Organoid Basics: Co-culture Models—An Essential Guide for Beginners

Since its emergence, organoid technology has become an indispensable tool in biomedical research, particularly in areas such as disease modeling and drug screening. This limitation restricts their ability to fully recapitulate the complex intercellular interactions that occur in vivo.

To overcome these challenges, organoid co-culture models have been developed as an advanced strategy to better mimic physiological and pathological conditions. By integrating multiple cell types into a single system, co-culture approaches enable more realistic modeling of tissue architecture and cellular crosstalk.

In this article, we provide a systematic introduction to organoid co-culture models, including their fundamental concepts, commonly used co-culture combinations, construction strategies, and key applications in modern research.

What is Organoid Co-culture?

Organoid technology involves the use of stem cells or tissue-derived cells to generate miniature, three-dimensional tissue structures in vitro that exhibit spatial organization and partial physiological functions resembling those of native organs. In simple terms, organoid co-culture builds upon conventional organoid systems by introducing additional cell types—such as immune cells, fibroblasts, or vascular endothelial cells—or even microorganisms, including specific microbiota. These components are cultured together within the same system, enabling more complex cellular interactions and a more physiologically relevant microenvironment.

Why is Co-culture Necessary?

Traditional single-lineage organoids exhibit notable limitations, primarily due to their incomplete recapitulation of the native physiological microenvironment. In particular, the absence of complex stromal networks and immune barriers found in vivo restricts their biological relevance. Within actual physiological tissues or tumor microenvironments, parenchymal cells are closely surrounded and regulated by immune cells, fibroblasts, vascular endothelial cells, and components of the extracellular matrix.

Without these non-parenchymal elements, conventional organoids are unable to accurately reproduce the dynamic crosstalk among diverse cell populations, often leading to discrepancies between in vitro experimental results and in vivo biological responses. As a result, organoid co-culture systems have emerged to address these critical limitations by incorporating multiple cellular components into a unified model system.

III. What are the various co-culture combinations, and what are their applications?

• Organoids + Immune Cells

Co-culturing tumor organoids with immune cells currently represents one of the most widely used applications of organoid co-culture systems. By integrating patient-derived tumor organoids (PDOs) with immune cell populations—such as T cells, natural killer (NK) cells, or macrophages—researchers can in vitro recapitulate the dynamic interactions between the immune system and tumor tissues. This platform has become an essential tool for evaluating tumor immunotherapies.

For example, in the context of immune checkpoint blockade therapy, this model enables the assessment of the effects of PD-1 inhibitors on tumor cell viability and immune-mediated cytotoxicity. In addition, by modulating the effector-to-target (E:T) ratio between CAR-T cells and tumor cells, the co-culture system is widely applied in high-throughput screening studies. It also facilitates the evaluation of CAR-T cell infiltration capacity, proliferative status, and tumor-killing efficacy in vitro.

Image adapted from *Pharm Biol*, 2025.

• Organoids + Stromal Cells

Stromal cells within the tumor microenvironment—particularly cancer-associated fibroblasts (CAFs)—represent a major cellular component and play a pivotal role in driving tumor progression and drug resistance. Co-culturing organoids with CAFs enables the simulation of in vivo extracellular matrix (ECM) remodeling processes, including the deposition of key components such as collagen and hyaluronic acid. The resulting dense ECM increases interstitial fluid pressure within the tissue, thereby forming a physical barrier that limits drug penetration.

For example, in pancreatic cancer co-culture models, CAF-derived collagen has been shown to significantly impede the delivery of the chemotherapeutic agent gemcitabine. Similarly, in breast cancer models, the presence of CAFs reduces intratumoral accumulation of targeted therapies such as lapatinib and trastuzumab, ultimately contributing to therapeutic resistance and treatment evasion.

Image adapted from Front Immunol, 2023.

• Organoids + Vascular Endothelial Cells

As in vitro organoids increase in size, the absence of functional blood perfusion often results in hypoxia-induced necrosis within their core regions, severely restricting tissue maturation and long-term culture stability. To address this limitation, endothelial cells (ECs) can be introduced, or vascular transcription factors such as ETV2 can be overexpressed, to promote the formation of microvascular-like networks within organoid structures.

Vascularized organoids not only enhance the diffusion of oxygen and nutrients, thereby improving overall tissue viability, but also serve as valuable platforms for studying vascular endothelial growth factor (VEGF)-mediated signaling pathways and tumor angiogenesis. In addition, they are widely used for screening anti-angiogenic therapeutics and evaluating their efficacy in more physiologically relevant in vitro models.

Image adapted from *Pharm Biol*, 2025.

• Organoids + Microorganisms

To investigate the pathogenic mechanisms of infectious diseases and the dynamics of microbial dysbiosis, specific bacteria (such as Escherichia coli, Salmonella, or Clostridioides difficile) or viruses can be introduced into intestinal, gastric, or respiratory organoid systems. This approach commonly utilizes microinjection to deliver microorganisms into the enclosed luminal space of conventional “basal-out” organoids.

In contrast, more recently developed “apical-out” organoid models expose the apical surface of epithelial cells directly to microbial suspensions, enabling more physiologically accessible host–pathogen interactions. This co-culture system allows researchers to better simulate key aspects of infection, including pathogen colonization, epithelial barrier disruption, and mucosal immune responses under physiologically relevant oxygen conditions.

Image adapted from *Foods*, 2022.

IV. What are the primary methods for constructing co-culture models?

Currently, driven by advancements in tissue engineering and biomaterials, the construction of organoid co-cultures has evolved into a variety of technical approaches. Based on the degree of physical cell-to-cell contact and the complexity of the microenvironment, these methods can be broadly categorized into the following four construction strategies:

• Direct Co-culture in Matrigel

This is currently the most widely used and technically straightforward co-culture approach. Parenchymal organoids are first enzymatically dissociated and then mixed with non-parenchymal cells—such as immune cells or fibroblasts—at defined ratios. The cell mixture is subsequently resuspended in a liquid extracellular matrix (e.g., Matrigel or basement membrane extract, BME), which solidifies at 37 °C to form a three-dimensional scaffold, after which culture medium is added to initiate 3D growth.

This method enables direct physical contact between heterogeneous cell populations, providing a more intuitive recapitulation of in vivo processes such as cell–cell recognition, adhesion, and extracellular matrix remodeling. It is frequently applied in tumor organoid–immune cell co-cultures, particularly with CAR-T cells, to evaluate antigen-specific cytotoxicity, as well as in fibroblast co-culture systems to investigate the formation of stromal barriers and tumor–stroma interactions.

• Transwell Indirect Co-culture

This method employs a Transwell insert equipped with a microporous membrane to divide the culture system into two distinct compartments: an upper and a lower chamber. Typically, Matrigel-embedded organoids are placed in one chamber, while other cell types—such as vascular endothelial cells or specific immune cell subsets—are seeded in the opposite compartment.

This configuration physically separates the different cell populations, preventing direct cell–cell contact while still allowing the exchange of soluble factors through the microporous membrane. Molecules such as chemokines, cytokines, and metabolic byproducts can freely diffuse between compartments. As a result, this system is primarily used to investigate paracrine signaling mechanisms and to evaluate chemokine-mediated long-range recruitment of immune cells.

• Air-Liquid Interface (ALI) Culture

Primary tissue fragments or organoid-containing hydrogels are directly placed onto the upper surface of a porous membrane insert. During culture, the medium in the upper compartment is removed, exposing the apical surface of the organoids to air, while the basal side remains in contact with nutrient-rich medium supplied from the lower compartment through the membrane.

This air–liquid interface (ALI) configuration enables the establishment of a physiologically relevant oxygen gradient, which is essential for the polarization and functional maturation of respiratory and gastrointestinal organoids. In addition, ALI culture allows for the in situ preservation of endogenous immune and stromal components—such as tumor-infiltrating lymphocytes (TILs)—that are inherently present in primary tissues, thereby eliminating the need for exogenous immune cell supplementation.

• Microfluidic Organ-on-a-Chip

This represents a highly sophisticated co-culture platform based on micro-engineering technology. Using micro-electro-mechanical systems (MEMS) techniques, microchannels and compartmentalized chambers are fabricated on miniaturized chips, typically constructed from polymeric materials such as polydimethylsiloxane (PDMS). Within these devices, organoids and other cell types are cultured in spatially defined regions, while integrated microfluidic pump systems enable continuous and controlled fluid perfusion.

This configuration allows for precise regulation of fluid shear stress, thereby more closely recapitulating in vivo microvascular circulation. It also enables the generation of well-defined biochemical gradients and mechanical forces, such as tensile stress, within the culture environment.

Its primary applications include the construction of dynamically perfused vascularized organoids, the development of multi-organ microphysiological systems (e.g., liver–gut axis models for drug metabolism studies), and high-throughput pharmacokinetic and toxicity screening.