Organoids are functional cell-tissue complexes that mimic structural and functional characteristics of organsin vitroin three dimensions (3D). Mimicking the natural extracellular matrix (ECM) environment is critical for guiding stem cell fate within organoid cultures. Current organoid cultures predominantly utilize animal- or tumor-derived ECMs such as dECMs and Matrigel. However, these materials introduce batch variability and uncertainty in composition, which hinders reproducibility. In contrast, naturally derived and synthetic hydrogels with excellent biocompatibility offer precise and adjustable compositions, along with tunable mechanical properties, thereby providing robust support for organoid development and maturation. We explore innovative hydrogel designs tailored specifically for organoid cultures, emphasizing the influence and meticulous control of functional hydrogels on organoid formation, differentiation, and maturation processes. Furthermore, the review highlights the potential of functionalized hydrogel scaffolds to advance both research and industrial applications in tissue and organ engineering. As research progresses, investigations will further concentrate on improving the adjustable properties, expanding their scope of application, and more biologically compatible gelation strategies of hydrogels.

Human pluripotent stem cells hold inherent properties, allowing researchers to recapitulate key morphogenetic processes. These characteristics, coupled with bioengineering techniques, have led to the definition of early procedures to derive organ-like cell cultures, the so-called organoids. With regard to kidney organoids, challenges stand ahead, such as the need to enhance cellular composition, maturation, and function to that found in the native organ. To this end, the kidney organoid field has begun to nourish from innovative engineering approaches aiming to gain control on the externally imposed biochemical and biophysical cues. In this review, we first introduce how previous research in kidney development and human pluripotent stem cells has informed the establishment of current kidney organoid procedures. We then discuss recent engineering approaches to guide kidney organoid self-organization, differentiation, and maturation. In addition, we present current strategies to engineer vascularization and promote in vivo-like physiological microenvironments as potential solutions to increase kidney organoid lifespan and functionality. We finally emphasize how working at the cusp of cell mechanics and computational biology will set the ground for successful translational applications of kidney organoids.

Diabetic Kidney Disease (DKD) represents a significant global health burden and is recognised as the leading cause of end-stage renal disease. Kidney organoids derived from human induced Pluripotent Stem Cells (hiPSCs) have the potential to transform how we model renal disease and may provide personalised replacement tissues for patients with renal failure. However, kidney organoids remain poorly reproducible, and are structurally and functionally immature. Three-dimensional cultures that more appropriately mimic the complexity of the in vivo microenvironment are required to improve organoid maturation and structural authenticity. Here, we describe the application of semi-synthetic Gelatin Methacryloyl (GelMA) hydrogels as extracellular support matrices for the differentiation of hiPSC-derived kidney organoids. Hydrogels of defined mechanical strengths were generated by varying the concentration of GelMA solution in the presence of low concentration photo-initiator. After confirming a high level of mechanical stability of the hydrogels over extended culture periods, their effect on kidney organoid maturation was investigated. Organoids differentiated within GelMA hydrogels generated typical renal cell types including podocytes, tubular epithelia, renal interstitial cells, and some nascent vascularisation. Interestingly, kidney organoids derived within hydrogels that closely approximate the stiffness of the adult human kidney (∼5000-10,000 Pa) demonstrated improved podocyte maturation and were shown to upregulate renal vesicle-associated genes at an earlier stage following encapsulation when compared to organoids derived within softer hydrogels (∼400 Pa). A model of TGFβ-induced injury was also developed to investigate the influence of the mechanical environment in propagating early, fibrotic-like features of DKD within organoids. Growth within the softer matrix was shown to reduce pSMAD3 expression following TGFβ1 treatment, and accordingly ameliorate the expression of the myofibroblast marker α-Smooth Muscle Actin (α-SMA). This work demonstrates the suitability of GelMA hydrogels as mechanically-stable, highly-tuneable, batch-to-batch reproducible three-dimensional supports for hiPSC-derived kidney organoid growth and differentiation, and further substantiates the role of the biophysical environment in guiding processes of cell fate determination and organoid maturation.

The kidney's endocrine function is essential for maintaining body homeostasis. Erythropoietin (EPO) is one of the key endocrine factors produced by the kidney, and kidney disease patients frequently experience anemia due to impaired EPO production. In the present study we explored the potential of human induced pluripotent stem cell (iPSC)-derived kidney organoids to restore EPO production.

EPO secretion by kidney organoids was examined under 1% and 20% oxygen levels. To increase the EPO secreting capacity of kidney organoids, iPSC were genetically engineered with a non-integrating scaffold/matrix attachment region (S/MAR) DNA vector containing the EPO gene and generated EPO-overexpressing (EPO+) kidney organoids. To assess the physiological effects of EPO + organoids, 2-8 organoids were implanted subcutaneously in immunodeficient mice.

Kidney organoids produced low amounts of EPO under 1% oxygen. EPO S/MAR DNA vectors persisted and continued to robustly express EPO during iPSC expansion and kidney organoid differentiation without interfering with cellular proliferation. EPO + iPSC demonstrated efficient differentiation into kidney organoids. One-month post-implantation, EPO + organoids displayed continuously elevated EPO mRNA levels and significantly increased endothelial cell numbers compared to control organoids. Hematocrit levels were notably elevated in mice implanted with EPO + organoids in an organoid number-dependent manner. EPO + organoids furthermore influenced bone homeostasis in their hosts, evidenced by a change in trabecular bone composition.

Kidney organoids modified by EPO S/MAR DNA vector allow stable long-term delivery of EPO. The observed physiological effects following the implantation of EPO + organoids underscore the potential of gene-edited kidney organoids for endocrine restoration therapy.

For several decades, research has studied the influence of the extracellular matrix (ECM) mechanical properties in cell response, primarily emphasising its elasticity as the main determinant of cell and tissue behaviour. However, the ECM is not purely elastic; it is viscoelastic. ECM viscoelasticity has now emerged as a major regulator of collective cell dynamics. This review highlights recent findings on the role of ECM viscoelasticity in development and pathology.

Organoids are a cutting-edge technology in the life sciences field, with applications in precision medicine, bionic organs, and toxicological evaluations of chemicals. Their 3D structure closely resembles that of real organs, allowing more accurate functional mimicry. The 3D organoid culture system can simulate the growth state of cells in vivo and establish a suspension culture system for organoid 3D culture by using scaffold-less or scaffold technology to avoid direct contact between cells and plastic culture vessels. Furthermore, organoids can simulate the pathophysiological state of tissues and organs in vitro. This paper primarily discusses the construction methodologies, as well as the advantages and disadvantages of 3D culture systems for both scaffold-free organoids and scaffolded organoids. This review also summarizes the application of organoid models in chemical toxicology evaluation, drug screening and functional evaluation, establishment of in vitro disease models, and research on disease occurrence and potential mechanisms. The aim is to provide a reference for the research and practical applications of organoid-related scientific fields.

The kidneys are vital for maintaining bodily homeostasis and are susceptible to various diseases that disrupt their function. Traditionally, research on kidney diseases has relied on animal models and simplistic two-dimensional cell cultures, which do not fully replicate human tissue pathology. To address this, recent advances focus on developing advanced 3D biomimeticin vitromodels using human-derived cells. These models mimic healthy and diseased kidney tissues with specificity, replicating key elements like glomerular and tubular structures through tissue engineering. By closely mimicking human physiology, they provide a promising platform for studying renal disorders, drug-induced nephrotoxicity, and evaluating new therapies. However, the challenges include optimizing scalability, reproducibility, and long-term stability to enhance reliability in research and clinical applications. This review highlights the transformative potential of 3D biomimeticin vitrokidney models in advancing biomedical research and clinical applications. By focusing on human-specific cell cultures and tissue engineering techniques, these models aim to overcome the limitations of conventional animal models and simplistic 2D cell cultures. The review discusses in detail the various types of biomimetic kidney models currently under development, their specific applications, and the innovative approaches used to construct them. It also addresses the challenges and limitations associated with these models for their widespread adoption and reliability in research settings.

As an emerging technology, organoids are promising new tools for basic and translational research in disease. Currently, the culture of organoids relies mainly on a type of unknown composition scaffold, namely Matrigel, which may pose problems in studying the effect of mechanical properties on organoids. Hydrogels, a new material with adjustable mechanical properties, can adapt to current studies. In this review, we summarized the synthesis of recent advance in developing definite hydrogel scaffolds for organoid culture and identified the critical parameters for regulating mechanical properties. In addition, classified by different mechanical properties like stiffness and viscoelasticity, we concluded the effect of mechanical properties on the development of organoids and tumor organoids. We hope this review enhances the understanding of the development of organoids by hydrogels and provides more practical approaches to investigating them.

While up to 50% of children requiring kidney replacement therapy have congenital anomalies of the kidney and urinary tract (CAKUT), they represent only a fraction of the total patient population with CAKUT. The extreme variability in clinical outcome underlines the fundamental need to devise personalized clinical management strategies for individuals with CAKUT. Better understanding of the pathophysiology of abnormal kidney and urinary tract development provides a framework for precise diagnoses and prognostication of patients, the identification of biomarkers and disease modifiers, and, thus, the development of personalized strategies for treatment. In this review, we provide a state-of-the-art overview of the currently known genetic causes, including rare variants in kidney and urinary tract development genes, genomic disorders, and common variants that have been attributed to CAKUT. Furthermore, we discuss the impact of environmental factors and their interactions with developmental genes in kidney and urinary tract malformations. Finally, we present multi-angle translational modalities to validate candidate genes and environmental factors and shed light on future strategies to better understand the molecular underpinnings of CAKUT.

The dynamic remodeling of the nascent vascular network into a mature hierarchy is essential for embryo survival. Cell behaviors and signaling mechanisms are often investigated with animal models and perfused microchannels, giving insights into this process. To support these studies and enrich our understanding, we demonstrate a complementary approach using vascular organoids. Organoids initially form a primitive endothelial plexus lined with NG2+/PDGFRβ+ mural cell progenitors containing immature pericytes, but there is no formation of large-diameter vessels covered with αSMA+ cells containing immature vascular smooth muscle cells (vSMCs). After transplantation to the chick chorioallantoic membrane, the network reorganizes into a branched architecture with large-diameter vessels covered by αSMA+ cells. We additionally show that blood flow from the host circulation perfuses the organoid. Compared with the developing skin vasculature in mouse embryos, organoids successfully recapitulate vascular morphogenesis, both in vitro and after transplantation. The model described here presents a further approach to enhance the study of vascular remodeling.

Kidney transplantation remains a pivotal treatment modality for kidney disease, yet its progress is significantly hindered by the scarcity of donor kidneys and ethical dilemmas surrounding their procurement. As organoid technology evolves and matures, the creation of bionic human kidney organoids offers profound potential for advancing kidney disease research, drug nephrotoxicity screening, and regenerative medicine. Nevertheless, current kidney organoid models grapple with limitations such as constrained cellular differentiation, underdeveloped functional structures, and a crucial absence of vascularization. This deficiency in vascularization, in particular, stunts organoid development, restricts their size, diminishes filtration capabilities, and may trigger immune inflammatory reactions through the resulting ischemic microenvironment. Hence, the achievement of vascularization within kidney organoids and the successful establishment of functional microvascular networks constitutes a paramount goal for their future progression. In this review, we provide an overview of recent advancements in biotechnology domains, encompassing organ-on-a-chip technology, biomimetic matrices, and bioprinting, with the aim of catalyzing technological breakthroughs that can enhance the vascularization of kidney organoids and broaden their applicability. These technologies hold the key to unlocking the full potential of kidney organoids as a transformative therapeutic option for kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent hereditary disorder characterized by distinct phenotypic variability that has posed challenges for advancing in-depth research. Recent advancements in kidney organoid construction technologies have enabled researchers to simulate kidney development and create simplified in vitro experimental environments, allowing for more direct observation of how genetic mutations drive pathological phenotypes and disrupt physiological functions. Emerging technologies, such as microfluidic bioreactor culture systems and single-cell transcriptomics, have further supported the development of complex ADPKD organoids, offering robust models for exploring disease mechanisms and facilitating drug discovery. Nevertheless, significant challenges remain in constructing more accurate ADPKD disease models. This review will summarize recent advances in ADPKD organoid construction, focusing on the limitations of the current techniques and the critical issues that need to be addressed for future breakthroughs. New and Noteworthy: This review presents recent advancements in ADPKD organoid construction, particularly iPSC-derived models, offering new insights into disease mechanisms and drug discovery. It focuses on challenges such as limited vascularization and maturity, proposing potential solutions through emerging technologies. The ongoing optimization of ADPKD organoid models is expected to enhance understanding of the disease and drive breakthroughs in disease mechanisms and targeted therapy development.

The increasing incidence of kidney diseases has highlighted the need for in vitro experimental models to mimic disease development and to test new therapeutic approaches. Traditional two-dimensional in vitro experimental models are not fully able to recapitulate renal diseases. Instead, kidney organoids represent three-dimensional models that better mimic the human organ from both structural and functional points of view. Human pluripotent stem cells (PSCs), both embryonic and induced, are ideal sources for generating renal organoids. These organoids contain all renal cell types and the protocols to differentiate PSCs into renal organoids consist of three different stages that recapitulate embryonic development: mesodermal induction, nephron progenitor formation, and nephron differentiation. Recently it has been establish a renal organoid model where collecting ducts are also present. In this case, the presence of ureteric bud progenitor cells is essential. Renal organoids are particularly useful for studying genetic diseases, by introducing the specific mutations in PSCs by genome editing or generating organoids from patient-derived PSCs. Moreover, renal organoids represent promising models in toxicology studies and testing new therapeutic approaches. Renal organoids can be established also from adult stem cells. This type of organoid, named tubuloid, is composed only of epithelial cells and recapitulates the tissue repair process. The tubuloids can be generated from adult stem or progenitor cells, obtained from renal biopsies or urine, and are promising in vitro models for studying tubular functions, diseases, and regeneration. Tubuloids can be derived from patients and permit the study of genetic diseases, performing personalized drug screening and modeling renal pathologies.

Human pluripotent stem cell-derived kidney organoids hold promise for future applications in regenerative medicine. However, significant biological hurdles need to be overcome to enable their use as a transplantable stem cell-derived therapeutic graft. Current kidney organoid protocols do not recapitulate a complete integrated developing kidney, but embryonic kidney transplantations have provided clues for advancing maturation and functionality of kidney organoids. Transplantation, subsequent vascularization, and blood perfusion of kidney organoids improve nephron patterning and maturation, suggesting a role for angiocrine factors as well as metabolic wiring in these processes. Transplanted organoids exhibit filtration, but the resulting filtrate has no apparent exit path for excretion. Improved in vitro patterning of kidney organoids may be required such that a more structurally correct tissue is formed before transplant. Here we review current progress with transplantation of kidney organoids, as well as their engraftment and integration, and identify the key obstacles to therapeutic success and how these might be achieved.

Developing disease models and screening for effective drugs are key areas of modern medical research. Traditional methodologies frequently fall short in precisely replicating the intricate architecture of bodily tissues and organs. Nevertheless, recent advancements in biomaterial-assisted organoid technology have ushered in a paradigm shift in biomedical research. This innovative approach enables the cultivation of three-dimensional cellular structures in vitro that closely emulate the structural and functional attributes of organs, offering physiologically superior models compared to conventional techniques. The evolution of biomaterials plays a pivotal role in supporting the culture and development of organ tissues, thereby facilitating more accurate disease state modeling and the rigorous evaluation of drug efficacy and safety profiles. In this review, we will explore the roles that various biomaterials play in organoid development, examine the fundamental principles and advantages of utilizing these technologies in constructing disease models, and highlight recent advances and practical applications in drug screening using disease-specific organoid models. Additionally, the challenges and future directions of organoid technology are discussed. Through continued research and innovation, we aim to make remarkable strides in disease treatment and drug development, ultimately enhancing patient quality of life.

Renal progenitor organoids have been proposed as a source of tissue for kidney regeneration; however, their clinical translatability has not been demonstrated due to an inability to mass-produce comprehensive renal progenitor organoids and the lack of an effective intra-renal delivery platform that facilitates rapid integration into functionally meaningful sites. This study addresses these shortcomings. Human-induced pluripotent stem cells were differentiated into renal progenitor cells using an established protocol and aggregated using a novel assembly method to produce high yields of organoids. Organoids were encapsulated in collagen-based scaffolds for in vitro study and in vivo implantation into mouse renal cortex. In vitro, the organoids demonstrated sustained cell viability and renal structure maturation over time. In vivo delivered organoids showed rapid integration into host renal parenchyma while showing tubular and glomerular-like structure development and maturity markers. This proof-of-concept study presents many promising results, providing a system of renal organoid formation and delivery that may support the development of clinically translatable therapies and the advancement of in vitro renal organoid studies.

Organoids are miniature organs developed through technology. Kidney organoids that originate from human inducible pluripotent stem cells (iPSCs) were developed to recreate renal diseases. However, it is impossible to simultaneously produce kidney organoids from iPSCs of multiple individuals and in a single medium. We herein report the development of adult renal tubular organoids, namely, "tubuloids," from primary renal epithelial cells from multiple human individuals in a single medium.

Kidneys from eight patients who underwent nephrectomy due to malignancy were sectioned, and primary renal epithelial tubule cells were cultured; four had normal kidney function, and four had mild chronic kidney disease (CKD). Growth factors and Matrigel were added to the primary culture.

Primary cultured renal epithelial cells from normal kidneys exhibited a fine and swollen epithelial appearance, whereas those from kidneys with mild CKD were smaller and slightly elongated. Growth was faster in normal kidney cells than in mild CKD cells. At the beginning of the three-dimensionalization (day 0), normal renal tubuloids grew faster than mild CKD tubuloids. The difference in size between normal and mild CKD tubuloids was not obvious by day 5. Both tubuloid types had comparable sizes by day 21.

Renal tubular organoids can be developed simultaneously and in a single medium. Our method is expected to be used as a human pathological model.

Organoids-on-chips is an emerging innovative integration of stem cell-derived organoids with advanced organ-on-chip technology, providing a novel platform for the in vitro construction of biomimetic micro-physiological systems. The synergistic merger transcends the limitations of traditional drug screening and safety assessment methodologies, such as 2D cell cultures and animal models. In this review, we examine the prevailing challenges and prerequisites of preclinical models utilized for drug screening and safety evaluations. We highlighted the salient features and merits of organoids-on-chip, elucidating their capability to authentically replicate human physiology, thereby addressing contemporary impediments. We comprehensively overviewed the recent endeavors where organoids-on-chips have been harnessed for drug screening and safety assessment and delved into potential opportunities and challenges for evolving sophisticated, near-physiological organoids-on-chips. Based on current achievements, we further discuss how to enhance the practicality of organoids-on-chips and accelerate the translation from preclinical to clinical stages in healthcare and industry by utilizing multidisciplinary convergent innovation.

Cells and tissues are often under some level of confinement, imposed by the microenvironment and neighboring cells, meaning that there are limitations to cell size, volume changes, and fluid exchanges. 3D cell culture, increasingly used for both single cells and organoids, inherently impose levels of confinement absent in 2D systems. It is thus key to understand how different levels of confinement influences cell survival, cell function, and cell fate. It is well known that the mechanical properties of the microenvironment, such as stiffness and stress relaxation, are important in activating mechanosensitive pathways, and these are responsive to confinement conditions. In this review, we look at how low, intermediate, and high levels of confinement modulate the activation of known mechanobiology pathways, in single cells, organoids, and tumor spheroids, with a specific focus on 3D confinement in microwells, elastic, or viscoelastic scaffolds. In addition, a confining microenvironment can drastically limit cellular communication in both healthy and diseased tissues, due to extracellular crowding. We discuss potential implications of extracellular crowding on molecular transport, extracellular matrix deposition, and fluid transport. Understanding how cells sense and respond to various levels of confinement should inform the design of 3D engineered matrices that recapitulate the physical properties of tissues.

The kidney plays a crucial role in maintaining the body's microenvironment homeostasis. However, current treatment options and therapeutic agents for chronic kidney disease (CKD) are limited. Fortunately, the advent of kidney organoids has introduced a novel in vitro model for studying kidney diseases and drug screening. Despite significant efforts has been leveraged to mimic the spatial-temporal dynamics of fetal renal development in various types of kidney organoids, there is still a discrepancy in cell types and maturity compared to native kidney tissue. The extracellular matrix (ECM) plays a crucial role in regulating cellular signaling, which ultimately affects cell fate decision. As a result, ECM can refine the microenvironment of organoids, promoting their efficient differentiation and maturation. This review examines the existing techniques for culturing kidney organoids, evaluates the strengths and weaknesses of various types of kidney organoids, and assesses the advancements and limitations associated with the utilization of the ECM in kidney organoid culture. Additionally, it presents a discussion on constructing specific physiological and pathological microenvironments using decellularized extracellular matrix during certain developmental stages or disease occurrences, aiding the development of kidney organoids and disease models.

The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform, called UniMat, which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered, permeable membrane-based platform. Using UniMat, we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably, kidney organoids within UniMat show improved maturation, showing increased expression of nephron transcripts, more in vivo-like cell-type balance, enhanced vascularization, and better long-term stability. Moreover, UniMat's design offers a more standardized organoid model for disease modeling and drug testing, as demonstrated by polycystic-kidney disease and acute kidney injury modeling. In essence, UniMat presents a valuable platform for organoid technology, with potential applications in organ development, disease modeling, and drug screening.

Lung cancer is emerging as a common malignancy worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Two-dimensional (2D) in vitro cell line cultures and animal models are currently used to study NSCLC. However, 2D cell cultures fail to replicate the medication response and neoplastic heterogeneity of parental tumors. Animal models are expensive and require lengthy modeling cycles. The generation of in vitro three-dimensional (3D) tissue cultures called organoids, which exhibit multicellular, anatomical, and functional properties of real organs, is now achievable owing to advancements in stem cell culturing. The genetic, proteomic, morphological, and pharmacological characteristics of tumors are largely preserved in tumor organoids grown in vitro. The design and physiology of human organs can be precisely reconstructed in tumor organoids, opening new possibilities for complementing the use of animal models and studying human diseases. This review summarizes the development of NSCLC organoids and their applications in basic research, drug testing, immunotherapy, and individualized treatments.

Organoids are emerging as a powerful tool to investigate complex biological structures in vitro. Vascularization of organoids is crucial to recapitulate the morphology and function of the represented human organ, especially in the case of the kidney, whose primary function of blood filtration is closely associated with blood circulation. Current in vitro microfluidic approaches have only provided initial vascularization of kidney organoids, whereas in vivo transplantation to animal models is problematic due to ethical problems, with the exception of xenotransplantation onto a chicken chorioallantoic membrane (CAM). Although CAM can serve as a good environment for vascularization, it can only be used for a fixed length of time, limited by development of the embryo. Here, we propose a novel lab on a chip design that allows organoids of different origin to be cultured and vascularized on a CAM, as well as to be transferred to in vitro conditions when required. Mouse embryonic kidneys cultured on the CAM showed enhanced vascularization by intrinsic endothelial cells, and made connections with the chicken vasculature, as evidenced by blood flowing through them. After the chips were transferred to in vitro conditions, the vasculature inside the organoids was successfully maintained. To our knowledge, this is the first demonstration of the combination of in vivo and in vitro approaches applied to microfluidic chip design.

Our knowledge of human biology is mainly originated from studies using animal models. However, interspecies differences between human and model organisms may lead to imprecise extrapolation of results obtained from model organisms. Organoids are three-dimensional cell clusters derived from pluripotent or adult stem cells that self-organize into organ-like structures reminiscent of the cognate organ. The establishment of human organoids makes it possible to study organ or tissue pathophysiology that is specific to human beings. However, most organoids do not have organ-specific vasculature, neurons, and immune cells, hence limiting their utility in emulating complex pathophysiological phenotypes. Among the various approaches to address these limitations, xenotransplantation represents a promising 'shortcut'. We will discuss recent advance in constructing tissue complexity in organoids, with a special focus on xenotransplantation.

Patient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient - in vitro PDOs - in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns.

The main liver metastasis of the CRC patient exhibited high 2-[18F]FDG uptake and moderate and focal [68Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[18F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient's liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient's sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [68Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [68Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking.

We successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient's tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [68Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[18F]FDG was avidly taken up in the patient's liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients.

The three-dimensional (3D) kidney organoid is a breakthrough model for recapitulating renal morphology and function in vitro, which is grown from stem cells and resembles mammalian kidney organogenesis. Currently, protocols for cultivating this model from induced pluripotent stem cells (iPSCs) and patient-derived adult stem cells (ASCs) have been widely reported. In recent years, scientists have focused on combining cutting-edge bioengineering and bioinformatics technologies to improve the developmental accuracy of kidney organoids and achieve high-throughput experimentation. As a remarkable tool for mechanistic research of the renal system, kidney organoid has both potential and challenges. In this review, we have described the evolution of kidney organoid establishment methods and highlighted the latest progress leading to a more sophisticated kidney transformation research model. Finally, we have summarized the main applications of renal organoids in exploring kidney disease.

Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.

To date, strategies aiming to modulate cell to extracellular matrix (ECM) interactions during organoid derivation remain largely unexplored. Here renal decellularized ECM (dECM) hydrogels are fabricated from porcine and human renal cortex as biomaterials to enrich cell-to-ECM crosstalk during the onset of kidney organoid differentiation from human pluripotent stem cells (hPSCs). Renal dECM-derived hydrogels are used in combination with hPSC-derived renal progenitor cells to define new approaches for 2D and 3D kidney organoid differentiation, demonstrating that in the presence of these biomaterials the resulting kidney organoids exhibit renal differentiation features and the formation of an endogenous vascular component. Based on these observations, a new method to produce kidney organoids with vascular-like structures is achieved through the assembly of hPSC-derived endothelial-like organoids with kidney organoids in 3D. Major readouts of kidney differentiation and renal cell morphology are assessed exploiting these culture platforms as new models of nephrogenesis. Overall, this work shows that exploiting cell-to-ECM interactions during the onset of kidney differentiation from hPSCs facilitates and optimizes current approaches for kidney organoid derivation thereby increasing the utility of these unique cell culture platforms for personalized medicine.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures.

Kidney disease is a global health crisis affecting more than 850 million people worldwide. In the United States, annual Medicare expenditures for kidney disease and organ failure exceed $81 billion. Efforts to develop targeted therapeutics are limited by a poor understanding of the molecular mechanisms underlying human kidney disease onset and progression. Additionally, 90% of drug candidates fail in human clinical trials, often due to toxicity and efficacy not accurately predicted in animal models. The advent of ex vivo kidney models, such as those engineered from induced pluripotent stem (iPS) cells and organ-on-a-chip (organ-chip) systems, has garnered considerable interest owing to their ability to more accurately model tissue development and patient-specific responses and drug toxicity. This review describes recent advances in developing kidney organoids and organ-chips by harnessing iPS cell biology to model human-specific kidney functions and disease states. We also discuss challenges that must be overcome to realize the potential of organoids and organ-chips as dynamic and functional conduits of the human kidney. Achieving these technological advances could revolutionize personalized medicine applications and therapeutic discovery for kidney disease.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

The formation of functional epithelial tubules is a central feature of many organ systems. Although the process of tubule formation by epithelial cells is well-studied, the way in which tubules connect with each other (i.e. anastomose) to form functional networks both in vivo and in vitro is not well understood. A key, unanswered question in the kidney is how the renal vesicles of the embryonic kidney connect with the nascent collecting ducts to form a continuous urinary system. We performed a ligand-receptor pair analysis on single cell RNA-seq data from embryonic mouse kidney tubules undergoing anastomosis to select candidates that might mediate this process in vivo. This analysis identified hepatocyte growth factor (HGF), which has known roles in cell proliferation, migration, and tubulogenesis, as one of several possible candidates. To test this possibility, we designed a novel assay to quantitatively examine epithelial tubule anastomosis in vitro using epithelial spheroids with fluorescently-tagged apical surfaces to enable direct visualization of anastomosis. This revealed that HGF is a potent inducer of tubule anastomosis. Tubule anastomosis occurs through a proliferation-independent mechanism that acts through the MAPK signaling cascade and matrix metalloproteinases (MMPs), the latter suggestive of a role in extracellular matrix turnover. Accordingly, treatment of explanted embryonic mouse kidneys with HGF and collagenase was sufficient to induce kidney tubule anastomosis. These results lay the groundwork for investigating how to promote functional interconnections between tubular epithelia, which have important clinical implications for utilizing in vitro grown kidney tissue in transplant medicine.

As an advance laboratory model, three-dimensional (3D) organoid culture has recently been recruited to study development, physiology and abnormality of kidney tissue. Micro-tissues derived from primary renal cells are composed of 3D epithelial structures representing the main characteristics of original tissue. In this research, we presented a simple method to isolate mouse renal clonogenic mesenchymal (MLCs) and epithelial-like cells (ELCs). Then we have done a full characterization of MLCs using flow cytometry for surface markers which showed that more than 93% of cells expressed these markers (Cd44, Cd73 and Cd105). Epithelial and stem/progenitor cell markers characterization also performed for ELC cells and upregulating of these markers observed while mesenchymal markers expression levels were not significantly increased in ELCs. Each of these cells were cultured either alone (ME) or in combination with human umbilical vein endothelial cells (HUVECs) (MEH; with an approximate ratio of 10:5:2) to generate more mature kidney structures. Analysis of 3D MEH renal micro-tissues (MEHRMs) indicated a significant increase in renal-specific gene expression including Aqp1 (proximal tubule), Cdh1 (distal tubule), Umod (loop of Henle), Wt1, Podxl and Nphs1 (podocyte markers), compared to those groups without endothelial cells, suggesting greater maturity of the former tissue. Furthermore, ex ovo transplantation showed greater maturation in the constructed 3D kidney.

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

Vascularized organoid-on-a-chip (VOoC) models achieve substance exchange in deep layers of organoids and provide a more physiologically relevant system in vitro. Common designs for VOoC primarily involve two categories: self-assembly of endothelial cells (ECs) to form microvessels and pre-patterned vessel lumens, both of which include the hydrogel region for EC growth and allow for controlled fluid perfusion on the chip. Characterizing the vasculature of VOoC often relies on high-resolution microscopic imaging. However, the high scattering of turbid tissues can limit optical imaging depth. To overcome this limitation, tissue optical clearing (TOC) techniques have emerged, allowing for 3D visualization of VOoC in conjunction with optical imaging techniques. The acquisition of large-scale imaging data, coupled with high-resolution imaging in whole-mount preparations, necessitates the development of highly efficient analysis methods. In this review, we provide an overview of the chip designs and culturing strategies employed for VOoC, as well as the applicable optical imaging and TOC methods. Furthermore, we summarize the vascular analysis techniques employed in VOoC, including deep learning. Finally, we discuss the existing challenges in VOoC and vascular analysis methods and provide an outlook for future development.

The 14th International Podocyte Conference took place in Philadelphia, Pennsylvania, USA from May 23 to 26, 2023. It commenced with an early-career researchers' meeting on May 23, providing young scientists with a platform to present and discuss their research findings. Throughout the main conference, 29 speakers across 9 sessions shared their insights on podocyte biology, glomerular medicine, novel technologic advancements, and translational approaches. Additionally, the event featured 3 keynote lectures addressing engineered chimeric antigen receptor T cell- and mRNA-based therapies and the use of biobanks for enhanced disease comprehension. Furthermore, 4 brief oral abstract sessions allowed scientists to present their findings to a broad audience. The program also included a panel discussion addressing the challenges of conducting human research within the American Black community. Remarkably, after a 5-year hiatus from in-person conferences, the 14th International Podocyte Conference successfully convened scientists from around the globe, fostering the presentation and discussion of crucial research findings, as summarized in this review. Furthermore, to ensure continuous and sustainable education, research, translation, and trial medicine related to podocyte and glomerular diseases for the benefit of patients, the International Society of Glomerular Disease was officially launched during the conference.

Stem cell-derived kidney organoids contain nephron segments that recapitulate morphological and functional aspects of the human kidney. However, directed differentiation protocols for kidney organoids are largely conducted using biochemical signals to control differentiation. Here, the hypothesis that mechanical signals regulate nephrogenesis is investigated in 3D culture by encapsulating kidney organoids within viscoelastic alginate hydrogels with varying rates of stress relaxation. Tubular nephron segments are significantly more convoluted in kidney organoids differentiated in encapsulating hydrogels when compared with those in suspension culture. Hydrogel viscoelasticity regulates the spatial distribution of nephron segments within the differentiating kidney organoids. Consistent with these observations, a particle-based computational model predicts that the extent of deformation of the hydrogel-organoid interface regulates the morphology of nephron segments. Elevated extracellular calcium levels in the culture medium, which can be impacted by the hydrogels, decrease the glomerulus-to-tubule ratio of nephron segments. These findings reveal that hydrogel encapsulation regulates nephron patterning and morphology and suggest that the mechanical microenvironment is an important design variable for kidney regenerative medicine.

The rising prevalence of kidney diseases urges the need for novel therapies. Kidney organoids and tubuloids are advanced in vitro models and have recently been described as promising tools to study kidney (patho)physiology. Recent developments have shown their application in disease modeling, drug screening, and nephrotoxicity. These applications rely on their ability to mimic (dys)function in vitro including endocrine activity and drug, electrolyte, and water transport. This review provides an overview of these emerging kidney models and focuses on the most recent developments that utilize their functional capabilities. In addition, we cover current limitations and provide future perspectives for this rapidly evolving field, including what these functional properties mean for translational and personalized medicine now and in the future.