Organoids can self-organize from tissue stem cells, primary cells, or pluripotent stem cells in a three-dimensional environment and partially reconstruct the cellular composition, spatial architecture, and functional characteristics of organs. They provide models that more closely resemble in vivo conditions for studying human development, disease mechanisms, and drug responses.

Keywords: organoid; three-dimensional culture; adult stem cell; pluripotent stem cell; extracellular matrix; growth factor; tissue-specific marker; disease modeling; drug screening; regenerative medicine

1 Biological Basis and Technical Characteristics of Organoid Models

1.1 Basic Definition of Organoids

Organoids are cellular structures formed under three-dimensional culture conditions from stem cells, progenitor cells, or primary cells with tissue-reconstitution capacity. Unlike conventional cell spheroids, organoids usually contain multiple tissue-associated cell types and exhibit a certain degree of cellular polarity, spatial compartmentalization, tissue-specific gene expression, and functional activity. However, their structural complexity and maturation level generally remain lower than those of intact organs.

1.2 Self-Organization and Lineage Differentiation

Organoid formation depends on cell proliferation, lineage differentiation, cell adhesion, polarity establishment, and morphogenesis. Under the combined influence of growth factor gradients and the extracellular matrix, cells can form lumen-like structures, epithelial buds, neuroepithelial regions, or tissue-like layers. Self-organization is not completely autonomous, and the signaling combinations used in the culture system strongly influence organoid identity and cellular composition.

1.3 Differences Between Organoids and Two-Dimensional Culture Models

Cells in two-dimensional culture are constrained by a planar substrate, resulting in substantial differences from in vivo tissues in cell polarity, intercellular junctions, and matrix signaling. Organoids retain more three-dimensional architecture, cellular heterogeneity, and tissue-specific functions and are therefore suitable for studying cell fate and tissue responses. However, imaging, nutrient diffusion, quantitative analysis, and batch standardization are also more complex.

1.4 Organoids, Organ-on-a-Chip Systems, and Xenotransplantation Models

Organoids emphasize cellular self-organization and tissue formation, whereas organ-on-a-chip systems focus on artificial control of fluid flow, mechanical forces, and multicellular tissue interfaces. After transplantation into animals, organoids may receive support from host vasculature, nerves, and immune environments, but host-derived signals are also introduced. These three model types differ in structural complexity, experimental controllability, and in vivo relevance.

2 Cellular Sources and Three-Dimensional Culture Systems for Organoids

2.1 Adult Stem Cell-Derived Organoids

Adult stem cell-derived organoids are generally established from stem cells and progenitor cells in tissues such as the intestine, stomach, hepatobiliary system, pancreas, lung, kidney, and reproductive tract. These models can be established relatively rapidly and retain the genetic background and some phenotypic characteristics of the donor tissue. They are suitable for patient-derived disease modeling and drug-sensitivity analysis, but their cellular diversity and developmental potential are usually constrained by the tissue of origin.

2.2 Pluripotent Stem Cell-Derived Organoids

Embryonic stem cells and induced pluripotent stem cells can sequentially undergo germ-layer induction, organ progenitor formation, and tissue maturation to generate cerebral, retinal, pulmonary, hepatic, pancreatic, cardiac, and renal organoids. This pathway can model early organogenesis and generate multiple lineages, but the culture cycle is relatively long, and differentiation batch variability and residual undifferentiated cells must be strictly controlled.

Figure 1 Generation, Characterization, and Research Applications of iPSC-Derived Organoids

2.3 Primary Cell- and Tumor-Derived Organoids

Primary cells isolated from normal tissue, tumor tissue, biopsy samples, or surgical specimens can form tissue-derived organoids. Patient-derived tumor organoids can retain certain genetic variants, histological features, and drug-response characteristics. However, differences in clonal growth advantage may cause selective changes in cellular composition during long-term culture.

2.4 Three-Dimensional Culture Methods

Basement membrane matrix embedding is suitable for many epithelial organoids. Suspension culture and rotating bioreactors facilitate the expansion of neural and pluripotent stem cell-derived organoids. Air-liquid interface culture enhances differentiation of airway and certain epithelial tissues, whereas microfluidic systems can introduce fluid shear, tissue interfaces, and immune cells. The culture method should be selected according to whether the organoid requires polarity, luminal structures, mechanical stimulation, or long-term perfusion.

Table 1 Major Cellular Sources and Culture Characteristics of Organoids

3 Regulation by Growth Factors, Small-Molecule Compounds, and the Extracellular Matrix

3.1 Wnt, R-Spondin, and Stem Cell Maintenance

Wnt signaling maintains the proliferation and undifferentiated state of multiple epithelial stem cells. R-Spondin supports the expansion of intestinal, gastric, hepatobiliary, and certain other epithelial organoids by enhancing Wnt receptor signaling. Dependence on Wnt signaling differs among tissues, and sustained high-level Wnt signaling may inhibit terminal differentiation. Expansion and differentiation media therefore generally use different signaling combinations.

3.2 EGF, FGF, and Expansion of Tissue Progenitors

EGF promotes the proliferation of multiple epithelial cell types, whereas members of the FGF family participate in the formation of neural, pulmonary, hepatic, pancreatic, cardiac, and urinary system progenitors. Factors such as FGF2, FGF7, and FGF10 have stage-dependent effects. For example, FGF10 supports the expansion of certain lung and pancreatic progenitors, but prolonged exposure may alter cellular maturation.

3.3 BMP, TGF-β, and Lineage Differentiation

BMP and TGF-β signaling have important roles in germ-layer formation, epithelial differentiation, and tissue maturation. Noggin inhibits BMP signaling and maintains certain tissue stem cells, whereas small molecules such as A83-01 and SB431542 inhibit TGF-β receptor-associated signaling and support epithelial expansion. Signal inhibition is not always beneficial, and reactivation of BMP or TGF-β at specific differentiation stages may be required for mature tissue formation.

3.4 Wnt Agonists and Differentiation-Regulating Small Molecules

CHIR99021 enhances canonical Wnt signaling by inhibiting GSK-3 and is commonly used for mesoderm induction, neural posteriorization, and the formation of certain organ progenitors. Retinoic acid regulates anteroposterior patterning and regional organ identity. SHH pathway agonists or inhibitors can control the patterning of neural and endodermal tissues, whereas Forskolin increases intracellular cAMP and is used in certain epithelial and cystic fibrosis functional assays.

3.5 ROCK Inhibitors and Cell Survival

Single-cell dissociation disrupts cell-cell junctions and induces anoikis. ROCK inhibitors such as Y-27632 can improve the survival of pluripotent stem cells and epithelial cells after passaging, cryopreservation and recovery, or re-embedding. Long-term or continuous use may alter the cytoskeleton, polarity, and differentiation and is therefore generally limited to a short period after dissociation.

3.6 Structural and Signaling Roles of the Extracellular Matrix

The extracellular matrix not only provides three-dimensional support for organoids but also regulates cell fate through integrins, focal adhesions, and mechanotransduction. Basement membrane matrices contain laminin, type IV collagen, and other matrix components and are suitable for organoid establishment and expansion. Type I collagen, laminin, vitronectin, and synthetic hydrogels can be used to regulate matrix stiffness, degradation rate, and adhesion signals.

4 Culture and Identification of Neural and Sensory System Organoids

4.1 Cerebral Organoids

Cerebral organoids are generally generated from pluripotent stem cells through neuroectoderm induction and formation of neuroepithelial structures, followed by regionalization into forebrain-, midbrain-, hindbrain-, or choroid plexus-like tissues. Early neural progenitor or forebrain markers include SOX2, PAX6, NESTIN, and FOXG1. After further differentiation, TBR1, BCL11B, SATB2, MAP2, and synapse-associated proteins can be detected. Cerebral organoids are suitable for studying neurodevelopment and genetic diseases, but the absence of a complete vasculature and stable microglial populations limits long-term maturation.

4.2 Retinal Organoids

Retinal organoids form after pluripotent stem cells pass through eye-field progenitor, optic cup-like, and neural retinal differentiation stages. RAX, PAX6, and VSX2 can be used to evaluate early eye-field and retinal progenitor formation, whereas CRX, RECOVERIN, RHO, and cone opsins can be used to evaluate photoreceptor differentiation. In addition to marker expression, maturation should be evaluated using outer segment-like structures, light responses, and electrophysiological activity.

4.3 Inner Ear Organoids

Inner ear organoids are generally generated by regulating BMP, FGF, and Wnt signaling to induce surface ectoderm, otic placode, and otic vesicle-like progenitors, followed by differentiation into sensory epithelium and hair cells. PAX2, PAX8, and SOX2 indicate otic progenitor formation, whereas ATOH1, POU4F3, and MYO7A are used to evaluate hair cell differentiation. Functional validation may include ion flux after mechanical stimulation and analysis of synapse-associated structures.

4.4 Olfactory and Other Sensory Organoids

Olfactory epithelial organoids can be established from nasal tissue stem cells or pluripotent stem cells and may generate basal cells, sustentacular cells, and olfactory neurons. KRT5 and TP63 are used to evaluate basal cells, SOX2 and ASCL1 reflect progenitor status, and OMP and specific olfactory receptors are used to evaluate mature olfactory neurons. However, receptor-expression heterogeneity makes a single marker insufficient to represent the overall degree of maturation.

5 Culture and Identification of Respiratory, Digestive, and Endocrine System Organoids

5.1 Airway Organoids

Airway organoids can be established from airway basal cells, secretory cells, or pluripotent stem cell-derived progenitors. During expansion, KRT5-, TP63-, and SOX2-positive progenitor cells are generally maintained. After differentiation, SCGB1A1-positive secretory cells, FOXJ1- and acetylated α-tubulin-positive ciliated cells, and MUC5AC-positive goblet cells may form. Air-liquid interface culture is commonly used to promote ciliogenesis and mucus secretion.

5.2 Alveolar Organoids

Alveolar organoids mainly focus on type II alveolar epithelial cells and their ability to differentiate into type I alveolar epithelial cells. NKX2.1 and SFTPC can be used to identify type II alveolar cells, whereas AGER, PDPN, and CAV1 can be used to evaluate type I alveolar-like differentiation. Detection of SFTPC alone is insufficient to establish alveolar maturation, and surfactant secretion, barrier function, and injury-repair capacity should also be assessed.

5.3 Gastric and Intestinal Organoids

Gastrointestinal organoids are commonly established from LGR5-positive adult stem cells, and Wnt, R-Spondin, EGF, and Noggin are frequently used to maintain expansion. LGR5 and OLFM4 indicate intestinal stem cells. MUC2, CHGA, LYZ, ALPI, and KRT20 are used to evaluate goblet cells, enteroendocrine cells, Paneth cells, absorptive cell function, and epithelial maturation, respectively. Gastric organoids may additionally be evaluated using regional markers such as MUC5AC, MUC6, TFF1, and pepsinogen.

5.4 Liver and Bile Duct Organoids

Liver organoids can be established from hepatic progenitor cells, cholangiocytes, hepatocytes, or pluripotent stem cells. HNF4A, ALB, and alpha-fetoprotein can be used to evaluate the hepatocyte lineage, whereas CYP3A4 activity, urea synthesis, and albumin secretion reflect functional maturation. KRT7, KRT19, and SOX9 are commonly used for cholangiocyte identification. Pluripotent stem cell-derived liver organoids often display fetal-like characteristics and require further optimization of metabolic and vascular environments.

5.5 Pancreatic Organoids

Pancreatic organoids can form ductal, acinar, or endocrine cell-like structures. PDX1 and NKX6.1 indicate pancreatic progenitors, KRT19 and SOX9 are used to identify ductal cells, and AMY2A and PRSS1 are used to evaluate acinar cells. INS and C-peptide indicate β-cell differentiation, GCG indicates α-cell differentiation, and SST indicates δ-cell differentiation. β-Cell maturation should be evaluated using glucose-stimulated insulin or C-peptide secretion.

5.6 Thyroid Organoids

Thyroid organoids can be established from pluripotent stem cells or thyroid tissue-derived cells. Coexpression of NKX2.1 and PAX8 indicates formation of the thyroid follicular lineage, whereas TG, TPO, TSHR, and SLC5A5 are used to evaluate follicular cell differentiation. Mature thyroid-like structures should also display follicular polarity, iodide uptake, and thyroid hormone synthesis.

6 Culture and Identification of Cardiovascular, Urinary, and Reproductive System Organoids

6.1 Cardiac Organoids

Cardiac organoids are generally generated from pluripotent stem cells through mesoderm and cardiac progenitor stages and may contain cardiomyocytes, endothelial cells, and mesenchymal cells. NKX2.5, GATA4, and TBX5 can be used to evaluate cardiac progenitors, whereas TNNT2, ACTN2, MYL7, and myosin heavy chains identify cardiomyocytes. Contraction frequency, calcium transients, action potentials, and drug responses reflect functional maturation more accurately than marker expression alone.

6.2 Vascular Organoids

Vascular organoids can model endothelial lumens, pericyte coverage, and partial microvascular networks. PECAM1/CD31, CDH5, VWF, and EMCN are used to identify endothelial cells, whereas PDGFRβ, CSPG4, and RGS5 reflect pericytes or vascular wall cells. Barrier permeability, perfusion capacity, and responses to angiogenic stimulation are important functional indicators.

6.3 Kidney Organoids

Kidney organoids are generally generated from pluripotent stem cells through intermediate mesoderm and nephron progenitor stages and may form glomerulus-like and tubule-like structures. PAX2, LHX1, and SIX2 indicate early renal progenitors. WT1, NPHS1, and NPHS2 are used to identify podocytes, LTL marks proximal tubules, EPCAM and CDH1 identify distal epithelium, and UMOD indicates thick ascending limb-like differentiation. Kidney organoids often show insufficient cellular maturation and contamination with off-target lineages.

6.4 Bladder and Urothelial Organoids

Bladder organoids can be established from urothelial stem cells or pluripotent stem cells. KRT5, KRT14, and TP63 indicate basal cells, whereas KRT8, KRT18, UPK1A, and UPK3A are used to evaluate superficial urothelial differentiation. Barrier integrity, urea tolerance, and tight-junction function reflect tissue maturation more accurately than expression of a single urothelial protein.

6.5 Endometrial and Fallopian Tube Organoids

Endometrial organoids are generally established from glandular epithelial cells and can retain long-term hormone responsiveness. EPCAM, KRT8, KRT18, and MUC1 are used for epithelial identification, FOXA2 reflects glandular differentiation, and ESR1 and PGR are used to evaluate estrogen and progesterone responses. Fallopian tube organoids may contain PAX8-positive secretory cells and FOXJ1-positive ciliated cells.

6.6 Ovarian, Testicular, and Prostate Organoids

Ovarian and testicular organoids can be used to study interactions between germ cells and supporting cells, but complete gametogenesis remains difficult to reconstruct in vitro. Prostate organoids can be evaluated using basal-cell markers such as KRT5 and TP63 and luminal epithelial markers including KRT8, KRT18, AR, and KLK3. They are suitable for studying androgen signaling and prostate tumors.

7 Evaluation of Organoid Differentiation Status and Tissue-Specific Markers

7.1 Principles for Marker Combination-Based Evaluation

Organoid identification should not rely on a single marker. Tissue identity, cellular lineage, differentiation stage, and spatial localization should be evaluated together. The same protein may be expressed in multiple tissues or developmental stages. For example, SOX2 is present in neural progenitors, airway progenitors, and certain sensory epithelia. Regional markers, morphological structures, and functional readouts are therefore required for interpretation.

7.2 Evaluation of Undifferentiated Cells and Progenitor Cells

Pluripotent stem cell-derived organoids require monitoring of undifferentiated markers such as OCT4, NANOG, SOX2, and TRA-1-60 to prevent excessive expansion of residual pluripotent cells. Tissue progenitors should be evaluated using lineage-specific markers, such as PAX6 and NESTIN for the nervous system, LGR5 and OLFM4 for the intestine, NKX2.1 and SOX9 for the lung, and SIX2 and PAX2 for the kidney.

7.3 Evaluation of Terminal Differentiation and Maturation

Terminal differentiation markers indicate that a specific expression program has been activated but cannot independently demonstrate that cells have reached the maturation level of adult tissues. Maturation should be evaluated using metabolic, secretory, electrophysiological, barrier, contractile, or transport functions and by comparing expression profiles with fetal tissue, adult tissue, or primary cells.

Table 2 Representative Identification Markers and Functional Readouts for Major Organ Organoids

8 Applications of Organoids in Disease Modeling, Drug Screening, and Regenerative Medicine

8.1 Modeling of Genetic Diseases and Developmental Abnormalities

Patient-derived induced pluripotent stem cell organoids can retain pathogenic variants and model neurodevelopmental abnormalities, inherited retinal diseases, cystic fibrosis, polycystic kidney disease, and metabolic disorders. Repairing a pathogenic variant through gene editing or introducing the same variant into normal cells can establish isogenic controls and distinguish gene effects from donor-background differences.

8.2 Infection and Inflammation Research

Respiratory, intestinal, hepatic, and neural organoids can be used to study pathogen entry, tissue tropism, cellular injury, and host immune responses. Organoids containing only epithelial cells cannot fully reproduce immune responses and generally require coculture with immune cells, fibroblasts, or microorganisms. The apical or basal direction of pathogen exposure should also be controlled.

8.3 Tumor Modeling and Personalized Drug Responses

Patient-derived tumor organoids can be used to compare responses to chemotherapy, targeted drugs, and combination treatments and to study the formation of drug-resistant clones. Organoid drug-sensitivity results are influenced by the matrix, culture factors, and the absence of immune and vascular environments and therefore cannot be directly equated with clinical responses. Results should be interpreted together with molecular classification and clinical information.

8.4 Drug Screening and Toxicity Evaluation

Organoids permit multidose and multitime-point screening while retaining a certain degree of tissue function. Liver, kidney, cardiac, and neural organoids are suitable for evaluating organ-specific toxicity, whereas multi-organ chips can further analyze metabolites and interorgan interactions. Screening systems should control organoid size, cellular composition, and readout timing to prevent morphological differences from producing false-positive or false-negative results.

8.5 Regenerative Medicine and Cell Replacement

Organoids can serve as a cellular source for tissue repair, cell transplantation, and biomaterial research. Transplantation applications must address vascularization, immune rejection, tissue integration, residual undifferentiated cells, and long-term genetic stability. At present, the value of organoids in regenerative medicine lies primarily in studying repair mechanisms and transplantation feasibility rather than directly replacing intact organs.

8.6 Biobanks and Precision Medicine

Organoid biobanks with clearly documented clinical information and molecular characteristics can be used to compare different patients, disease subtypes, and treatment regimens. Biobank quality depends not only on the number of organoids but also on the completeness of source tissue information, pathological diagnosis, culture conditions, passage history, cryopreservation batches, and molecular quality-control data.

9 Products Related to Organoid Culture, Differentiation, and Identification

9.1 Products for Three-Dimensional Matrices, Tissue Dissociation, and Culture Supplementation

9.2 Small Molecules for Organoid Signal Regulation and Lineage Differentiation

9.3 Growth Factors and Products for Culture Quality Evaluation

Whether an organoid can represent a human organ cannot be determined solely from its three-dimensional appearance or a small number of molecular markers. A reliable organoid model should have a clearly defined cellular source, a reproducible culture system, an appropriate cellular composition, tissue-specific spatial architecture, and verifiable physiological functions. The required degree of maturation and model complexity should be selected according to the research objective.

For more related articles, please see below:

[1] Overview of Organs-on-Chips

[2] New Uses for Organoids