The evolving paradigm of organoids: advances in cell line-derived organoids with 3-dimensional printing and emerging technologies

Hyungjin Mun; Min Heui Yoo

doi:10.51335/organoid.2025.5.e9

Organoid > Volume 5; 2025 > Article

Mun and Yoo: The evolving paradigm of organoids: advances in cell line-derived organoids with 3-dimensional printing and emerging technologies

Review Article

Organoid 2025;5:e9.

Published online: September 25, 2025

DOI: https://doi.org/10.51335/organoid.2025.5.e9

The evolving paradigm of organoids: advances in cell line-derived organoids with 3-dimensional printing and emerging technologies

Hyungjin Mun

, Min Heui Yoo

Division of Next-Generation Nonclinical Research, Korea Institute of Toxicology, Daejeon, Korea

Correspondence to: Min Heui Yoo, DVM, PhD Division of Next-Generation Nonclinical Research, Korea Institute of Toxicology, 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, Korea E-mail:minheui.yoo@kitox.re.kr

Received August 8, 2025 Revised August 29, 2025 Accepted August 29, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Organoid research has rapidly emerged as a prominent field within the life sciences, particularly in biomedical research. Stem cells represent the primary source of human organoids due to their exceptional ability to reproduce organ-specific functions and differentiation pathways. Organoids derived from pluripotent stem cells or other stem cells have proven to be powerful platforms for studying developmental biology, disease mechanisms, and therapeutic responses. Nonetheless, stem cell-derived organoids face several critical limitations, including batch-to-batch variability, lengthy maturation periods, and ethical concerns—particularly with the use of embryonic stem cells. In response to these challenges, alternative approaches have gained attention, especially the generation of organoids from established cell lines. Organoids derived from immortalized or cancer cell lines present distinct advantages, including greater genetic stability, rapid expansion, reproducibility, and scalability. These features make them particularly suitable for high-throughput applications such as toxicological screening and drug discovery. Furthermore, recent technological advancements—including 3-dimensional bioprinting, microfluidic systems, and co-culture platforms—have enhanced the architectural fidelity and functional complexity of cell line-derived organoids. Such innovations are driving the evolution of organoid platforms beyond conventional stem cell models. Ultimately, the full translational potential of cell line-derived organoids in both preclinical and clinical contexts will depend on the establishment of standardized experimental protocols and consistent regulatory frameworks.

Keywords: Cell culture techniques; Bioprinting; Organoids; Ecotoxicology

Introduction

Organoids are 3-dimensional (3D) cell aggregates cultured in vitro to reproduce the microarchitecture and functions of specific organs. Their structural fidelity arises from the spatial organization of multiple cell types—such as epithelial, stromal, and endothelial cells—that collectively replicate the organ-specific microenvironment [1]. In addition, organoids mimic tissue-level functionality by performing physiological activities characteristic of native organs, including hormone secretion, metabolic activity, and electrical signal transmission [2]. Owing to these properties, organoids have become widely used in life science research, including disease modeling, drug screening, and regenerative medicine [3,4]. Organoids are most commonly derived from pluripotent stem cells (PSCs) (embryonic stem cells [ESCs] and induced PSCs [iPSCs]) or adult stem cells (ASCs), which possess self-renewal and self-organization capabilities that enable them to form organ-like structures with comparable cellular composition, architecture, and physiological function [4–6]. However, stem cell-derived organoids have several drawbacks. They frequently display poor reproducibility between batches and require prolonged differentiation times [7]. Due to their complex protocols, organoid quality can vary depending on the researcher’s expertise. In the case of ESCs, ethical concerns are unavoidable, and the need for costly reagents—such as specialized media, growth factors, and matrix supports like Matrigel—represents another significant limitation [3,8].

To overcome these challenges, researchers have pursued new strategies for organoid development. With advances in 3D bioprinting, microfluidics, and engineered extracellular matrix (ECM) or hydrogel systems, organoids derived from established cancerous and non-cancerous cell lines—cultured under 3D conditions to enable self-organization—are now being actively explored [9]. These cell line-derived organoids provide key advantages, including high reproducibility, scalability, cost- and time-efficiency, and compatibility with genome-editing technologies [10]. As a result, their application in high-throughput screening (HTS) for drug discovery and toxicological testing continues to expand [11,12]. How are cell line-derived organoids reshaping the landscape of biomedical research? This review addresses that question by examining the transition away from stem cell-based systems and emphasizing the enabling role of technologies such as microfluidics and 3D bioprinting in unlocking new efficiencies and applications.

Stem cell-derived organoids

Ethics statement: This study was a literature review of previously published studies and was therefore exempt from institutional review board approval.

1. Definition and characteristics

Stem cell-derived organoids are 3D culture models generated from ESCs, iPSCs, or ASCs, all of which are capable of differentiating into multiple cell types (Fig. 1) [13]. Through self-directed organization, these cells develop into small tissue-like structures that resemble real organs in morphology, cellular composition, and physiological activity. Consequently, they are widely employed to investigate disease mechanisms and to explore strategies for tissue repair and regeneration [14,15]. Their intrinsic capacity to differentiate into diverse cell types and establish spatial organization through intercellular communication and morphogen gradients enables researchers to study tissue-specific functions in vitro under conditions that approximate physiological environments [16,17].

A key strength of stem cell-derived organoids lies in their ability to model organ development and replicate pathological processes. Cerebral organoids generated from PSCs, for example, can form distinct brain-like regions, thereby providing experimental access to complex neurodevelopmental disorders such as microcephaly [18]. Similarly, ASC-derived intestinal organoids give rise to crypt-villus structures that closely mimic the architecture of the human gut epithelium, supporting research on gastrointestinal disorders and host–microbe interactions [19]. These platforms have proven invaluable for modeling genetic diseases, infectious processes, and tumorigenesis in controlled experimental settings.

Beyond disease modeling, applications in regenerative medicine are expanding rapidly. Organoids are being explored as potential sources for tissue reconstruction and cell-based therapies in fields such as neuroscience, hepatology, and gastroenterology [20–23]. However, significant technical and ethical challenges remain. Variability in differentiation efficiency between batches, lengthy culture periods, and persistent debates surrounding the use of ESCs continue to limit broader clinical translation and standardization [24].

2. Limitations of stem cell-derived organoids

Despite their transformative contributions to disease modeling, drug screening, and regenerative medicine, stem cell-derived organoids face persistent limitations that hinder their widespread adoption in clinical and industrial contexts (Fig. 1). Among the most pressing concerns is the substantial heterogeneity between organoid batches. This variability arises from differences in stem cell source material, subtle changes in culture conditions, and inconsistencies in differentiation protocols [25]. Because these systems depend heavily on intrinsic self-organization, even minor perturbations in signaling pathways can result in divergent phenotypic outcomes [26]. This unpredictability poses major obstacles to reproducibility, particularly in applications where consistency is essential, such as high-throughput pharmacological screening or patient-specific therapeutic modeling [27].

Another critical limitation is the slow pace of organoid maturation. Depending on the organ being modeled, differentiation may require several weeks or even months [28]. For example, the generation of cerebral organoids necessitates extended culture to achieve region-specific patterning, often at the cost of cellular stability and viability. Prolonged cultivation increases the risk of hypoxia, metabolic stress, and apoptosis within organoid cores, thereby delaying experimental timelines and complicating data interpretation [29]. This lengthy maturation process restricts scalability and limits the suitability of stem cell-derived organoids for applications that demand rapid and reproducible tissue generation.

In addition to technical hurdles, ethical and economic considerations further constrain routine use. The derivation of ESC-based organoids raises persistent moral concerns associated with embryo destruction, while iPSC-based models, though less ethically contentious, still require costly and labor-intensive reprogramming protocols [30]. The financial burden is compounded by the dependence on expensive supplements, including specialized growth factors, ECM components such as Matrigel, and bioreactor systems—all of which substantially increase the overall cost of production [31].

To address these challenges, research efforts are increasingly focused on organoids derived from established cell lines. By employing immortalized or cancer-derived cell lines, researchers can generate standardized 3D tissue models without the need for extended differentiation. Owing to their genetic stability and rapid proliferation, such models can be produced in large quantities, making them well-suited for toxicology testing, drug development, and routine laboratory use. Moreover, recent advances in 3D bioprinting, microfluidic platforms, and synthetic ECM scaffolds have significantly enhanced the structural fidelity and functional complexity of organoids. These technologies are expected to drive the development of next-generation organoid platforms that combine scalability with physiological relevance [32].

Cell line-derived organoids: a paradigm shift

1. Concept and advantages

Organoids generated from established cell lines are increasingly recognized as a practical and reliable alternative to stem cell-derived systems. These models—derived from immortalized, tumor-derived, or primary human cell lines—possess favorable properties such as enhanced reproducibility, genetic stability, and reduced experimental complexity (Fig. 2) [33,34]. Unlike stem cell-derived organoids, which often require lengthy and variable differentiation protocols, cell line-based systems offer a more uniform cellular baseline that minimizes variability across experiments [35].

Their reliability makes them especially attractive for high-throughput applications, including drug discovery and toxicological profiling [36]. This advantage is largely due to the genetic stability and thorough characterization of the underlying cell lines, which result in minimal inter-batch variability and consistent phenotypic responses [37]. In contrast, stem cell-based models often exhibit inconsistencies in differentiation that complicate the interpretation of experimental outcomes.

Beyond reproducibility, the practical benefits of using cell line-derived organoids are also noteworthy. While stem cell-derived models may require weeks or even months to mature, organoids from established cell lines can be generated and expanded within days, significantly improving experimental throughput and scalability [28,35]. This efficiency is particularly valuable for large-scale drug screening initiatives, time-sensitive toxicological assessments, and environmental exposure studies.

The integration of gene-editing technologies—most notably CRISPR/Cas9—further enhances the versatility of cell line-derived organoids [38]. These tools enable precise genetic modifications, including targeted mutations, insertions, deletions, and reporter gene integration, thereby allowing the creation of highly customizable disease models [39]. By engineering cell lines in this way, researchers can dissect oncogenic signaling pathways, explore drug resistance mechanisms, and evaluate therapeutic responses in controlled settings. Such engineered organoids are also well suited to monitor dynamic changes in tumor biology, providing insights into heterogeneity and evolutionary trajectories [40]. In oncology, patient-specific tumor organoids derived from cell lines serve as particularly valuable platforms for personalized medicine. They facilitate preclinical evaluation of drug efficacy and resistance, improving treatment predictions while minimizing off-target effects [41]. The compatibility of these organoids with gene-editing approaches strengthens their role in both basic research and translational applications, paving the way for innovative strategies in disease modeling and drug discovery [42].

Nevertheless, cell line-derived organoids should not be viewed as complete replacements for stem cell-based models. The same modifications that confer indefinite propagation—such as telomerase activation or altered checkpoint control—may compromise their ability to fully replicate native tissue physiology. Thus, while they serve as powerful tools for many applications, findings must be interpreted cautiously when extrapolated to in vivo systems.

2. Notable examples in research

Cell line-derived organoids are emerging as versatile platforms in oncology, toxicology, and pharmacology. Their reproducibility and scalability make them particularly well-suited for preclinical applications, from drug screening to mechanistic studies of disease. Unlike conventional 2-dimensional (2D) cultures, which fail to capture the complexity of tissue architecture, organoids generated from immortalized cancer or normal cell lines preserve essential genetic and phenotypic characteristics of native tissues [43–45]. This distinction is especially significant in cancer research. Traditional 2D cancer models have long been criticized for their inability to represent intratumoral heterogeneity, a limitation that reduces translational relevance. In contrast, tumor organoids retain oncogenic mutations and patient-specific genomic alterations, enabling detailed exploration of tumor progression, metastasis, and therapy resistance [42,44]. With the growing emphasis on precision oncology, patient-matched tumor organoids are becoming central to individualized therapy design. Their use in ex vivo drug screening has shown promise for improving treatment response prediction and minimizing adverse effects [46]. Collectively, these developments highlight the expanding role of cell line-derived organoids as bridges between experimental models and clinical decision-making.

Role of 3D printing and emerging technologies

1. 3D bioprinting in organoid fabrication

The introduction of 3D bioprinting has revolutionized organoid engineering by enabling precise spatial control of cells, scaffolding materials, and signaling molecules within defined architectures. Traditional self-organizing organoid systems, although highly valuable, are often limited by structural variability and poor reproducibility across batches. By contrast, 3D bioprinting offers a deterministic approach in which cells are positioned with accuracy, resulting in constructs with greater functional complexity and consistent morphology (Table 1) [9,47–55].

This degree of spatial control is particularly advantageous in contexts that demand reproducibility and scalability, including drug discovery pipelines, patient-specific tissue modeling, and regenerative medicine [48,49]. In recent years, multiple bioprinting platforms have been refined for organoid fabrication. Extrusion-based bioprinting remains widely used because of its ability to deposit high-viscosity bioinks and generate dense, cell-rich constructs that maintain viability during extended culture. Inkjet bioprinting, by contrast, enables rapid, high-resolution patterning of cells and biomolecules, making it suitable for throughput-oriented applications. Laser-assisted techniques achieve even higher resolution, facilitating the meticulous placement of individual cells or ECM components [50,51].

Beyond spatial precision, 3D bioprinting also allows the incorporation of biochemical signals—such as growth factors, adhesion peptides, and ECM proteins—directly into bioinks. These inclusions guide cellular behavior, promoting differentiation, organization, and maturation during organoid development [52]. One of the most significant outcomes of this approach has been the fabrication of vascularized organoids. By pre-patterning vascular channels or co-printing with endothelial cells, researchers have begun to address the diffusion limitations that often impair nutrient and oxygen delivery in traditional spheroid models. Bioprinted kidney, liver, and heart organoids incorporating vascular networks demonstrate improved physiological activity and are increasingly used in disease modeling, drug metabolism assays, and regenerative therapies [53–55]. Nevertheless, challenges remain before bioprinted organoids can be broadly implemented. Optimization of bioink formulations, refinement of print resolution without compromising cell viability, and the development of dynamic culture systems to support maturation remain active areas of research. Addressing these bottlenecks will be essential for the clinical and industrial translation of bioprinted organoid systems.

2. Microfluidics and organ-on-a-chip technologies

The integration of microfluidic technology—commonly referred to as organ-on-a-chip platforms—has introduced a new dimension of physiological relevance to organoid systems by simulating dynamic in vivo conditions such as fluid shear stress, nutrient perfusion, and mechanical cues [56]. In contrast to static culture systems, which often suffer from uneven nutrient delivery and limited gas exchange, microfluidic platforms provide continuous perfusion, thereby improving cellular viability, differentiation efficiency, and tissue maturation [57].

Microfluidic technologies offer sophisticated platforms capable of generating tightly controlled microenvironments that closely replicate the physiological conditions experienced by cells and tissues in vivo (Table 2) [56–62]. This control allows researchers to investigate cellular behaviors and tissue-level interactions with greater fidelity than conventional static cultures [58]. One of the most valuable features of these systems is their ability to replicate biomechanical forces, including cyclic stretching and shear stress, while establishing finely regulated biochemical gradients that guide cellular functions and sustain tissue homeostasis. For example, lung-on-a-chip devices mimic the rhythmic mechanical expansion and contraction associated with breathing, offering a robust platform for studying pulmonary pathophysiology and evaluating therapeutic interventions under physiologically relevant conditions [59,60]. Similarly, gut-on-a-chip models reproduce peristaltic movements and intestinal fluid flow, facilitating improved analysis of barrier integrity, immune responses, and host–microbiome interactions that are difficult to capture in static systems. The incorporation of sensors and real-time monitoring capabilities further improves these platforms, enabling researchers to track dynamic changes and responses in organotypic models and thereby advancing the study of human health and disease [61].

The development of multi-organ-on-a-chip systems has further extended the utility of microfluidic platforms by enabling systemic toxicity and pharmacokinetic studies. These configurations allow simulation of drug distribution and metabolism across interconnected tissue interfaces [62]. Despite these advances, technical challenges remain, particularly in achieving standardized device fabrication, maintaining long-term culture stability, and scaling platforms for widespread clinical translation.

Looking ahead, the integration of artificial intelligence-driven monitoring systems and robotic automation is expected to enhance the precision, reproducibility, and scalability of microfluidic organoid models. These innovations may usher in a new era of high-content, high-throughput organoid research that bridges laboratory science with clinical application.

3. Cell line-derived organoids cultured in ECM-based 3D systems

Embedding cell line-derived organoids within ECM-based 3D systems represents a major advance in in vitro tissue modeling and toxicology research (Fig. 3) [43]. These platforms employ immortalized or tumor-derived cell lines seeded into ECM scaffolds, such as Matrigel, collagen, or engineered hydrogels, to promote the self-assembly of structured, organoid-like formations that more closely mimic the spatial organization of native tissues [63,64]. This 3D configuration creates a microenvironment that supports both cell–cell and cell–matrix interactions, which are largely absent in conventional 2D monolayer cultures.

Compared to traditional flat cultures, ECM-integrated systems facilitate the emergence of tissue-relevant morphology and stimulate the expression of lineage-specific markers, thereby improving the physiological relevance of in vitro assays [65]. Furthermore, the use of established cell lines ensures high reproducibility and scalability—attributes that are essential for large-scale drug discovery and toxicological testing. These models retain the core phenotypic characteristics of the parental cell lines while providing improved experimental control.

In toxicology and pharmacology, ECM-based organoid systems offer a practical and cost-efficient platform for evaluating a broad spectrum of compound-induced effects, including cytotoxicity, metabolic responses, and phenotypic transitions such as epithelial–mesenchymal transition [43,44]. The ECM scaffold not only enhances structural integrity and cellular viability but also supports long-term functional stability, making these systems particularly valuable for studies involving chronic or low-dose chemical exposures.

By striking a balance between experimental complexity and feasibility, ECM-supported cell line-derived organoids provide a critical intermediate model that bridges the simplicity of 2D cultures and the physiological relevance of in vivo animal models. Consequently, they are increasingly recognized as indispensable tools for mechanistic studies as well as preclinical applications.

Challenges and future perspectives

1. Current limitations

Although cell line-derived organoids offer numerous advantages, they also face critical limitations that restrict their capacity to fully emulate human tissue microenvironments [10]. A major drawback is the absence of intrinsic vascular networks. In most existing systems, perfusable vasculature is lacking, resulting in poor oxygen and nutrient diffusion, inefficient waste clearance, and eventual central necrosis during extended culture. These conditions induce hypoxic stress and create non-physiological gradients, undermining the reproducibility and interpretability of experimental outcomes (Table 3) [66].

Another significant limitation involves cellular diversity. Many cell line-derived organoids fail to capture the full multicellular complexity of native tissues. Essential elements such as stromal fibroblasts, endothelial cells, and immune populations are often missing or only partially integrated. This omission limits the ability to model dynamic biological processes, including immune surveillance, angiogenesis, and fibrotic remodeling, which are central to understanding complex pathologies such as tumor–immune interactions and chronic inflammation [67].

Moreover, maintaining phenotypic and functional stability over long-term culture also remains challenging. Without precise regulation of biochemical and mechanical cues, organoids may undergo drift, leading to altered gene expression, loss of functional markers, or inconsistent morphologies. These problems highlight the ongoing need to optimize extracellular matrices, media formulations, and biophysical conditions to preserve organoid integrity and reproducibility [68].

While advances in 3D bioprinting and organ-on-a-chip technologies have begun to mitigate some of these issues, the field still requires substantial innovation in both biological and technological domains. Only through such progress will organoid systems achieve the fidelity and functional complexity required for widespread translational use in biomedicine and toxicology.

2. Future directions

Efforts to improve the translational relevance of cell line-derived organoids are now at a pivotal stage. While monoculture systems have been valuable, they cannot replicate the cellular interplay characteristic of native tissues. A transition toward co-culture models—integrating epithelial, stromal, immune, and endothelial cells—is not simply a technical refinement but a necessary evolution. Such multicellular environments more closely approximate human physiology and allow researchers to study complex processes, including immune evasion in cancer, fibrotic remodeling, and chronic inflammatory responses.

Importantly, these next-generation systems already demonstrate practical utility. For example, tumor organoids enriched with autologous or hematopoietic-derived immune cells enable studies of immune checkpoint function and T cell infiltration under conditions that are far more physiologically relevant than 2D assays. Likewise, hepatic or renal cell co-cultures incorporated into organoid scaffolds show promise in detecting early-stage toxicity signals that traditional assays often miss.

When genome-editing tools such as CRISPR/Cas9 are applied, the potential expands further. Patient-specific mutations can be engineered into organoid models, allowing personalized disease modeling and therapeutic screening. These genetically customized systems hold particular promise for rare disease research and precision medicine, where accurate genotype-to-phenotype modeling opens up a new tier of clinical relevance.

However, technical advances alone will not be sufficient. Progress must be accompanied by systemic infrastructure. Without standardized protocols, reproducibility will remain fragile. Similarly, regulatory consensus—particularly from agencies such as the Food and Drug Administration and European Medicines Agency—will be essential to validate organoid-based assays for clinical application. Analytical interoperability also represents a key challenge: organoid models must integrate seamlessly with HTS platforms, single-cell transcriptomics, and other modern analytical pipelines if they are to be fully integrated into modern research ecosystems.

Ultimately, the question is no longer whether organoid technologies can transform biomedical research, but whether the supporting frameworks—technical, regulatory, and analytical—can evolve rapidly enough to keep pace. Bridging the gap between in vitro innovation and clinical practice depends on building these connective systems to fully unlock the promise of organoid science.

NOTES

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Funding

This work was supported by the Korea Institute of Toxicology (grant number 2710086923; KK-2502) and by the Rural Development Administration (grant RS-2024-00400007).

Data availability

Not applicable.

Fig. 1.

Stem cell-derived organoids are 3-dimensional (3D) tissue models generated through the self-organization of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells. They replicate the structure and function of real organs and are widely applied to study development and disease. However, limitations such as lengthy differentiation, variability in outcomes, high production costs, and ethical concerns restrict their broader use. Their relatively low throughput further reduces their suitability for large-scale screening.

Fig. 2.

Organoid technology is shifting from stem cell-derived models to those based on established cell lines. Stem cell organoids are limited by long differentiation times, high costs, and ethical concerns. In contrast, organoids from immortalized or cancer cell lines provide greater consistency, faster growth, and scalability. Advances such as 3-dimensional bioprinting and organ-on-a-chip systems have further improved organoid architecture and functionality. These innovations enable applications in drug screening, precision medicine, and toxicology by integrating multi-lineage co-cultures of epithelial, stromal, immune, and vascular endothelial cells.

Fig. 3.

Development of a thyroid cancer organoid model using a cell line-based approach and a novel extracellular matrix (ECM) system. (A) Schematic illustration depicting the process of generating thyroid cancer organoids from the human thyroid carcinoma cell line SNU790. The workflow involves embedding dissociated cells in a newly formulated ECM, which supports 3-dimensional growth and self-organization into organoid structures. This model recapitulates key features of thyroid cancer tissue and offers a reproducible platform for investigating tumor biology and therapeutic responses. (B) Confocal microscopy z-stage imaging of established thyroid cancer organoid cultures for morphological and biomarker (TSHR, F-actin) analysis. Scale bar=100 µm. (C) Immunofluorescence microscopy images of thyroid cancer organoids expressing thyroglobulin (Tg), captured using the Operetta CLS high-content imaging system. Scale bar=100 µm. (D) E-cadherin expression: confocal microscopy z-stage imaging of human thyroid cancer organoids following 21-day culture. Scale bar=100 µm. (E) Immunofluorescence microscopy images of thyroid cancer organoids exhibiting expression of vimentin captured with the Operetta CLS high-content imaging system. Scale bar=100 µm. Reproduced from Yoo et al. J Hazard Mater 2024;479:135771, according to the Creative Commons license [43].

Table 1.

Applications and advantages of 3D bioprinting in organoid engineering

Feature/technology	Description	Advantages	Example applications
Spatial precision and architecture	Controlled placement of cells, scaffolds, and signaling cues within 3D structures	Enables reproducible, anatomically organized organoids [9,47]	Patient-specific tissue models, organoid standardization
Extrusion-based bioprinting	Deposition of high-viscosity bioinks for dense, cell-rich constructs	High viability, long-term culture stability [48]	Hepatic, renal, and tumor organoids
Inkjet bioprinting	Droplet-based, high-resolution patterning of cells or biomolecules	High throughput, suitable for screening applications [49]	Drug testing, cancer microarrays
Laser-assisted bioprinting	Non-contact, high-precision placement of individual cells or ECM components	Ultra-fine resolution, minimal mechanical stress [50,51]	Neural or vascular niche modeling
Biochemical integration	Bioinks embedded with growth factors, peptides, and ECM proteins	Promotes directed differentiation and maturation [52]	Endocrine, gut, and neural organoids
Vascularization strategies	Co-printing endothelial cells or pre-patterning vascular channels	Enhances oxygen/nutrient delivery, supports larger tissue volumes [53–55]	Vascularized liver, kidney, and heart organoids
Challenges ahead	Issues with bioink formulation, print fidelity, and maturation protocols	Requires optimization for clinical scalability	Standardized regenerative implants, toxicology models

3D, 3-dimensional; ECM, extracellular matrix.

Table 2.

Applications and advantages of microfluidic-integrated organoids (organ-on-a-chip systems)

Functional feature	Description	Key advantages	Example applications
Dynamic fluid perfusion	Continuous flow of nutrients and oxygen through microchannels	Enhances cell viability, differentiation, and maturation [57]	Liver-on-a-chip, kidney-on-a-chip
Biomechanical cue simulation	Recreates in vivo forces (e.g., shear stress, stretching, peristalsis)	Improves physiological relevance and mechanotransduction studies [56,59,60]	Lung-on-a-chip (breathing), gut-on-a-chip (peristalsis)
Biochemical gradient control	Establishes spatial/temporal concentration gradients	Allows analysis of morphogen signaling and chemotactic responses [58]	Embryonic development modeling, cancer invasion assays
Patient-specific drug testing	Integration of patient-derived organoids for drug response evaluation	Enables precision medicine and personalized therapy screening	Oncology-on-a-chip, inflammatory bowel disease treatment testing
Multi-organ connectivity	Interlinked organoid compartments simulating systemic physiology	Allows pharmacokinetic and systemic toxicity studies [62]	Multi-organ-on-a-chip (e.g., liver–heart–kidney axis)
Automation & AI integration	Real-time monitoring, feedback control, and high-throughput screening using AI	Improves reproducibility, scalability, and predictive power [61]	Robotics-integrated screening platforms

AI, artificial intelligence.

Table 3.

Summary of key limitations and strategic solutions for cell line-derived organoids

Category	Major limitation	Description and impact	Potential solutions and technological approaches
Lack of vascularization	Absence of vascular networks limits oxygen and nutrient supply	Leads to hypoxia and central necrosis during long-term culture, reducing physiological fidelity	Integration of microfluidic vasculature, 3-dimensional bioprinting
Limited cellular diversity	Incomplete incorporation of immune, stromal, and endothelial cells	Impairs modeling of tumor–immune interactions, fibrosis, and chronic inflammation	Development of advanced co-culture systems
Phenotypic instability	Decline in functional and structural stability over time	Causes loss of marker expression and morphological heterogeneity, compromising experimental reproducibility	Optimization of the extracellular matrix, culture media, and mechanical stimuli
Structural complexity	Simplified architecture due to a 2-dimensional or mono-cellular origin	Fails to replicate native tissue histology and complexity; limits disease modeling capability	Use of biomimetic scaffolds and tissue-guided self-assembly
Limited technological scalability	Lack of standardized equipment and interoperable analytics	Hinders reproducibility and industrial application	Incorporation of automated platforms and artificial intelligence-assisted analytics

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