Organoids, defined as 3-dimensional (3D) organ-like structures grown in vitro, represent a significant breakthrough in both biological and clinical research. These structures offer a near-physiological reproduction of native tissues and have emerged as transformative alternatives to traditional monolayer cell cultures and animal models [1,2]. The limitations of 2-dimensional (2D) cell monolayers—namely, the lack of complexity of tissue interactions and the often-inconsistent human relevance of animal models—have highlighted the need for more accurate in vitro 3D models that can replicate human organ structures and functions [3]. Organoids are primarily constructed from cells capable of self-renewal and self-organization, including embryonic stem cells, induced pluripotent stem cells, primary tissues, adult stem cells, and cancer stem cells [4]. These cells undergo proliferation, differentiation, and self-organization to form structures that closely resemble in vivo organ systems [5]. With advancements in stem cell biology and biomaterials, a variety of human organoids have been developed that effectively simulate the complex structures and functionalities of organs, such as the brain, intestine, liver, kidney, lung, and various tumors [6-11].

Organoid research has been significantly influenced by two key advancements: the exploration of decellularized extracellular matrix (dECM) hydrogels and their integration with 3D bioprinting technology [12]. dECM hydrogels, which are vital for organ development and function, provide a platform for cultivating organoids that replicate physiological conditions more closely and facilitate appropriate cell behavior, replication, and disease modeling [12,13]. Furthermore, 3D bioprinting technology marks a transformative phase in organoid research by enhancing the structural and functional complexity of organoid models and expanding their application in pharmacology and regenerative medicine [14].

Our review explores the transformative potential of dECM hydrogels for advanced organoid culture and the integration of 3D bioprinting technology into organoid research (Fig. 1). We delve into the diverse applications of dECM hydrogels in various organ systems, highlighting their crucial role in maintaining the structural integrity and enhancing the functional attributes of tissues and organs. Moreover, 3D bioprinting technology has been shown to mimic human tissue architecture, augmenting the biological relevance and functionality of organoids. These advancements not only represent a paradigm shift in tissue engineering and regenerative medicine, but also open new avenues for precise disease modeling, drug testing, and personalized therapeutic strategies [15].

At the forefront of this revolution has been the synergistic application of tissue-specific dECM hydrogels and 3D bioprinting technology, which play a crucial role in creating more physiologically accurate organoids [16]. The convergence of these technologies not only advances our understanding of complex medical conditions, but also opens doors to new possibilities for effective and personalized healthcare solutions [17]. Hence, this integration signifies a promising future in medical research with significant implications for disease modeling, drug discovery, and the development of patient-specific treatments [14].

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

Previous methodologies for organoid culture primarily relied on the influence of biochemical signals from the composition of the culture medium and intercellular interactions to regulate growth [18]. Recent research has underscored the crucial role of the chemical composition of the extracellular matrix (ECM), as well as mechanical signals related to the expansion and differentiation of organoids [19]. During organoid formation, the ECM provides niches for stem cells through cell-matrix interactions. The chemical and mechanical characteristics of the ECM significantly affect the cellular phenotype. However, in certain circumstances, the ECM can inadvertently induce aberrant cellular states, such as those observed in diseases or tumors, illustrating the complex interplay between the ECM and cellular behavior [20]. Therefore, the stem cell signaling pathways involved in organoid formation require a highly specific ECM environment [5]. The ECM encloses cells within a complex fibrous network composed of various macromolecules such as glycoproteins, proteoglycans, and glycosaminoglycans. Advancements in our understanding of the extracellular milieu have facilitated the development of sophisticated 3D culture systems for organoids supported by ECM scaffolds. Providing proper ECM cues to cells for organoid formation and culture is crucial [21]. Diverse scaffold systems, including Matrigel, ECM, natural polymers, and synthetic polymers, have been tailored to meet the requirements of organoid cultures for application in clinical settings [22].

Each system has its advantages and disadvantages for organoid culture. Matrigel is one of the most common substrates for organoid culture because it can be readily obtained as a commercialized product and is easy to handle [23]. Matrigel is composed of various elements, including laminin, collagen type IV, and growth factors, which are essential for growth of organoids [24]. However, Matrigel is derived from a mouse tumor, raising concerns about the potential transmission of animal pathogens that could impact the immune system [25]. Additionally, Matrigel has ECM components associated with tumors, such as laminin, and is considerably different from matrisomes in normal tissues. Therefore, it could provide improper tissue-specific cues for targeting human organs in organoid culturing systems [26]. Thus, numerous researchers have attempted to find and develop alternatives to Matrigel [27]. Natural polymers, including collagen, fibrin, gelatin, and alginate, are preferred as alternatives to Matrigel because of their bioactivity and similarity to the human ECM. Their microarchitectures, such as microfibers and microspheres, promote the attachment and proliferation of organoids [28]. However, natural polymers may display variable gelation kinetics, complicating the attainment and preservation of the intended scaffold architecture. This inconsistency can affect the uniformity and reproducibility of organoid cultures, which often fail to replicate precise tissue-specific ECM compositions [29]. Synthetic hydrogels have also emerged to support organoid cultures, offering a solution for the complexity and variability of natural matrices [30]. Synthetic polymers, such as polyethylene glycol (PEG), polyacrylamide (PAAM), and polyvinyl alcohol (PVA) are usually chemically defined, and their mechanical properties and chemical composition can be accurately modulated to enhance organoid differentiation [31]. However, synthetic polymers still exhibit low bioactivity and insufficient tissue-specific ECM composition. Therefore, the incorporation of a dECM hydrogel in organoid culture increases the physiological relevance of these models by providing a biomimetic microenvironment and facilitates the investigation of cell behavior in a context that closely resembles native tissue, enabling more accurate and insightful research outcomes.

dECM, which contains tissue-specific compositions and molecules, plays a vital role in organoid culture by providing appropriate biochemical and biophysical signals that stand out from other biomaterials used in organoid cultures, such as Matrigel and synthetic polymers [32]. The tissue-specific nature of the dECM delivers unique biochemical, biophysical, and biomechanical cues to organoids akin to in vivo cell-matrix communication systems [29]. Considering the diverse composition and organization of ECM across different organs, the use of tissue-specific dECM hydrogels offers a customized approach for organoid culture. This method provides tissue-specific dECM content, creating niche environments for stem cells and a biomimetic microstructure that closely mimics the natural organ context [21]. Furthermore, dECM hydrogels have a native tissue-oriented architecture with topographical cues and elastic properties [29]. These cues activate or deactivate the cellular mechanisms underlying cell adhesion, migration, proliferation, and differentiation. Furthermore, Saldin et al. [33] investigated the cellular responses of survival, proliferation, differentiation, and tissue-specific functions using a dECM hydrogel as an organoid culture scaffold. Prior studies have investigated the efficacy of dECM hydrogels as scaffolds for organoid culture across various organoid culture systems. As detailed in Table 1, liver organoids [34], brain organoids [35], endoderm-derived human organoids [16], and islet organoids [36], when combined with a dECM, exhibit improved functions resembling those of native tissues.

Saheli et al. [34] investigated whether a sheep liver dECM hydrogel could promote the physiological function of liver organoids formed with human hepatocarcinoma cells, human endothelial cells, and mesenchymal cells. Self-organized liver organoids expressed albumin and cytochrome P450 3A4 (CYP3A4), key markers of liver function, when cultured in the liver dECM hydrogel (Fig. 2A). The 3D liver dECM gel significantly enhanced the functional activity of these organoids compared to traditional 2D culture and collagen gel-based culture at both the gene (e.g., albumin) and protein levels (albumin secretion and urea production). Similarly, Simsa et al. [35] demonstrated that human embryonic stem cells (hESCs) cultured within brain-derived dECM hydrogels exhibited gene expression patterns and differentiation profiles remarkably similar to those observed in hESCs grown in Matrigel. This finding suggests that brain dECM hydrogels possess the necessary biochemical and structural properties to effectively support hESC culture.

Consequently, these dECM hydrogels have emerged as promising alternatives to Matrigel, particularly for applications requiring a more physiologically relevant ECM environment. This equivalence in performance highlights the potential of brain dECM hydrogels in stem cell research and opens new avenues for exploring tissue-specific interactions in neurodevelopmental studies [35]. Giobbe et al. [16] explored the potential of a small intestine (SI) mucosa/submucosa dECM hydrogel as a culture environment for endoderm-derived human organoids, including gastric, hepatic, pancreatic, and SI organoids. Gastric organoids cultured in this hydrogel expressed epithelial markers, such as zonula occludens-1, epithelial cadherin, and actin, along with gastric-specific markers such as ezrin and mucin-5AC (Fig. 2B). SI organoids were successfully maintained for eight consecutive passages over two months, as shown by morphological analyses (Fig. 2C). Additionally, Giobbe et al. [16] demonstrated the angiogenic potential of endoderm-derived human organoids cultured in SI dECM gel using a chick chorioallantoic membrane assay. Bi et al. [36] investigated the ability of pancreatic dECM hydrogels to induce the self-assembly of human islet organoids via pluripotent stem cell differentiation. Islet organoids cultured on Matrigel-coated substrates supplemented with pancreatic dECM expressed all hormone-secreting pancreatic islet cell types, detected using C-peptide, glucagon, somatostatin, and pancreatic polypeptide expression (Fig. 2D). Therefore, tissue-specific dECM hydrogels hold significant potential as culture scaffolds for organoids. They not only maintain the structure of these organoids but also promote crucial functions specific to each tissue and organ.

The integration of 3D bioprinting technology into organoid development represents a groundbreaking advance in biomedical research. This technology offers a unique combination of meticulous precision and control over cell and biomaterial placement, along with the ability to produce complex large-scale structures [37]. As highlighted by Murphy and Atala, this precise control enables the creation of highly organized and structured organoid models that surpass the traditional methods in accurately mimicking the human tissue architecture [12]. The capabilities of 3D bioprinting extend beyond the micrometer and millimeter scales. Kang et al. [38] demonstrated the ability to bioprint complex structures at the centimeter scale, including lumens, branched vasculature, and tubular intestinal epithelia with in vivo-like features. The scalability of 3D bioprinting is crucial for high-throughput screening and clinical applications. Furthermore, 3D bioprinting enables the customization of organoids to cater to specific patient needs or disease characteristics, thereby advancing personalized medical applications, as discussed by Zhang et al. [39]. This aspect of customization is a key feature of bioprinting-assisted tissue emergence, a concept introduced by Brassard et al. [40] that utilizes a syringe-based extrusion system paired with a manually controlled stage. The integration of these capabilities, ranging from micro-to macroscales, coupled with customization potential, highlights the revolutionary role of 3D bioprinting technology in organoid research, which broadens our knowledge and abilities in fields such as development, regeneration, and disease modeling [41].

These advancements are aligned with significant progress in organoid research. For example, in pharmacology, significant strides have been made in bioprinting liver organoids that mimic the critical functions of human liver tissue, primarily for drug toxicity assessments [42]. This achievement exemplifies the potential of 3D bioprinting to create complex organ structures. Additionally, skin organoids with integrated vascular structures have been developed to improve wound healing in animal models [43]. Similarly, previous cancer research demonstrated the use of bioprinted breast cancer organoids to study tumor growth and drug resistance, marking a significant advancement in the understanding of cancer biology [44,45]. Moreover, the development of a hydrogel-in-hydrogel live bioprinting system that guides neural axon directionality in organotypic spinal cords demonstrates the potential of 3D bioprinting in neural applications, particularly in spinal cord regeneration [41]. Collectively, these diverse applications showcase the broad spectrum of 3D bioprinting in organoid research, impacting fields ranging from pharmacology to personalized medicine. This technology opens new avenues for treatment development and offers a deeper understanding of complex biological processes.

The integration of 3D bioprinting into organoid research has led to a series of innovative advancements across various medical fields, reflecting the vast potential and impact of this technology [46]. This was exemplified by a groundbreaking vascularized lung cancer (LC) model by Choi et al. [47]. This model integrated patient-derived LC organoids (LCOs), lung fibroblasts, and perfusable vessels using 3D bioprinting (Fig. 3A). Notably, the use of a lung-derived dECM hydrogel in this model played a critical role in creating an authentic LC microenvironment, particularly when incorporating lung fibroblasts derived from idiopathic pulmonary fibrosis (Fig. 3B). This approach boosted cell proliferation and increased the expression of drug resistance genes in LCOs, offering insights into developing targeted therapies and identifying biomarkers for fibrosis in LC patients [47].

Simultaneously, a breakthrough in gastric cancer research was achieved with the development of a vascularized organoid model (VOM) (Fig. 4A). Kim et al. integrated patient-derived gastric cancer organoids, functional endothelium, and stomach-derived dECM (Fig. 4B) [48]. This innovative VOM exhibited the potential to contribute to personalized medicine efforts by accurately predicting individual patient responses to therapies targeting the vascular endothelial growth factor receptor 2 (VEGFR2). A key example is the response to ramucirumab, a U.S. Food and Drug Administration-approved therapy that inhibits VEGFR2. This treatment has been effective in curbing cancer cell growth and improving survival rates in gastric cancer patients, reflecting real-world treatment outcomes (Fig. 4C).

Table 2 presents the use of 3D bioprinting technology, which has substantially advanced the biological accuracy and functional capacity of organoids, leading to more realistic in vitro models that reflect the complex microenvironments of living human tissues [41,47-50]. These advancements have enabled the more accurate replication of tissue microenvironments, leading to more precise disease modeling and effective drug testing, as described by Wang et al. [51]. Furthermore, these 3D bioprinted organoids have demonstrated improved longevity and stability, facilitating longer-term studies and experiments, and marking a notable advancement over traditional organoid cultures [5]. The integration of 3D bioprinting with innovative technologies like microfluidics and biosensors has further increased the sophistication and functional relevance of these 3D organoid models [52-54].

The integration of 3D bioprinting technologies and dECM hydrogels in the field of organoid research represents a significant leap toward revolutionizing our understanding of human biology and advancing medical practices. The synergy between these advanced techniques dramatically enhances the precision, complexity, and fidelity of bioprinted structures, thereby expanding the potential applications of organoids in biomedical research, regenerative medicine, and development of personalized therapeutic strategies. Although dECM hydrogels provide a biomimetic scaffold that facilitates organoid growth, their inherent limitations in mechanical strength and printability pose challenges. These challenges can be addressed by incorporating innovative materials such as gelatin methacrylate, hyaluronic acid, alginate, and gelatin [55]. Such modifications not only increase the structural integrity and longevity of organoid models but also facilitate the creation of more complex and faithful representations of native tissue architecture and microenvironments [5,56].

However, advancing these technologies entails navigating the intricate tradeoffs between the precision of 3D bioprinting technology and the complex microenvironments that organoid cultures aim to mimic. As previously mentioned, the integration of vascular networks and functional components within a bioprinted organoid is essential for mimicking dynamic tissue interactions, whereas preserving natural organoid development within 3D bioprinting constraints poses significant challenges [57-60]. Furthermore, the introduction of new materials (e.g., other natural and synthetic polymers) into dECM hydrogels can alter their tissue-specific properties, requiring careful adjustments to balance their benefits and potential drawbacks [61]. The scalability of bioprinting for high-throughput applications such as drug discovery and personalized medicine presents significant opportunities and challenges [12,62,63]. Overcoming limitations related to the resolution, scalability, and slow maturation processes of current 3D bioprinting technologies is essential [64,65]. Moreover, as the field matures, the development of standardized protocols and regulatory guidelines is crucial to ensure the safety, efficacy, and ethical deployment of bioprinted products [66,67].

Blatchley and Anseth have emphasized the importance of middle-out strategies in organoid development. These strategies integrate the macroscale control of top-down approaches with the precise cellular and extracellular assembly characteristics of bottom-up methods. This ensures precise guidance for organoids to develop with specific geometries, complex structures, and functions. [68]. This 3D bioprinting-technology-based approach to spatiotemporal tissue engineering achieves an unparalleled level of biomimicry, increasing the clinical relevance of organoids by ensuring precise control over engineered cellular environments and providing a balanced framework for cellular self-organization [68]. This combination of structural advantages and detailed modular construction advances the field toward addressing the challenges and maximizing the potential of 3D bioprinting technology and organoid research to revolutionize healthcare. Continued investment in translational research is essential to validate the therapeutic potential of bioprinted organoids in clinical applications, paving the way for a new era in precision medicine and personalized healthcare solutions that offer profound insights into human biology and innovative therapeutic strategies.