epithelia, ALI culture allows airway epithelial cells to develop into pseudostratified layers with tight junctions, functional cilia, and mucin-producing capacities, faithfully recapitulating their native phenotypes. This approach has also been adapted to investigate interactions between organoids and TME.

Additionally, various co-culture models have been employed in cancer research, providing critical insights into the complexity of tumor behavior.

Table 1. Technological overview of co-culture models^[2].

Applications of Organoid-Immune Co-Culture

Tumor organoid-immune co-culture models have broad applications in oncology research and serve as indispensable experimental systems for investigating immune evasion mechanisms, elucidating the mechanisms of immunotherapy, and developing novel treatment strategies.

This figure illustrates the application scenarios of co-culturing tumor organoids with immune cells, including modeling interactions between tumor cells and immune cells, investigating TME mechanisms (such as immune responses and drug resistance), screening personalized therapeutics (e.g., high-throughput drug screening), and developing novel immunotherapies (e.g., immune checkpoint inhibitor development).

Investigation of Tumor Immune Evasion Mechanisms

Ko et al. established 32 genetically engineered esophageal organoids and utilized single-cell RNA sequencing to profile their transcriptomic signatures and chemokine secretion patterns. Their study identified the concurrent deletion of TP53, CDKN2A, and NOTCH1 as a critical genetic determinant driving esophageal tumorigenesis and immune escape. These findings deepen our understanding of tumor immune evasion mechanisms and provide a theoretical foundation for the development of new immunotherapeutic approaches^[8].

Immunotherapeutic Drug Screening

Furthermore, tumor organoid-immune cell co-culture models enable the efficient screening and evaluation of new immunotherapeutic drugs. Efficacy can be directly assessed by monitoring immune cells activation and subsequent tumor cell cytotoxicity in vitro. For example, Norkin et al. employed patient-derived colorectal cancer (CRC) organoids to screen several drug classes not traditionally used for CRC, including antipsychotic phenothiazines, cholesterol-lowering statins, antimycotic conazoles, selective estrogen receptor modulators, and antihistamines. They identified ifenprodil, opipramol, perphenazine, and toremifene as promising candidates with a favorable therapeutic window for targeting human CRC, warranting further in vivo validation^[9]. Such findings provide significant preclinical data to support future clinical trials of these drugs.

Personalized Immunotherapy Assessment

Additionally, tumor organoid-immune cell co-culture models can be applied to personalized immunotherapy. By isolating tumor cells and autologous immune cells from a single patient, researchers can establish patient-specific co-cultures to empirically evaluate individual immune responses and the efficacy of tailored immunotherapeutic regimens. Van de Wetering et al. constructed patient-derived CRC organoids to test the activity of cetuximab in KRAS wild-type organoids. Their results, consistent with clinical observations, highlighted the potential of tumor organoids to directly assess the drug sensitivity of tumors in a personalized treatment approach^[10].​​​​

Collectively, these research findings hold significant potential for informing personalized immunotherapy strategies for patients. The innovative integration of tumor organoids with immune cells offers a powerful tool to simulate the TME and interrogate complex tumor-immune interactions. This methodology not only deepens our understanding of tumor immune evasion but also establishes a vital experimental basis for discovering new immunotherapeutic strategies and personalized immunotherapy plans.

Challenges and Future Directions

Current Status and Challenges in Tumor Organoid Culture

While tumor organoid-immune cell co-culture provides invaluable models for simulating the TME, their current methodologies heavily rely on matrices and culture media that present significant limitations:

• Media-associated constraints
  The reliance on purified growth factors, conditioned media, and animal-derived sera results in high costs, substantial batch-to-batch variability, and biological heterogeneity, thereby complicating patient-specific modeling.

• Matrix inconsistencies
  Conventional Engelbreth-Holm-Swarm (EHS) matrices (e.g., Matrigel) are animal-derived and harbor undefined impurities, exhibiting batch-to-batch protein homology of merely ~53%. While Type I collagen is more economical, it remains susceptible to batch variations and exogenous contaminants. Conversely, chemically synthesized hydrogels offer a highly controlled environment with minimal variability, making them superior candidates for high-throughput drug screening and novel therapeutic development^[11].

• Stringent technical demands
  The establishment of organoids and the expansion of autologous immune cell demand advanced technical proficiency and rigorous culture conditions. Consequently, substantial time investment, lack of standardization, and limited reproducibility remain critical bottlenecks.

Complexity and Technical Advancements in Co-Culture Models

Although current co-culture systems partially mimic the TME, achieving holistic physiological relevance remains challenging:

• Multiple microenvironmental parameters must be faithfully reproduced, including cell-cell and cell-matrix interactions, oxygen gradients, and nutrient supply.

• Conventional models frequently omit crucial stromal components such as vascular cells, fibroblasts, and diverse cytokines, rendering complete replication of complex in vivo conditions difficult.

• Emerging technologies offer potential solutions: Organ-on-chip technology provides vascular perfusion and fluid dynamics; 3D bioprinting allows precise replication of tumor physical architecture, enhancing biological fidelity from macroscopic to microscopic scales[12].

• Further improvements can be achieved by incorporating immune cell types and optimizing culture media. However, experimental findings still require in vivo validation to confirm reliability.

Future Directions and Potential

Despite existing challenges, tumor organoid-immune cell co-culture systems hold substantial potential:

• Enhanced model complexity: Incorporation of multiple cell types and intricate cytokine networks enables more authentic simulation of the TME.

• Technological integration: Combination of organ-on-chip technology, 3D bioprinting, and dynamic culture methods facilitate reconstruction of the tumor immune microenvironment.

Figure 3. Future co-culture models^[2].

• Next-generation co-culture systems: Increasingly sophisticated models are being developed to better recapitulate in vivo conditions, including the integration of additional immune and stromal components, reconstruction of intercellular signaling networks, and replication of dynamic physical conditions. These advanced systems are likely to offer more accurate platforms for studying tumor–immune interactions and for drug development (Figure 3).

• Data-driven and intelligent analysis: Integration of big data and artificial intelligence will facilitate multi-omics analysis, identification of critical factors, prediction of therapeutic efficacy, and optimization of experimental conditions.

Summary

Tumor organoid-immune cell co-culture systems substantially improve the biological fidelity of in vitro models by incorporating key cellular components of the TME. These models enable the investigation of tumor immune evasion mechanisms, support the screening of immunotherapeutic agents, and facilitate the development of personalized treatment strategies.

Recommended Tumour Organoid Culture Media

For researchers working with tumour organoids, immune co-culture systems or advanced 3D cell culture models, Bio-Connect can help you find the right MedChemExpress products for your work in the Benelux and Europe. Whether you are setting up a new model, comparing culture conditions or selecting reagents for drug screening, our team is happy to think along and point you towards suitable options.

References

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