Advances in the Development and Application of Human Organoids: Techniques, Applications, and Future Perspectives

Abstract

Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.

Graphical Abstract.

Introduction

Over the past hundred years, animal models and two-dimensional (2D) cell lines have played crucial roles in biomedical research, leading to significant breakthroughs in understanding developmental processes, disease mechanisms, and drug effects. Despite their contributions, these models have notable limitations that impact their effectiveness. Cell lines, while practical and cost-efficient, are inherently limited by their simplistic, single-layer structure. This approach fails to accurately mimic the complex three-dimensional (3D) environments and cell–cell interactions present in living organisms. Consequently, important aspects of cell behavior, such as heterogeneity and in vivo characteristics, are lost during in vitro culture^1,2. Animal models, though more reflective of human physiology, face their own set of challenges. They are often affected by confounding variables, practical constraints, and differences between species, which can limit their accuracy and applicability to human health. These limitations highlight the need for continued refinement and development of research models to better simulate and understand complex biological systems^3,4.

In recent years, organoid models have revolutionized biomedical research. The term “organoid” originally came from studies of dermoid cysts ⁵ and was first used in the 1960s to describe organ cultures where cells aggregate to form structures similar to organs^6,7. This early research focused on understanding organ formation and development ⁸ . Despite their promise, early organoid cultures faced significant challenges. Researchers lacked a deep understanding of how the stem cell environment influences self-renewal and differentiation. As a result, organoid cultures often required large numbers of starting cells, had low viability in vitro, and could not be sustained for long periods ⁹ . These issues limited their practical applications. A major breakthrough came in 2009 when Sato et al. successfully cultured intestinal organoids from intestinal stem cells (ISCs) without the need for stromal cells. This advancement marked the beginning of a new era in organoid research ¹⁰ . Today, scientists have successfully developed 3D organoids that mimic a range of organs, including the colon^11–13, esophagus^14,15, pancreas^16,17, liver^18,19, prostate^20,21, and mammary gland^22,23, as well as corresponding tumor organoids.

As organoid technology has matured, it has developed into distinct research areas. In 2014, Lancaster et al. precisely defined organoids as collections of organ-specific cell types derived from stem cells or progenitors, which self-organize through cell sequencing and spatially restricted lineage differentiation, mimicking processes in the body ⁶ . This review aims to explore various types of organoids and their origins, offering a comprehensive overview of their main categories. We evaluate their potential applications in regenerative medicine, disease modeling, drug discovery, toxicity assessment, and microecology, while also addressing the current limitations and challenges that need to be addressed. As a leading innovation in biomedical research, organoids are opening new avenues for scientific exploration with their distinctive advantages.

Organoid Models Derived From Different Tissues and Cells

The development of organoids in vitro represents a major advancement in biomedical research, driven by our growing understanding of stem cell biology and tissue regeneration. This journey began in 1907 when Henry Van Peters Wilson demonstrated that sponge cells could regenerate an entire organism through self-organization ²⁴ (Fig. 1). This early work laid the foundation for studying biological regeneration in the laboratory. Following Wilson’s discovery, researchers conducted dissociation-reaggregation experiments with cells from amphibian pronephros and chicken embryos ⁷ . These experiments showed that cells could be reassembled into different types of organs, advancing our knowledge of cell behavior and tissue formation. In 1964, Malcolm Steinberg proposed the differential adhesion hypothesis, explaining how cells sort and arrange themselves based on their adhesive properties. This theory provided crucial insights into cell organization and supported the development of stem cell research. A significant milestone occurred in 1981 with the successful isolation of pluripotent stem cells (PSCs) from mouse embryos. This achievement opened new possibilities in stem cell research and regenerative medicine^25,26. In 1998, scientists further advanced the field by isolating and culturing human embryonic stem cells (hESCs) from blastocysts, marking a new era in studying human development and disease ²⁷ . The development of induced pluripotent stem cells (iPSCs) in 2006 was another breakthrough. By reprogramming adult cells to become pluripotent, researchers could create patient-specific stem cells without the ethical issues associated with embryonic cells^28–30. Today, organoid research leverages these advancements to recreate the stem cell microenvironment in the lab. This approach allows scientists to guide stem cells into forming specific organs, enhancing our ability to study tissue development, model diseases, test drugs, and explore regenerative medicine. This progression from early experiments to modern techniques highlights the significant strides made in mimicking complex biological systems in vitro.

Figure 1.

Timeline of organoid development. The key milestones in organoid technology are presented chronologically from left to right, with each point highlighting a significant advancement in the field.

Stem cells are fundamental to the creation of organoids, as they can differentiate into various cell types necessary for organ development and can self-renew within tissues. Organoids can be generated from different types of stem cells, including ESCs, iPSCs, and adult stem cells (ASCs)^8,31,32. The choice of stem cell type significantly influences the characteristics of the resulting organoids. Currently, organoids are often derived from specific types of stem cells. For instance, optic cups ³³ and cerebral organoids^34,35 are predominantly generated from PSCs, including both ESCs and iPSCs. In contrast, mesodermal kidney organoids are also mainly derived from PSCs^36,37. Organoids originating from the surface ectodermal lineage are usually produced from ASCs or isolated adult tissues^38–41. In addition, organoids of endodermal lineage can be derived from both PSCs and ASCs.

ESCs are pluripotent and can develop into various cell types due to their ability to differentiate. Despite their potential, the clinical application of ESCs is constrained by ethical concerns surrounding their use from early embryos. Creating organoids from ESCs involves several essential steps. First, researchers must identify the biological signals that guide organ development ⁸ . Understanding these signals helps replicate the conditions needed for ESCs to transform into specific organ structures. Next, a 3D culture system is used to support ESC growth. This system mimics the natural environment within the body, allowing ESCs to organize into structures that resemble actual organs. Initially, ESCs form clusters known as embryoid bodies (EBs), which serve as a precursor to organoids. By adding specific signaling factors and adjusting the culture conditions, researchers can guide these clusters to develop into more complex organ structures ⁶ . Notable studies have demonstrated this potential. For instance, Eiraku et al. ³⁴ showed that mouse ESCs cultured in a 3D system could develop into structures similar to the cerebral cortex, including components such as the olfactory bulb and medulla oblongata. Similarly, Taguchi et al. ³⁷ used Wnt signaling to cultivate kidney organoids from mouse ESCs, resulting in structures with functional glomeruli and renal tubules. These advances highlight the significant promise of ESCs in organ development research. As technology progresses, it is anticipated that ESC-derived organoids will achieve complete physiological functions, offering valuable insights into organ development and potential clinical applications.

iPSCs are PSCs obtained by reprogramming transcription factors (Oct4, Sox2, Klf4, and C-Myc) into differentiated somatic cells. This groundbreaking method, developed by Shinya Yamanaka in 2006, offers a less ethically controversial alternative to ESCs because iPSCs do not involve embryos. iPSCs share similar gene expression profiles and developmental potential with ESCs and are valuable for human disease modeling, drug development, and cell therapy. To create organoids from iPSCs, somatic cells are first collected and reprogrammed into iPSCs through the introduction of specific transcription factors. The subsequent culture and induction processes closely resemble those used for ESC-derived organoids ⁴² . Significant advancements have been made in iPSC-derived organoids. Lancaster et al. ³⁵ successfully developed human brain organoids from iPSCs obtained from patients with microcephaly, revealing insights into early neuronal differentiation. Qian et al. ⁴³ used iPSCs to create organoids that model neurodevelopmental disorders caused by the Zika virus, providing a platform to study complex human brain development and hereditary neurological diseases. Also in 2017, Lancaster et al. ⁴⁴ combined iPSC technology with bioengineering to produce brain organoids of varying sizes, enhancing capabilities for high-throughput analysis and drug testing. Mansour et al. ⁴⁵ transplanted iPSC-derived brain organoids into mouse brains, observing the growth of axons, functional blood vessels, and mature neurons and glial cells, thereby establishing a model for studying brain tissue vascularization and potential regenerative treatments.

ASCs are precursor cells found in organs that, while not fully differentiated, are essential for tissue renewal and repair. They can differentiate into one or two closely related cell types and form organoids that closely resemble their tissue of origin^46,47. To develop these organoids, ASCs are extracted from adult tissues and cultured in 3D environments with specific signaling factors that mimic the natural conditions of organ development ⁴⁸ . A notable breakthrough came in 2009 when Sato et al. ¹⁰ identified Lgr5⁺ ISCs that can self-organize into intestinal organoids, demonstrating their ability to migrate and differentiate along the crypt-villus axis. In 2014, Simmini et al. ⁴⁹ found that the protein Cdx2 is crucial for maintaining the physiological function of these intestinal organoids; without it, the organoids’ genetic characteristics are disrupted, making them valuable for studying disease mechanisms. Compared to ESCs and iPSCs, ASCs provide a more direct and specific method for creating organoids, offering significant potential for researching both normal tissue functions and pathological processes.

Different Types of Organoids

Gastrointestinal Organoids

The gastrointestinal (GI) system plays crucial roles in digestion, absorption, excretion, and protection ⁵⁰ . It originates from the endoderm, which develops into an epithelial tube. This tube then differentiates into three distinct regions: the foregut, midgut, and hindgut. The foregut forms essential structures including the oral cavity, pharynx, respiratory tract, pancreas, stomach, and liver. The midgut gives rise to the small intestine and the ascending colon, while the hindgut develops into the remaining sections of the colon and the rectum. GI organoids are engineered to model these different parts of the GI system. This allows for comprehensive studies of their development and function.

A significant breakthrough in GI organoids was achieved by Hariro Sato and his team in 2009, who developed the first intestinal organoid model ¹⁰ . This model utilized Lgr5+ ASCs from mouse intestinal crypts, embedded in an extracellular matrix (ECM) and provided with key growth factors like R-spondin-1, epidermal growth factor (EGF), and the bone morphogenetic protein (BMP) inhibitor Noggin. Building on this progress, researchers introduced the first gastric organoid culture system a year later ⁵¹ . This system used Lgr5⁺ stem cells from antrum glands and was supplemented with fibroblast growth factor 10 (FGF10) and the hormone gastrin to facilitate the growth of gastric organoids.

However, the initial organoid models only replicated epithelial cells of the stomach or intestine. To create a more accurate cell culture model that includes multiple cell types and germ layers, researchers developed intestinal and gastric organoids from PSCs in 2011 ⁵² and 2014 ⁵³ , respectively. Notably, gastric organoids can now be directly derived from human gastric samples^54,55. These PSC-derived organoids contain mesenchymal cells, unlike the earlier models that were based on ASCs and lacked this feature^56,57. Despite this advancement, these models still fall short by missing other cell types like nerve and immune cells. To address these gaps, recent research has focused on new techniques such as co-culture ⁵⁸ , biomaterial-based methods^59,60, and 3D bioprinting ⁶¹ , to enhance the development and functionality of gastric and intestinal organoids.

Liver Organoids

The liver, situated in the right upper abdomen, performs crucial biological functions including metabolism, detoxification, and synthesis of proteins, and it possesses remarkable regenerative abilities following severe injury ⁶² . Liver development begins with the formation of the liver bud from the epithelial tissue of the foregut endoderm. This initial structure gives rise to hepatoblasts, which are precursor cells that differentiate into two main cell types: hepatocytes, the primary functional cells of the liver, and biliary epithelial cells, which line the bile ducts ⁶³ . Concurrently, the surrounding ectomesenchyme contributes to the formation of mesoderm-derived hepatic fibroblasts and stellate cells, which play roles in liver structure and function.

Research into liver regeneration has demonstrated its impressive capabilities since as early as 1960 ⁷ . Early experiments demonstrated that isolated embryonic liver tissue could reassemble in the lab and form functional bile ducts. Advances continued with the creation of liver organoids from ASCs, specifically Lgr5⁺ cells. These organoids could develop into mature liver cells when cultured in 3D environments using Matrigel ⁶⁴ . Further studies identified that bile duct-derived progenitor cells could also be used to generate liver organoids. These organoids had the ability to differentiate into both liver cells and bile duct cells ⁶⁵ . By 2018, researchers successfully expanded human and mouse liver cells in 3D cultures, showing high efficiency in transplantation experiments^66,67. To address the limited growth potential of mature liver cells, scientists have turned to generating liver organoids from hESCs, which have higher proliferation rates ⁶⁸ . In addition, new methods are being explored to improve the vascularization of liver organoids using human iPSCs ⁶⁹ . Overall, these advances in liver organoid technology are expected to enhance organ modeling, liver transplantation, and drug testing^70,71.

Brain Organoids

The vertebrate central nervous system originates from the neuroectoderm ⁷² , a specialized tissue that initially forms the neural plate. This plate then folds and fuses to create the neural tube, an epithelial structure with an internal fluid-filled chamber, which eventually evolves into the brain ventricles. During development, the neural tube divides into four major regions: the forebrain, midbrain, hindbrain, and spinal cord. The forebrain develops into key brain structures, including the neocortex, hippocampus, and various components of the ventral telencephalon, such as the amygdala and hypothalamus. The midbrain contributes to the formation of the tectum, while the hindbrain gives rise to the cerebellum, pons, medulla, and brainstem.

The development of the brain is a highly intricate process and has been a central focus of scientific research due to its complexity. However, ethical issues and limitations in model complexity mean that animal models and 2D cell cultures often fail to create suitable brain models ⁷³ . In contrast, brain organoids are capable of replicating several key aspects of early human brain development, including molecular, cellular, structural, and functional dimensions ⁶ . Early studies utilizing chicken neural progenitor cells (NPCs) demonstrated that these cells could self-organize into clusters of neuroepithelial cells around neural tube-like cavities, indicating a natural propensity for self-organization in brain development ⁷⁴ . Similarly, NPCs have shown the ability to aggregate and form neurospheres in suspension cultures. These neurospheres can further differentiate into neurons and astrocytes ⁷⁵ , highlighting their potential for modeling brain development. Advances in technology have allowed researchers to build more sophisticated brain models. For example, neural aggregates can be derived from PSCs in the form of EBs ⁷⁶ . Notably, PSCs have facilitated the creation of neural rosettes, where NPCs are arranged around a central lumen, resembling the structure of the neural tube ⁷⁷ . These rosettes can be further differentiated into various mature cell types characteristic of different brain regions^78–82. Despite these advancements, most current models remain based on 2D cultures or simple aggregations, which often fail to capture the full complexity of brain development and function.

To overcome this limitation, Watanabe and his team made significant advancements in brain research by developing 3D culture systems using mouse or human stem cells to better replicate the complex architecture of brain tissues ⁸³ . Initially, they created forebrain tissue using 2D cultures, but transitioning to 3D aggregate cultures allowed for the formation of more intricate structures that closely mimic the development of the dorsal forebrain ⁸³ . They further refined this method to enable the neuronal layer to self-organize in a manner similar to early cortical development and sustain cultivation for up to 112 days ⁸⁴ . Building on this foundation, Lancaster et al. developed 3D cerebral organoid models incorporating various brain regions, marking a significant improvement over earlier models. By manipulating growth factors such as Hedgehog, Fgf, Bmp, and Wnt^85–87, they created organoids that represent different brain regions, such as the mesencephalon ⁸⁸ , hippocampus ⁸⁹ , and cerebellum ⁹⁰ . In addition, the use of 3D printing technology has enabled the efficient and cost-effective generation of detailed brain structures from human iPSCs, providing valuable tools for studying brain development and function ⁴³ .

Kidney Organoids

The kidney, a vital organ in the human body, comprises the nephron, which develops from the metanephric mesenchyme, and the collecting duct and ureter, which arise from the ureteric bud ⁹¹ . Its intricate structure includes over 25 specialized cell types, many of which are essential for eliminating harmful metabolites and maintaining homeostasis ⁹² . As a result, the kidney is particularly susceptible to toxins. Its unique function in urine concentration and its close integration with complex vascular networks contribute to its vulnerability to toxic effects ⁹³ .

Early research on chicken embryo kidneys indicated that kidney tissue might have the ability to self-organize^7,94. In 2013, scientists successfully developed ureteric bud organoids from human PSCs by culturing them with specific growth factors and then applying additional compounds to guide their development into ureteric bud cells ⁹⁵ . The following year, researchers successfully generated kidney organoids from both mouse embryonic stem cells (mESCs) and human iPSCs ³⁷ . Building on this progress, Takasato and colleagues improved this method by producing kidney organoids with well-organized nephrons, endothelial cells, and renal interstitium ³⁶ . This breakthrough marked the first creation of complex kidney organoids containing both ureteric buds and metanephric mesenchyme, with transcriptional profiles closely resembling human fetal kidneys, thus validating their biological characteristics^96,97.

In addition, researchers have recently made significant strides in the maturation of kidney organoids, achieving advancements in glomerular vascularization and renal tubular function. These improvements have brought kidney organoids closer to mimicking real kidney tissue in both structure and function^98,99. This progress offers a valuable tool for modeling the complex kidney environment in vivo and for pharmacological screening. Notably, the activation of Notch signaling in these organoids has been shown to promote the differentiation of proximal tubules, underscoring the potential of organoids as effective platforms for drug screening ¹⁰⁰ . In addition, kidney organoids have been used to assess the toxic effects of various compounds, including doxorubicin, interleukin-1β, aminoglycosides drug, and cisplatin, providing new avenues for drug development and toxicity testing^97,101–104. Moreover, transplanted kidney organoids have demonstrated improved maturity and vascularization within host mice. This suggests that kidney organoids can serve as a promising source for autologous transplantation, offering new hope for treating kidney diseases^105,106.

Lung Organoids

The lung, originating from the developing ventral pre-gut endoderm ¹⁰⁷ , is a complex organ with conducting tubes that end in highly vascularized distal sacs for efficient gas exchange between air and blood. The airway epithelium consists of four major cell types: goblet cells, ciliated cells, club cells (or Clara cells), and basal cells ¹⁰⁸ . In comparison, the alveolar epithelium, which covers over 99% of the lung’s inner surface, is primarily made up of two types of cells: alveolar epithelial cell type I (AECI), responsible for gas exchange, and alveolar epithelial cell type II (AECII), which secretes surfactants ¹⁰⁹ .

In 1980, Yoshida et al. ¹¹⁰ endeavored to replicate lung function in vitro by culturing mouse lung organoids. They used tissue from full-term mouse embryos and a substrate of sterile porcine skin dermal collagen, observing various ductal structures. Three decades later, Kotton et al. advanced the field by deriving purified endoderm precursors from mESCs. They achieved this by precisely modulating transforming growth factor β (TGF-β)/BMP and BMP/FGF signaling pathways, successfully guiding these precursors to differentiate into primitive lung progenitors. Their work demonstrated the ability to replicate the lung-development process ¹¹¹ , laying a crucial foundation for the creation of lung organoids.

In 2012, Rossant and colleagues generated lung organoids through human induced pluripotent stem cells (hiPSCs) for the first time ¹¹² . They developed mature airway epithelial cells through air-liquid interface culture, offering a new approach to model diseases like cystic fibrosis (CF). Following this, Huang et al. ¹¹³ and Dye et al. ¹¹⁴ and their teams created similar stepwise differentiation processes, successfully generating both airway and AECs from hiPSCs. Their work not only identified four types of airway epithelial cells but also included functional types AECI and AECII. Then, Chen et al. ¹¹⁵ produced lung bud organoids containing both pulmonary endoderm and mesoderm, enhancing our understanding of lung development. In addition, Sachs and team ¹¹⁶ cultivated airway organoids from lung cancer biopsy samples, which preserved the tumor’s genetic mutations and histopathological features, thereby offering valuable tools for drug screening.

The lungs possess a remarkable capacity for regeneration and repair, even after injury. This regenerative ability allows for the creation of lung-like tissues from adipose-derived stem cells (AdSCs) ¹¹⁷ . Basal stem cells in the airway can self-renew, while secretory club cells exhibit significant plasticity following injury, whereas AECIIs of the alveolar epithelium are able to replenish lost AECIIs and generate new AECIs after proliferative injury^118–121. Leveraging this regenerative potential, researchers have successfully developed organoids from airway basal cells^122,123, secretory club cells^124,125, and AECIIs ¹²⁶ in both mice and humans. These lung organoids are invaluable for studying respiratory diseases, as they effectively replicate lung functions such as gas exchange. One of the most significant applications of lung organoid models in recent years has been in the study of respiratory diseases caused by viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent research has shown that lung organoids serve as an essential tool for understanding the biological mechanisms underlying COVID-19 infection and the respiratory damage it causes^127–129. These models have been instrumental in elucidating the cellular interactions between SARS-CoV-2 and the lung epithelium, providing important insights into viral replication, immune response, and the pathology of COVID-19. Furthermore, lung organoids are being actively utilized in drug-discovery efforts, aiding in the identification of potential therapeutic agents that may mitigate the disease’s impact on lung function ¹³⁰ . The continued development and application of these organoid models hold great promise for advancing our understanding of lung diseases and improving therapeutic strategies for conditions like COVID-19 and beyond.

New Players in the Organoid Field

Advancements in scientific research have led to the successful development of various organ models, fueling new research opportunities. Skin, being one of the largest and most complex organs, presents unique challenges due to its disease diversity and differences in healing mechanisms between mice and humans. This makes human skin organoid models particularly valuable for scientific research and drug development ¹³¹ . The skin’s multi-layered structure includes the epidermis, dermis, and subcutaneous fat, along with accessory structures like hair follicles, sebaceous glands, and sweat glands, which are crucial for regulating body temperature and fluid retention, resisting external pressure, and regulating touch and pain sensation^132,133. Compared with previous skin organoid models ¹³⁴ , recent studies highlight the importance of creating complex human skin organoids that incorporate these accessory structures ¹³⁵ . Researchers have developed a multi-stage culture strategy that emulates embryonic skin development, starting from PSCs and progressing to fully formed skin. By precisely regulating signaling pathways such as TGF-β and FGF, researchers can induce the growth and differentiation of skin cells, resulting in a detailed human skin model. This model features a keratinized epidermis and includes sensory neurons, dermal fibroblasts, adipose tissue, cartilage, melanocytes, and Schwann cells. These advanced skin organoids are pivotal for improving disease modeling and drug discovery for skin genetic disorders and skin cancer, as well as offering innovative transplant options for burn victims.

In recent years, muscle organoids have also emerged as a promising tool for investigating muscle development, regeneration, and disorders such as muscular dystrophies ¹³⁶ . Advances in this field have led to the creation of more sophisticated skeletal muscle organoids that closely resemble the complex architecture of in vivo muscle tissue, including striated fibers and functional contractility. These improvements have been made possible by the development of 3D culture systems, innovative biomaterials, and the integration of muscle progenitor cells (myoblasts) into the organoid cultures. Notably, studies by Rao et al. ¹³⁷ demonstrated the successful generation of human skeletal muscle tissues from engineering human PSCs, while Lin et al. ¹³⁸ reported muscle organoids capable of functional contraction, providing a model for studying neuromuscular junctions. More recently, Shahriyari et al. ¹³⁹ utilized muscle organoids to investigate the regenerative capacity of muscle in vitro, showing how they can serve as models for diseases like Duchenne muscular dystrophy. These muscle organoids not only offer a platform for disease modeling but also hold promise for drug screening and cell-based therapies in the context of muscle degenerative disorders.

In addition, a significant advancement in regenerative medicine is the creation of lacrimal gland organoids from ASCs ¹⁴⁰ and PSC ¹⁴¹ . The lacrimal gland plays a crucial role in eye lubrication and protection by secreting tears in response to neurotransmitters released by parasympathetic nerves. Dysfunction in this gland can lead to various eye diseases. For example, the dysfunction of lacrimal gland will lead to a decrease in tear production, resulting in poor maintenance of ocular surface and increasing the risk of infection ¹⁴² . Furthermore, the disorder of tear secretion may cause dry eye disease, which can even lead to vision loss in severe cases ¹⁴³ . Currently, treatments such as artificial tears, anti-inflammatory drugs, and punctal occlusion generally cannot prevent the atrophy of lacrimal gland, so there is a great demand for alternative treatment options. Recent research has successfully developed lacrimal gland organoids that not only mimic natural gland swelling but also produce secretions when stimulated with norepinephrine. The Pax6 gene has been identified as crucial for the differentiation of lacrimal duct cells. These organoids can form tear tube-like structures and have the potential to mature into fully functional lacrimal glands when transplanted into mice. Thus, lacrimal gland organoids hold promise as a tool for developing treatments for conditions like dry eye.

Application of Organoids

Regenerative Medicine

Currently, allogeneic transplantation remains a cornerstone of organ replacement therapy, but challenges such as donor matching and immune rejection limit its efficacy, and thousands of patients die each year while waiting for a transplant ¹⁴⁴ . In this context, organoid technology presents a promising alternative due to its ability to self-expand and maintain genetic stability, making it an exciting tool for generating transplantable tissues and functional cell types in regenerative medicine. Recent advancements in organoid technology have shown significant promise. For example, researchers have demonstrated that mouse colon organoids can be effectively expanded in vitro and successfully transplanted into damaged mice ¹⁴⁵ , where they remodel functional crypt units. Similarly, fetal progenitor-derived small intestinal organoids have exhibited the ability to integrate and function in vivo ¹⁴⁶ . When intestinal organoids derived from PSCs were implanted under the kidney capsule of mice, they displayed good permeability and peptide uptake, highlighting their potential in treating conditions like short bowel syndrome ¹⁴⁷ . At the same time, in 2022, a Japanese research team used intestinal organoids cultured from healthy intestinal mucosal stem cells of patients with ulcerative colitis to perform autologous transplantation ¹⁴⁸ . Furthermore, combining organoids with synthetic or bioacellular (decellularized) scaffolds offers new possibilities for enhancing intestinal transplantation. This integration may improve the functionality and viability of transplanted tissues, opening up innovative pathways for treating GI diseases and other conditions^149–151.

Liver organoids, including those derived from adult mice and PSCs, show significant promise in addressing liver failure. These organoids have demonstrated the ability to improve survival rates in various liver disease models, such as fumarylacetoacetate hydrolase mutant mice and tyrosine disease type I models, as well as in cases of chemical liver injury^64,66. PSC-derived liver organoids have been particularly effective in treating acute liver failure, restoring liver function effectively ¹⁵² . In addition, engineered bile duct organoids have shown potential in reconstructing the extrahepatic bile duct tree, including repairing the gallbladder wall and bile duct epithelium ¹⁵³ . This innovation offers a new approach for treating common bile duct diseases, expanding the possibilities for regenerative therapies in liver-related conditions. In addition, great progress has been made in the transplantation of various organs, including the retina^154,155, pancreas ¹⁵⁶ , brain ⁴⁵ , and lungs ¹⁵⁷ .

The promise of organoid technology in regenerative medicine is substantial, but it presents several safeties, ethical, and legal challenges that must be addressed before it can be applied clinically. First, it is essential to ensure that patients provide informed consent, fully understanding how their cells will be used and any potential implications of this use. Ownership and management of patient-derived organoids and the associated data must be clearly defined. This includes determining who owns the organoids and establishing protocols for how data will be handled, shared, and protected. Given the differences in medical regulations worldwide, it is important to create global standards for stem cell products and treatments to ensure safety and consistency. In addition, establishing ethical guidelines is crucial to address how closely organoids can be linked to their donors. Ensuring ethical practices in organoid development and use is vital. Finally, fostering open dialogue among scientists, policymakers, and the public is necessary to set appropriate boundaries for the clinical application of these technologies. This will help ensure that advancements are made responsibly and align with societal values.

Disease Modeling

Organoid culture represents a significant advancement over traditional single-cell culture models, especially in the field of disease modeling. The key advantage of organoids is their ability to accurately replicate the pathological processes at an organ level, providing a more realistic and comprehensive model of human diseases. Organoids derived from ASCs or iPSCs are particularly valuable for creating clinically relevant human disease models. These organoids effectively capture and translate specific disease characteristics, allowing for more precise studies of various conditions. They are extensively used in research on genetic diseases, host-pathogen interactions, and cancer, facilitating a deeper understanding of disease mechanisms and potentially leading to more effective treatments. Moreover, the ability of organoids to mimic complex tissue architecture and function enhances their utility in advancing medical research and therapeutic development.

Organoid technology has revolutionized the modeling of genetic and neurodegenerative diseases. CF is an autosomal recessive genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel ¹⁵⁸ . In 2013, Dekkers et al. ¹⁵⁹ utilized organoid technology to recreate this disease in vitro, successfully generating human intestinal organoids with the F508del CFTR mutation. Their innovative swelling assays demonstrated the significant responses of these CF organoids to CFTR modulators. Beyond CF, organoids have shown considerable promise in modeling neurological diseases. Their 3D structure enables accurate simulations of various conditions, including genetic brain disorders like microcephaly ³⁵ and agyria ¹⁶⁰ , as well as neurodegenerative diseases such as Alzheimer’s disease. This capability provides a valuable platform for drug screening and the development of treatment strategies. In addition, Raja et al. ¹⁶¹ developed a scaffold-free 3D culture system that produced iPSC-derived brain organoids from familial Alzheimer’s patients. These organoids replicated typical Alzheimer’s pathology and validated the therapeutic potential of β- and γ-secretase inhibitors. Collectively, these findings underscore the advantages of 3D organoid culture systems for studying genetic diseases and advancing drug discovery.

Organoid technology has emerged as a powerful tool in infectious disease research, offering valuable models for studying host-pathogen interactions across various infectious agents, including viruses, bacteria, and protozoan parasites^162–164. For instance, brain organoids have been employed to investigate the mechanisms of microcephaly associated with Zika virus infection. The infected organoids exhibited reduced size compared to controls, aligning with the pathological findings observed in affected patients^43,165,166. Similarly, intestinal organoids serve as important models for studying infectious diseases. Researchers using human primary gut organoids have proposed that the human gut may act as an alternative infection route for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) ¹⁶⁷ . In addition, small intestine organoids have been utilized to study norovirus, revealing that nidazolamide can inhibit norovirus replication by activating the cellular antiviral response ¹⁶⁸ .

Moreover, organoids have emerged as invaluable tools in cancer research, enabling the in-depth study of a variety of tumors while preserving their inherent heterogeneity and complex tissue architecture. Their application spans across multiple cancer types, including colorectal^169–171, liver^18,172,173, pancreatic ¹⁷⁴ , ovarian ¹⁷⁵ , and gastric^176–178 cancers. In these contexts, organoids facilitate the exploration of molecular mechanisms underlying tumor biology, provide a platform for assessing drug sensitivity, and aid in the development of personalized treatment strategies. This capacity to mimic the unique characteristics of individual tumors not only enhances our understanding of cancer progression but also significantly advances the field of precision medicine. By tailoring therapies based on the specific responses observed in organoid models, researchers can identify more effective treatment options, ultimately improving patient outcomes and paving the way for more targeted therapeutic approaches in oncology.

In summary, organoid culture systems significantly enhance research across genetic, infectious, and cancer diseases, injecting new vitality into medical science and fostering innovative therapeutic strategies. Organoid technology stands as a transformative advancement in disease modeling, accurately replicating human tissue and disease characteristics. This capability facilitates deeper insights into disease mechanisms, improves drug testing, and supports personalized treatments. Ultimately, organoids hold the promise of accelerating our understanding of complex diseases and pave the way for improved patient outcomes in precision medicine.

Drug Discovery and Toxicity Assessment

Careful drug screening and thorough evaluation of efficacy and toxicity are essential for effective clinical strategies. A drug’s efficacy serves as a key measure of its potential for widespread use, while its toxicity determines the safe range for administration. Compared to traditional animal models and 2D cell cultures, organoids provide a more accurate representation of organ structure and maintain the genetic diversity of original tissues ¹⁷⁹ . This ability to closely mimic real organs enhances both the scope and detail of drug testing. Organoids allow for personalized assessments of how different patients may respond to various medications. By using organoids in research, scientists can significantly shorten the drug-development timeline and reduce costs, facilitating more efficient and targeted therapeutic solutions.

A new and exciting advancement in this area is the creation of organoid factories, which are specialized platforms designed for the high-throughput generation and culture of organoids. These innovative facilities offer a scalable solution for advancing organoid-based drug discovery and toxicity testing. According to recent research, organoid factories have the potential to revolutionize the drug discovery process by providing a consistent and reproducible source of organoids derived from a variety of tissues ¹⁸⁰ . These factories utilize automated systems and advanced bioreactors to efficiently produce large quantities of organoids, which can then be used for drug screening, personalized medicine, and safety testing. The scalability provided by organoid factories can significantly accelerate the pace of research, allowing for high-throughput drug screening and better identification of promising therapeutic candidates. Furthermore, these platforms can integrate bioengineering and artificial intelligence technologies to optimize drug-testing processes, ultimately bridging the gap between laboratory research and clinical application.

In 2017, Broutier et al. ¹⁸ successfully targeted extracellular signal-regulated kinase (ERK) inhibitors using human cancer organoids, paving the way for new cancer treatment approaches. Crespo et al. ¹⁸¹ also developed colonic organoids from patients with familial adenomatous polyposis (FAP) and identified two compounds that significantly inhibited excessive organoid proliferation, with effects extending to wild-type organoids. Organoids have also been valuable in drug screening for neurological conditions. Cortical organoids infected with the Zika virus exhibit symptoms similar to congenital Zika syndrome, including neuronal layer atrophy and ventricular dilation^43,166,182. Some studies have used virus-infected brain organoids to evaluate the effectiveness of antiviral drug candidates such as duramycin, ivermectin, and azithromycin^183,184. In the area of GI organoids, high-throughput drug screening has shown remarkable advancements. Kozuka et al. ¹⁸⁵ rapidly screened nearly 2,000 potential drug compounds by culturing mini colon organoids in 96-well plates, highlighting the efficiency of organoid models in drug research and development. Microplastics refer to plastic fragments and particles less than 5 mm in diameter. As the awareness of the hazards of microplastics is increasing, endometrial organoids have been used to investigate microplastic pollution and its potential impact on reproductive health ¹⁸⁶ . In addition, researchers have realized the combination of bioengineering and artificial intelligence technologies with organoid technologies for more efficient drug screening and toxicity assessment^187–189.

While the potential of organoids in drug research is immense, toxicity assessment and preclinical studies still grapple with substantial challenges. Many toxic reactions remain undetected until clinical trials, complicating the drug-development process. Organoid models, due to their physiological relevance, could serve as powerful platforms for early toxicity testing. By constructing long-term toxicity screening models that mimic human responses, researchers can gain deeper insights into drug safety profiles earlier in the development pipeline. This approach not only enhances the predictability of adverse reactions but also aligns more closely with human biology, reducing reliance on animal models. As we delve into this innovative strategy, the prospects for safer and more effective drug development become increasingly promising, potentially transforming the landscape of pharmaceutical research.

Host-Microbiota Interactions

Over 100 trillion microorganisms reside within and around the human body ¹⁹⁰ , representing a complex and integral component of our biological ecosystem. An individual’s health is shaped not only by genetic factors but also by the delicate balance and activities of the microbiota. The GI tract is a major reservoir for these microbes, hosting around 1,000 different bacterial species that play essential roles in maintaining overall health and well-being^191,192. In humans, both the stomach and gut are closely linked to the microbiota, with microorganisms typically found on the mucosal surfaces and lumen of the GI tract ¹⁹³ . These microbes significantly influence the GI biology of the host. The gut microbiota can act as a natural barrier, helping to maintain the integrity of the intestinal lining and preventing the invasion of harmful pathogens. In addition, it plays a crucial role in immune regulation and competitive inhibition^194,195. Research has demonstrated that the gut microbiota can significantly impact drug metabolism and effectiveness ¹⁹⁶ . Often referred to as the “second genome” of the human body, the gut microbiota has a wide-ranging regulatory role in overall health. However, the precise relationship between host-microbe interactions and the development of GI diseases remains unclear^197,198.

The advent of organoid technology has revolutionized the study of GI microecology. Researchers have discovered that co-culturing intestinal symbiotic bacteria, such as lactic acid bacteria, with GI organoids enhances organoid proliferation and differentiation into Paneth cells ¹⁹⁹ . This technology effectively simulates host-microbe interactions in the gut, serving as a robust platform for developing intestinal microbial infection models. Current cutting-edge research is leveraging Lgr5⁺ ISC and hiPSC-derived intestinal organoid models to explore interactions between pathogens like Clostridium difficile, Salmonella typhimurium, enterohemorrhagic Escherichia coli, and rotavirus with the intestinal epithelium ²⁰⁰ . For instance, Hou et al. ²⁰¹ established a pioneering co-culture system incorporating Lactobacillus, intestinal lamina propria lymphocytes, and Lgr5⁺ ISC organoids, revealing that Lactobacillus stimulates the secretion of interleukin-22 (IL-22) from intestinal lymphocytes. In addition, studies have shown that human small intestinal organoids derived from Lgr5⁺ ISC can be infected with human norovirus, allowing for insights into the evolution, immunity, and pathogenesis of the virus ¹⁶⁸ . Research has identified susceptibility factors for host infections and explored preventive measures. In specific experiments, microinjection of Clostridium difficile into small intestinal organoids derived from human iPSCs demonstrated downregulation of the sodium/hydrogen exchange factor 3 (NHE3), leading to diarrhea ²⁰² . Similarly, microinjection of Salmonella typhimurium into these organoids revealed damage to the intestinal epithelial barrier and altered transcription profiles, showcasing the organoid model’s utility in studying the molecular mechanisms underlying Salmonella-associated enteritis ²⁰³ . Furthermore, organoids have been instrumental in investigating the pathogenesis of Helicobacter pylori infections. For example, McCracken and colleagues found that injecting Helicobacter pylori into gastric organoids resulted in the binding of GagA to the c-Met receptor on epithelial cells, causing their overexpression ⁵³ . This research underscores the potential of organoid technology in developing effective treatments for GI infections.

In summary, organoids signify a groundbreaking development in biomedical research, providing various applications that greatly improve our understanding of human biology (Fig. 2). Organoids play a crucial role in regenerative medicine, enabling research into tissue regeneration and stem cell therapies. Their capability to accurately model diseases like cancer and serve as dependable platforms for drug discovery and toxicity testing highlights their significance. Moreover, organoids offer valuable insights into the interactions between human cells and microbiota, enhancing our understanding of their roles in health and disease. Overall, the versatility and adaptability of organoids make them essential tools in the quest for innovative therapeutic strategies, driving advancements in precision medicine and personalized healthcare. Their potential to bridge the gap between basic research and clinical application underscores their transformative impact on biomedical science.

Figure 2.

Applications of organoid technology. This schematic diagram illustrates the diverse applications of organoids across multiple fields, including regenerative medicine, genetic diseases, infectious diseases, cancer research, drug discovery, toxicity assessment, and host-microbiota interactions.

Conclusion and Prospectives

Since its birth, human organoid technology has demonstrated significant potential across diverse fields, from basic scientific research to biomedical applications. These organoids, derived from various stem cell types, differ in development, maturity, and complexity. They have successfully established unique culture systems, paving the way for advancements in disease modeling and drug screening. However, several challenges persist within current organoid frameworks. While researchers have made substantial progress in elucidating the survival and functional dynamics of organs such as the brain ⁴⁵ , liver ²⁰⁴ , and bile duct ⁷¹ in animal models, critical issues remain. These include limitations in organoid maturity and cellular diversity, insufficient vascularization, and constraints in the composition of ECM. Overcoming these challenges is crucial for enhancing the effectiveness and applicability of organoids in both research and clinical settings (Fig. 3).

Figure 3.

Challenges and perspectives of organoid technologies. While organoids cannot fully replicate the structure and maturity of in vivo organs, advances are being made to create highly mature organoids by precisely controlling the substances required for their growth. Limitations arise from the absence of blood vessels and vascular structures, which restrict the size of organoids. Researchers are actively investigating strategies to enhance oxygenation and nutrient delivery, with the integration of vascular networks being a crucial step forward. In addition, identifying alternatives to animal-derived extracellular matrices could facilitate the clinical application of organoids. Co-culture techniques allow for the exploration of interactions among different cell populations over time and space. In the future, organoids are expected to evolve by integrating diverse emerging technologies to overcome current limitations.

Currently, organoids cultured in vitro do not accurately replicate the cell types and maturity of organs found in vivo, which makes it challenging to simulate the complex conditions of living tissues. This limitation arises mainly from the dynamic nature of cell development, which requires the precise release of various growth factors to support the differentiation of different cell types. To achieve the goal of creating highly mature organoids, researchers are focusing on overcoming these challenges. Significant progress has been made in brain organoid research, particularly using human PSCs that express the Sonic Hedgehog (SHH) protein. Researchers have successfully developed a system that allows for the controlled release of SHH, enabling precise regulation of its concentration over distance. This advancement marks an important step forward in the field, bringing us closer to the goal of creating more accurate organoid models ²⁰⁵ .

Most organoids produced today are limited in size, largely due to the absence of blood vessels and vascular structures, which are essential for adequate nutrient and oxygen uptake. Without a functional vascular system, organoids can suffer from insufficient supply, leading to necrotic cores in larger organoids ²⁰⁶ . To address this challenge, researchers are actively investigating various strategies to enhance oxygenation and nutrient delivery within organoids. A key approach is the introduction of a vascular network. This development not only improves the efficiency of nutrient and oxygen supply ⁴⁵ but also provides critical signals that guide cell migration and differentiation during organoid development ²⁰⁷ .

In addition, the dependence on animal-derived ECM significantly hinders the clinical translation of organoid models. The most widely used gel, Matrigel, is derived from Engelbreth-Holm-Swarm (EHS) sarcoma and contains various ECM components, including laminin and collagen IV, along with growth factors like insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), EGF, and TGF-β. However, its complexity, characterized by thousands of proteins, results in batch variations and inconsistent mechanical properties, leading to unreliable experimental outcomes ²⁰⁸ . Moreover, the production of Matrigel involves growing tumors in mice, raising ethical concerns. To overcome these challenges, researchers are exploring alternative materials. Potential substitutes include de-cellularized ECM from organs, isolated ECM components, graphene oxide, and synthetic hydrogels. These alternatives aim to provide more consistent and ethically acceptable options for organoid culture, thereby enhancing their clinical applicability^208–211.

At the same time, the inability to create multi-organ pathological models restricts the application of organoids in regenerative medicine. The human organoid system fundamentally mimics parts of the human body, rather than the whole body. However, this challenge can be partially addressed through co-culture methods. For instance, researchers have successfully co-cultured human stem cell–derived intestinal organoids with neural crest cells, facilitating interaction between different cell types ²¹² . At the same time, scientists have connected multiple organoids to study the communication between the liver, pancreas, and GI tract ²¹³ . In addition, integrating organ chip technology has allowed scientists to develop 3D systems that enable connection and communication between multiple pre-formed “organs”^214,215. This innovative approach enhances the potential for studying complex biological processes and diseases, paving the way for more effective regenerative therapies.

The future of organoid technology hinges on overcoming the limitations of individual approaches by integrating real-time imaging, gene editing, organ chip technology, bioprinting, and microfluidics^216,217. For instance, Geyer et al. ²¹⁸ utilized a microfluidics-based organoid system to study the effects of hypoxia on pancreatic ductal adenocarcinoma, while Tran et al. ²¹⁹ employed real-time imaging to screen 247 protease inhibitors that prevent cyst formation without hindering organoid growth. These innovations highlight the significant potential of organoid technology to enhance our understanding of complex biological processes. However, many of these applications are still in the exploratory phase, indicating that further research is necessary to fully realize their capabilities. As this field evolves, 3D organoid systems are expected to complement existing models, enriching the research landscape and strengthening the foundation for both basic and clinical studies. This integration of technologies promises to advance our knowledge of disease mechanisms and improve the development of targeted therapies, ultimately leading to more effective treatments and better patient outcomes.