The degree to which engineered cell-based in vitro models, such as organoids, should faithfully replicate the structures and functions of their in vivo organ counterparts is a subject of debate. While one approach is to strive for a high level of similarity to in vivo tissue architecture and function in order to demonstrate the physiological relevance of increasingly complex models, bioengineers often only need to replicate specific features of the in vivo tissue that are relevant to their research on physiological or diseased functions. There is a hopeful outlook among researchers that highly complex models can accurately mimic the organ of origin, but for most users, simpler models with one or two cells in a monolayer or 3D culture are more practical and effective for mechanistic studies and applications, compared to more intricate models like assembloids or other multicellular models.
Cell Source
The origin of cells used in organoid cultures varies depending on the desired tissue type. Cells can be derived from iPSCs, adult or fetal cells, and can be either stem/progenitor cells or differentiated cells. The selection of the starting cellular population is crucial as it impacts both the variability and heterogeneity of the resulting structures and the function of the modeled tissue. Different methods are employed to obtain tissue-resident stem/progenitor/differentiated cells or tumor cells for tissue-derived organoids or cancer organoids, respectively. iPSC-derived organoids require the establishment and characterization of iPSC lines. Patient/tissue-derived stem cells are obtained through optimized tissue dissociation methods and embedded into a 3D matrix that mimics stem cell niches. The isolation and seeding of primary cells from tissue fragments play a critical role in generating tissue-derived organoids, such as intestinal organoids, as an example.
Matrix
After cell isolation, cells are typically seeded into matrices such as Matrigel, natural extracellular matrix (ECM) like collagen, or synthetic hydrogels. Matrigel, a biologically derived matrix, is commonly used and composed of laminin, collagen IV, entactin, perlecan, and growth factors, resembling the basement membrane. However, matrices like Matrigel offer limited control over the biochemical and biophysical cues necessary for improving organoid culture. Therefore, alternative matrices with defined compositions have been explored, such as recombinant human collagen, fibrin, or synthetic hydrogels. Natural matrices can be recombinantly produced to address the variability of Matrigel, while synthetic hydrogels provide the ability to manipulate both biochemical and biophysical properties. The ideal organoid matrix should be stress-relaxing, dynamically adjustable, and capable of accommodating or controlling changes in organoid structure during culture.
Soluble Factors
Organoid cultures rely on the presentation of soluble cues to cells in a spatio-temporally controlled manner. Soluble factors include proteins like growth factors and small-molecule drugs that activate or inhibit signaling pathways. Combining both biologics and small-molecule drugs in organoid protocols has been explored. The use of conditioned medium from engineered cell lines producing biologically active growth factors or the development of surrogate molecules are emerging strategies. The presentation of soluble cues in a spatio-temporally relevant manner can be achieved through different tissue engineering approaches, such as encapsulation within nanoparticles, tethering onto cell surfaces, or using microfluidic systems.
Physical Cues
Besides biochemical cues, providing appropriate physical cues to cultured organoids is crucial. Nutrient supply and waste removal, which are diffusion-dependent, become less efficient as organoids grow into larger tissue structures. Bioreactors, shaking cultures, microfluidic chips, or perfusable microfluidic devices can address this challenge by enhancing nutrient diffusion and waste removal. Topographical cues can also be considered by manipulating the substrate's topography to control organoid culture.
Integrating Cues
Integrating cues is a strategy used in tissue engineering to exert greater control over organoid morphogenesis. By integrating environmental cues, such as mechanical and biochemical cues, researchers can achieve precise control over the morphogenesis process. Biomaterials engineering, bioprinting, microfluidic platforms, and organ-on-a-chip devices are examples of techniques that facilitate the integration of cues and enable better control over organoid structure and function. These approaches have shown promise in enhancing organoid growth, maturation, and modeling of specific tissue characteristics and physiological features.
Organoids are complex three-dimensional structures that can be created in a laboratory setting to mimic the structure and function of specific tissues. In 2009, Sato and his team made a significant breakthrough by developing a 3D culture system that allowed the growth of mouse intestinal epithelial organoids from single Lgr5+ stem cells (Sato et al., 2009). These organoids, often referred to as "mini guts," closely resemble the architecture and function of the intestinal tissue and can be expanded and differentiated in vitro. Since then, researchers have developed various protocols to create human organoid lines using pluripotent or tissue-resident stem cells (Clevers, 2016; Fatehullah et al., 2016) (Figure 1). Organoids have several advantages over traditional 2D culture systems. They exhibit greater cellular diversity, organization, and tissue-like structures, making them a more relevant in vitro model for studying tissue development, function, and personalized therapies (Li et al., 2019; Corrò et al., 2020). Consequently, organoids have become an invaluable experimental model for investigating the underlying mechanisms of genetic and acquired diseases, as well as for developing patient-specific drug screening (Rossi et al., 2018; Tam et al., 2022). This review focuses on the recent advancements in the field of primary human stem/progenitor cell-derived epithelial organoids, particularly their applications in bioengineering, including culture, analysis, and practical uses.
Derivation and Culture of Primary Organoids
Researchers have successfully generated primary organoids from a variety of human tissues obtained from both fetal and adult sources. The process involves isolating stem/progenitor cells from the tissue sample and culturing them in a 3D environment using an embedding agent that mimics the extracellular matrix. The growth medium containing specific molecules and growth factors is essential for maintaining stemness and inducing tissue-specific processes. These conditions support the division and formation of organoids that closely resemble the original tissue in terms of structure and function.
The culture conditions for each tissue-specific stem/progenitor cell aim to replicate the signals present in their natural environment. The Wnt/b-catenin signaling pathway, activated by Wnt ligands like Wnt-3a and R-Spondin one, plays a crucial role in the growth of epithelial organoids. LGR5+ stem cells, found in many epithelial organoids, have the ability to divide and differentiate into mature cell types when Wnt signaling is activated. Other components in the culture medium include serum- and xeno-free supplements, Wnt activators, tyrosine kinase receptor signaling activators, and BMP/TGF-β signaling inhibitors. Tissue-specific growth factors and small molecules are also added to support organoid growth and self-renewal.
Chemically defined culture conditions are preferred for growing organoids as they offer reproducibility and are compatible with good manufacturing practices. Bioengineering approaches are being explored to enhance the reproducibility of organoid generation and facilitate applications such as drug screening and clinical translation. Characterizing organoids through RNA profiling techniques, such as bulk and single-cell RNA sequencing, is crucial for confirming their molecular similarity to native tissue.
Lung, Upper Airways, and Tonsils
The lung epithelium consists of airway and alveolar regions and contains different pools of stem cells. Early studies identified a subset of P63+ basal cells as multipotent stem cells in the human lung that could form bronchospheres containing differentiated cells. However, these models had limited culture lifespan. In a groundbreaking study, self-renewing primary lung organoids were successfully expanded from human embryonic and fetal lung epithelial tips. These organoids branched and maintained the expression of specific transcription factors. Distal lung organoids partially differentiated into bronchioalveolar structures using a specialized differentiation medium. These organoids have been used to study lung development, molecular mechanisms, and diseases such as cystic fibrosis, lung cancer, and viral infections including SARS-CoV-2 and influenza. Alveolar organoids have also been derived, but their establishment required a co-culture system, and long-term expansion was challenging. The activation or inhibition of the Wnt pathway promoted the differentiation of alveolar organoids into specific cell types. Organoids derived from upper respiratory tract tissues such as oral mucosa and nasal airway cells have been used to study herpes simplex virus (HSV) and human papillomavirus (HPV) infections and model genetic conditions like primary ciliary dyskinesia (PCD). Primary airway organoids have been bioengineered to investigate respiratory diseases, such as ciliopathies. Tonsil organoids resembling human tonsil epithelium have also been established and can be infected with SARS-CoV-2, providing a platform for disease modeling and drug testing. These primary organoid models offer valuable insights into lung biology and disease mechanisms.
Oesophagus:
The development of epithelial organoids from human biopsy samples has provided insights into the study of Barrett's esophagus, a condition associated with dysplasia and adenocarcinoma in the esophageal tissue. Organoids derived from patients with Barrett's esophagus exhibited cellular characteristics of the condition, such as the presence of intestinal goblet cells. FGF10 supplementation enabled the long-term maintenance of these organoids, whereas healthy esophageal organoids could not be passaged. Another study focused on the transitional epithelium found in the human gastro-esophageal junction, which expands during disease progression. Additionally, organoids derived from esophageal biopsies were used to model inflammatory conditions like eosinophilic esophagitis. Co-culturing organoids with non-epithelial cells, such as immune or mesenchymal cells, could enhance the usefulness of these models and provide insights into the disease's pathogenesis.
Stomach:
LGR5+ stem cells play a crucial role in the self-renewal and regeneration of gastric glands. As a result, primary gastric organoids can be derived from isolated gastric glands or stem cells, mimicking the structure and function of the stomach. These organoids retain the morphological characteristics of the human gastric mucosa and contain differentiated cell types found in the stomach. They have been used to model stomach infections like Helicobacter pylori, as well as enteropathogens such as Salmonella enterica and Lysteria monocytogenes. Gastric organoids derived from infants and pediatric biopsies have been established to study gastric epithelial homeostasis and diseases in children. Furthermore, gastric organoids derived from fetal, pediatric, and adult stages have been infected with SARS-CoV-2 to study its effects and transmission in the gastrointestinal tract. The challenge of obtaining fully differentiated parietal cells, which produce and secrete acid, in gastric organoid cultures remains. However, efforts are being made to develop more complex 3D systems that incorporate multiple cellular types, including the mesenchymal component, to address this limitation.
Intestine:
Intestinal organoids were the first type of organoids to be developed and extensively studied. These organoids, derived from adult stem cells, can replicate the crypt-villus axis of the intestine and contain differentiated cells. They have been derived from healthy and diseased human gastrointestinal tissues and have proven valuable in studying gut development, regeneration, diseases, and tissue engineering. Intestinal organoids have been used to investigate age-related diseases by analyzing mutational signatures acquired by adult stem cells through aging. They have also been utilized to model genetic diseases such as cystic fibrosis and inflammatory bowel diseases (IBD) like ulcerative colitis and Crohn's disease. Intestinal organoids can be infected with viral and bacterial pathogens, making them useful for studying microbiology and pharmacology. Bioengineering approaches, such as micro-engineered cultures and bioprinting technology, have been employed to enhance the complexity and functionality of intestinal organoids. These advances have enabled the generation of functional and perfusable tube-like structures that mimic the native crypts of the intestine and preserve its homeostatic and regenerative capacity. Intestinal organoids have also shown promise in regenerative medicine, as they can be used to reconstruct the colon epithelium and generate a functional small intestinal mucosa for transplantation in patients with short bowel syndrome (SBS) and intestinal failure (IF).
Liver and biliary tree:
The culture and expansion of primary liver organoids have been achieved using Lgr5+ stem cells found near the bile ducts. These organoids, derived from intrahepatic bile duct cells, exhibit characteristics of both biliary and hepatic cells. They can differentiate into functional hepatocytes and have been successfully transplanted into damaged mouse livers. Liver organoids have been used to model hepatobiliary disorders such as α1-antitrypsin (A1AT) deficiency and Alagille syndrome (AGS), showcasing disease-specific features. Additionally, organoids derived from extrahepatic tissues, such as the gallbladder and extrahepatic ducts, have been generated and used to study biliary tree disorders. Bioengineering strategies, including the use of microfluidic chips, have been employed to create liver-on-a-chip platforms that mimic the physiological conditions and interactions with other tissues, such as the pancreas. These platforms have shown promise in monitoring parameters and investigating cross-talk between liver and pancreas. Notably, primary cholangiocyte organoids have been used to regenerate intrahepatic bile ducts using ex vivo perfusion systems, offering potential for bioengineering human biliary epithelium in a translational setting.
Pancreas:
Primary organoids have been extensively used to study human pancreatic development and disease. Mouse pancreatic duct organoids were the first to be established, followed by the adaptation of the protocol for generating organoids from human fetal and adult pancreatic progenitors. Human fetal pancreatic organoids exhibit 3D duct-like structures and maintain fetal characteristics, while adult pancreatic organoids contain progenitor cells capable of incomplete differentiation into endocrine cells. These organoids have been used to model cystic fibrosis-related diabetes and have the potential to advance β cell replacement therapy for diabetes. However, the challenge lies in generating mature and functional endocrine cells within the organoids.
Prostate and bladder:
Prostate organoids have been derived from normal and diseased human prostate samples. These organoids can be generated from single luminal or basal cells, and they closely resemble the structure and function of glandular tissue. Similarly, bladder organoids have been successfully established from human urinary bladder epithelium, allowing the study of urothelial cancer. The primary biopsy samples serve as a valuable source for modeling these tissues and investigating their respective diseases.
Thyroid:
Recent studies have demonstrated the generation of thyroid organoids from human primary thyroid gland tissue. These self-renewing organoids have been used to investigate the stemness of thyroid gland cells and can be maintained in culture for extended periods. Thyroid follicular organoids exhibit the characteristics of follicular epithelium and contain differentiated follicular cells. Furthermore, these organoids have the potential to form functional hormone-producing thyroid-like follicles when transplanted into a mouse model of hypothyroidism. Thyroid organoids also offer a platform for modeling autoimmune disorders such as Graves' disease and studying thyroid gland development.
Kidney:
Human kidney organoids, known as tubuloids, have been derived from cortical kidney tissue. Although they lack glomerular cells, these organoids consist of various nephron compartments, including proximal tubules, loop of Henle, distal tubules, and collecting ducts. Tubuloids have been used to model kidney diseases, such as viral infections and Wilms tumors, and have shown potential for personalized disease modeling and drug screening. Bioengineering approaches, including the use of microfluidic chips, have been employed to enhance tubuloid formation and create perfusable kidney tubules. Recent advancements have also focused on generating renal organoids from human fetal kidneys, providing insights into kidney branching morphogenesis and congenital defects.
Endometrium, cervix, and fallopian tube:
Endometrial organoids derived from adult endometrial specimens can be cultured long-term and generate glandular structures that closely resemble the human endometrium. These organoids exhibit functional responsiveness to ovarian hormones and have been used to study menstrual cycle, pregnancy, and endometrial diseases. Similarly, organoids derived from the cervix and fallopian tubes have been successfully generated, allowing the modeling of cervical tissue homeostasis, sexually transmitted infections, and uterine microenvironment during fetal development and early pregnancy.
Ovary and testis:
Organoids from human ovaries have been cultured long-term, enabling the study of ovarian cancer and the derivation of healthy organoids from ovarian surface epithelium. However, the long-term expansion and functional maturation of human testicular organoids in chemically defined media and 3D matrices remain challenging. Further development of physiological culture systems and bioengineering approaches are necessary to improve the maturation of these organoids and generate haploid cells in vitro.
Salivary and lacrimal glands:
Human salivary gland organoids have been derived from submandibular gland biopsies and shown to contain differentiated salivary gland cell types. These organoids have the potential for cell therapy in hyposalivation conditions. Similarly, lacrimal gland organoids have been successfully derived from lacrimal gland biopsies, maintaining the epithelial identity of the primary tissue. These organoids can differentiate into functional lacrimation markers, offering a potential model for studying lacrimal gland function and related disorders.
Mammary gland:
Organoids resembling the ductal and lobular units of the mammary gland have been generated from human mammary epithelial cells. These organoids exhibit contractility and contain specialized differentiated cells, providing insights into lactation and branching morphogenesis. Recent studies have highlighted the potential of mammary organoids in investigating complex developmental processes and the use of bioengineered strategies to enhance their efficiency and reproducibility.
Placenta:
Trophoblast organoids derived from human placenta closely resemble the complex branched morphology of the placental epithelium. These organoids can model placental development and diseases, exhibiting characteristics of different trophoblast lineages. Further development of more complex systems is necessary to fully recapitulate the cell heterogeneity and function of the placenta, allowing a comprehensive investigation of the fetal-maternal interface.
Primary cellular models, such as organoids, have revolutionized research by providing a more accurate representation of human tissue compared to traditional 2D cell cultures. Organoids, which were introduced in 2009, offer a unique modeling tool that allows the study of tissue homeostasis, disease, and drug screening. They have the advantage of better resembling tissue-like features both morphologically and functionally. Unlike 2D systems, organoids can capture the complexity of cellular interactions and represent functional differentiation. They have been successfully derived from various human tissues, including endoderm, mesoderm, ectoderm, and extraembryonic tissues.
Primary organoids have proven invaluable in disease modeling, as they closely recapitulate patient-specific pathological cues and allow for the investigation of specific abnormal cell types. They offer a platform for studying stem/progenitor cell biology, disease mechanisms, and experimental therapies. Additionally, the combination of single-cell profiling, bioengineering, and organoid modeling has provided insights into human organ development and regeneration.
Translating organoid technology to regenerative medicine and clinical applications holds great potential. Developing clinically relevant organoids that can be transplanted as transplantable tissues in a dish requires the use of innovative extracellular matrices (ECMs) and chemically defined media. However, reproducibility of organoid generation, morphology, and function remains a challenge. Establishing robust pipelines for the development, characterization, and clinical translation of organoids is crucial. The organoid research community is striving to establish standards for data quality and reproducibility, addressing issues such as variability between organoids derived from different patients and intra-organoid heterogeneity.
Bioengineering approaches, including co-culture, microfluidics, and bioprinting, offer ways to improve organoid technology. Engineering the stem cell niche and the ECM microenvironment is essential for the growth and maturation of functional organoid models. Incorporating a vascular compartment within organoids through co-culture systems, microfluidics, or in vivo transplantation is also a promising avenue of research. Furthermore, efforts to generate 3D blood vessel models from primary endothelial cells are underway.
The ultimate goal is to achieve standardized and validated organoid systems through international collaborations, such as the Organoid Cell Atlas, to advance clinical and basic research. Despite challenges, primary organoids have proven to be a valuable tool for in vitro disease modeling, and their clinical application is expected to expand in the coming years.
