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The mesolimbic pathway connects ventral tegmental area dopaminergic neurons and striatal medium spiny neurons, playing a critical role in reward and stress behaviors. Exposure to substances of abuse during development and adulthood has been linked to adverse outcomes and molecular changes. The rise of human cell repositories and whole-genome sequences enables human functional genomics ‘in a dish’, offering insights into human-specific responses to substances of abuse. Continued development of new models is needed, and the characterization of in vitro models is also necessary to ensure appropriate experimental designs and the accurate interpretation of results. This study introduces new culture conditions for generating medium spiny neurons and dopaminergic neurons with an early common media, allowing for coculture and assembloid generation. It then provides a comprehensive characterization of these and prior models and their responses to substances of abuse. Single-cell analysis reveals cell-type-specific transcriptomic responses to dopamine, cocaine, and morphine, including compound and cell-type-specific transcriptomic signatures related to neuroinflammation and alterations in signaling pathways. These findings offer a resource for future genomics studies leveraging human stem cell-derived models.

Human noroviruses (HuNoVs) are the main pathogens that cause acute gastroenteritis and lead to huge economic losses annually. Due to the lack of suitable culture systems, the pathogenesis of HuNoVs and the development of vaccines and drugs have progressed slowly. Although researchers have employed various methods to culture HuNoVs in vitro in the last century, problems relating to the irreducibility, low viral titer, and non-infectiousness of the progeny virus should not be ignored. In 2016, researchers achieved the cultivation and successive passaging of some HuNoV genotypes using human intestinal enteroids, initially demonstrating the potential use of organoids in overcoming this challenge. This paper reviews the efforts made in the last 20 years to culture HuNoVs in vitro and discusses the superiority and limitations of employing human intestinal enteroids/organoids as an in vitro culture model for HuNoVs.

The incidence of liver diseases is high worldwide. Many factors can cause liver fibrosis, which in turn can lead to liver cirrhosis and even liver cancer. Due to the shortage of donor organs, immunosuppression, and other factors, only a few patients are able to undergo liver transplantation. Therefore, how to construct a bioartificial liver that can be transplanted has become a global research hotspot. With the rapid development of three-dimensional (3D) bioprinting in the field of tissue engineering and regenerative medicine, researchers have tried to use various 3D bioprinting technologies to construct bioartificial livers in vitro. In terms of the choice of bioinks, liver decellularized extracellular matrix (dECM) has many advantages over other materials for cell-laden hydrogel in 3D bioprinting. This review mainly summarizes the acquisition of liver dECM and its application in liver 3D bioprinting as a bioink with respect to availability, printability, and biocompatibility in many aspects and puts forward the current challenges and prospects.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Biophysical factors play a fundamental role in human embryonic development. Traditional in vitro models of organogenesis focused on the biochemical environment and did not consider the effects of mechanical forces on developing tissue. While most human tissue has a Young’s modulus in the low kilopascal range, the standard cell culture substrate, plasma-treated polystyrene, has a Young’s modulus of 3 gigapascals, making it 10,000–100,000 times stiffer than native tissues. Modern in vitro approaches attempt to recapitulate the biophysical niche of native organs and have yielded more clinically relevant models of human tissues. Since Clevers’ conception of intestinal organoids in 2009, the field has expanded rapidly, generating stem-cell derived structures, which are transcriptionally similar to fetal tissues, for nearly every organ system in the human body. For this reason, we conjecture that organoids will make their first clinical impact in fetal regenerative medicine as the structures generated ex vivo will better match native fetal tissues. Moreover, autologously sourced transplanted tissues would be able to grow with the developing embryo in a dynamic, fetal environment. As organoid technologies evolve, the resultant tissues will approach the structure and function of adult human organs and may help bridge the gap between preclinical drug candidates and clinically approved therapeutics. In this review, we discuss roles of tissue stiffness, viscoelasticity, and shear forces in organ formation and disease development, suggesting that these physical parameters should be further integrated into organoid models to improve their physiological relevance and therapeutic applicability. It also points to the mechanotransductive Hippo-YAP/TAZ signaling pathway as a key player in the interplay between extracellular matrix stiffness, cellular mechanics, and biochemical pathways. We conclude by highlighting how frontiers in physics can be applied to biology, for example, how quantum entanglement may be applied to better predict spontaneous DNA mutations. In the future, contemporary physical theories may be leveraged to better understand seemingly stochastic events during organogenesis.

Porcine hemagglutinating encephalomyelitis virus (PHEV) replicates in the upper respiratory tract and tonsils of pigs. Using an air–liquid interface porcine respiratory epithelial cells (ALI-PRECs) culture system, we demonstrated that PHEV disrupts respiratory epithelia homeostasis by impairing ciliary function and inducing antiviral, pro-inflammatory cytokine, and chemokine responses. This study explores the mechanisms driving early innate immune responses during PHEV infection through host transcriptome analysis. Total RNA was collected from ALI-PRECs at 24, 36, and 48 h post inoculation (hpi). RNA-seq analysis was performed using an Illumina Hiseq 600 to generate 100 bp paired-end reads. Differential gene expression was analyzed using DeSeq2. PHEV replicated actively in ALI-PRECs, causing cytopathic changes and progressive mucociliary disruption. Transcriptome analysis revealed downregulation of cilia-associated genes such as CILK1, DNAH11, LRRC-23, -49, and -51, and acidic sialomucin CD164L2. PHEV also activated antiviral signaling pathways, significantly increasing the expression of interferon-stimulated genes (RSAD2, MX1, IFIT, and ISG15) and chemokine genes (CCL5 and CXCL10), highlighting inflammatory regulation. This study contributes to elucidating the molecular mechanisms of the innate immune response to PHEV infection of the airway epithelium, emphasizing the critical roles of the mucociliary, interferon, and chemokine responses.

Intensive efforts to develop anti-cancer agents have been made for over 60 years. However, cancer is still considered a lethal disease. To study the best anti-cancer agents for improving the survival rates of cancer patients, many researchers have focused on establishing advanced experimental applications reflecting on the biomimetics of cancer patients involving the heterogeneity of cancer cells. The heterogeneity of cancer cells, which are derived from various clones and affected by different environments, presents different genetic backgrounds and molecular characteristics attributed to the differential responses to cancer therapies, and these are responsible for the resistance to cancer therapies, as well as for recurrence following cancer treatments. Therefore, the development of advanced applications for the cancer patient is expected to help the development of more effective anti-cancer agents. The present review evaluates recently developed cancer models encompassing the heterogeneity of cancer cells, which present similar morphological architecture, genetic backgrounds, and molecular characteristics to corresponding patient tumor tissues.

Intestinal absorption is a complex process involving the permeability of the epithelial barrier, efflux transporter activity, and intestinal metabolism. Identifying the key factors that govern intestinal absorption for each investigational drug is crucial. To assess and predict intestinal absorption in humans, it is necessary to leverage appropriate in vitro systems. Traditionally, Caco-2 monolayer systems and intestinal Ussing chamber studies have been considered the ‘gold standard’ for studying intestinal absorption. However, these methods have limitations that hinder their universal use in drug discovery and development. Recently, there has been an increasing number of reports on complex in vitro models (CIVMs) using human intestinal organoids derived from intestinal tissue specimens or iPSC-derived enterocytes plated on 2D or 3D in microphysiological systems. These CIVMs provide a more physiologically relevant representation of key ADME-related proteins compared to conventional in vitro methods. They hold great promise for use in drug discovery and development due to their ability to replicate the expressions and functions of these proteins. This review highlights recent advances in gut CIVMs employing intestinal organoid model systems compared to conventional methods. It is important to note that each CIVM should be tailored to the investigational drug properties and research questions at hand.

Salivary gland dysfunction affects millions globally, and tissue engineering may provide a promising therapeutic avenue. This review delves into the current state of salivary gland tissue engineering research, starting with a study of normal salivary gland development and function. It discusses the impact of fibrosis and cellular senescence on salivary gland pathologies. A diverse range of cells suitable for tissue engineering including cell lines, primary salivary gland cells, and stem cells are examined. Moreover, the paper explores various supportive biomaterials and scaffold fabrication methodologies that enhance salivary gland cell survival, differentiation, and engraftment. Innovative engineering strategies for the improvement of vascularization, innervation, and engraftment of engineered salivary gland tissue, including bioprinting, microfluidic hydrogels, mesh electronics, and nanoparticles, are also evaluated. This review underscores the promising potential of this research field for the treatment of salivary gland dysfunction and suggests directions for future exploration.

Cataract, the opacification of the lens, is the leading cause of blindness worldwide. Although effective, cataract surgery is costly and can lead to complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined three-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization and biology. These organoids can be rapidly produced in large amounts. High-throughput RNA sequencing (RNA-seq) on specific organoid regions isolated via laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display a spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to the induction of opacities. Finally, the knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes that are functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataracts.

Common prostate diseases such as prostatitis and benign prostatic hyperplasia (BPH) have a high incidence at any age. Cellular stresses, such as reactive oxygen species (ROS) and chronic inflammation, are implicated in prostate enlargement and cancer progression and development. Kaempferol is a flavonoid found in abundance in various plants, including broccoli and spinach, and has been reported to exhibit positive biological activities, such as antioxidant and anti-inflammatory properties. In the present study, we introduced prostate organoids to investigate the protective effects of kaempferol against various cellular stresses. The levels of COX-2, iNOS, p-IκB, a pro-inflammatory cytokine, and ROS were increased by LPS treatment but reversed by kaempferol treatment. Kaempferol activated the nuclear factor erythroid 2-related factor 2(Nrf2)-related pathway and enhanced the mitochondrial quality control proteins PGC-1α, PINK1, Parkin, and Beclin. The increase in mitochondrial ROS and oxygen consumption induced by LPS was stabilized by kaempferol treatment. First, our study used prostate organoids as a novel evaluation platform. Secondly, it was demonstrated that kaempferol could alleviate the mitochondrial damage in LPS-induced induced prostate organoids by reducing the production of mitochondrial ROS.

One major challenge associated with lung cancer organoids (LCOs) is their predominant derivation from surgical specimens of patients with early-stage lung cancer. However, patients with advanced lung cancer, who are in need of chemotherapy, often cannot undergo surgery. Therefore, there is an urgent need to successfully generate LCOs from biopsy specimens. Conventional lung biopsy techniques, such as transthoracic needle biopsy and forceps biopsy, only yield small amounts of lung tissue, resulting in a low success rate for culturing LCOs from biopsy samples. Furthermore, potential complications, like bleeding and pneumothorax, make it difficult to obtain sufficient tissue. Another critical issue is the overgrowth of normal lung cells in later passages of LCO culture, and the optimal culture conditions for LCOs are yet to be determined. To address these limitations, we attempted to create LCOs from cryobiopsy specimens obtained from patients with lung cancer (n = 113). Overall, the initial success rate of establishing LCOs from cryobiopsy samples was 40.7% (n = 46). Transbronchial cryobiopsy enables the retrieval of significantly larger amounts of lung tissue than bronchoscopic forceps biopsy. Additionally, cryobiopsy can be employed for peripheral lesions, and it is aided via radial endobronchial ultrasonography. This study significantly improved the success rate of LCO culture and demonstrated that the LCOs retained characteristics that resembled the primary tumors. Single-cell RNA sequencing confirmed high cancer cell purity in early passages of LCOs derived from patients with advanced lung cancer. Furthermore, the three-dimensional structure and intracellular components of LCOs were characterized using three-dimensional holotomography. Finally, drug screening was performed using a specialized micropillar culture system with cryobiopsy-derived LCOs. LCOs derived from cryobiopsy specimens offer a promising solution to the critical limitations of conventional LCOs. Cryobiopsy can be applied to patients with lung cancer at all stages, including those with peripheral lesions, and can provide sufficient cells for LCO generation. Therefore, we anticipate that cryobiopsy will serve as a breakthrough strategy for the clinical application of LCOs in all stages of lung cancer.

Gastric organoids are biological models constructed in vitro using stem cell culture and 3D cell culture techniques, which are the latest research hotspots. The proliferation of stem cells in vitro is the key to gastric organoid models, making the cell subsets within the models more similar to in vivo tissues. Meanwhile, the 3D culture technology also provides a more suitable microenvironment for the cells. Therefore, the gastric organoid models can largely restore the growth condition of cells in terms of morphology and function in vivo. As the most classic organoid models, patient-derived organoids use the patient’s own tissues for in vitro culture. This kind of model is responsive to the ‘disease information’ of a specific patient and has great effect on evaluating the strategies of individualized treatment. Herein, we review the current literature on the establishment of organoid cultures, and also explore organoid translational applications.

Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. Moreover, most drug tests are restricted to whole-well viability as the sole read-out, thereby losing important information about key biological aspects that might be impacted due to the use of administered drugs. These bulk read-outs also discard potential inter-organoid heterogeneity in drug responses. To tackle these issues, we developed a systematic approach for processing organoids from prostate cancer (PCa) patient-derived xenografts (PDXs) for viability-based drug testing and identified essential conditions and quality checks for consistent results. In addition, we generated an imaging-based drug testing procedure using high-content fluorescence microscopy in living PCa organoids to detect various modalities of cell death. Individual organoids and cell nuclei in organoids were segmented and quantified using a dye combination of Hoechst 33342, propidium iodide and Caspase 3/7 Green, allowing the identification of cytostatic and cytotoxic treatment effects. Our procedures provide important insights into the mechanistic actions of tested drugs. Moreover, these methods can be adapted for tumor organoids originating from other cancer types to increase organoid-based drug test validity, and ultimately, accelerate clinical implementation.

Since the dawn of the past century, landmark discoveries in cell-mediated immunity have led to a greater understanding of the innate and adaptive immune systems and revolutionised the treatment of countless diseases, including cancer. Today, precision immuno-oncology (I/O) involves not only targeting immune checkpoints that inhibit T-cell immunity but also harnessing immune cell therapies. The limited efficacy in some cancers results mainly from a complex tumour microenvironment (TME) that, in addition to adaptive immune cells, comprises innate myeloid and lymphoid cells, cancer-associated fibroblasts, and the tumour vasculature that contribute towards immune evasion. As the complexity of TME has called for more sophisticated human-based tumour models, organoids have allowed the dynamic study of spatiotemporal interactions between tumour cells and individual TME cell types. Here, we discuss how organoids can study the TME across cancers and how these features may improve precision I/O. We outline the approaches to preserve or recapitulate the TME in tumour organoids and discuss their potential, advantages, and limitations. We will discuss future directions of organoid research in understanding cancer immunology in-depth and identifying novel I/O targets and treatment strategies.

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, with a grave prognosis and few effective treatment options. Organoids represent revolutionary three-dimensional cell culture models, derived from stem or differentiated cells and preserving the capacity to differentiate into the cell types of their tissue of origin. The current review aims at studying the potential of patient-derived TNBC organoids for drug sensitivity testing as well as highlighting the advantages of the organoid technology in terms of drug screening. In order to identify relevant studies, a literature review was conducted using the MEDLINE and LIVIVO databases. The search terms “organoid” and “triple-negative breast cancer” were employed, and we were able to identify 25 studies published between 2018 and 2022. The current manuscript represents the first comprehensive review of the literature focusing on the use of patient-derived organoids for drug sensitivity testing in TNBC. Patient-derived organoids are excellent in vitro study models capable of promoting personalized TNBC therapy by reflecting the treatment responses of the corresponding patients and exhibiting high predictive value in the context of patient survival evaluation.

Bovine coronavirus (BCoV) is one of the major viral pathogens of cattle, responsible for economic losses and causing a substantial impact on animal welfare. Several in vitro 2D models have been used to investigate BCoV infection and its pathogenesis. However, 3D enteroids are likely to be a better model with which to investigate host–pathogen interactions. This study established bovine enteroids as an in vitro replication system for BCoV, and we compared the expression of selected genes during the BCoV infection of the enteroids with the expression previously described in HCT-8 cells. The enteroids were successfully established from bovine ileum and permissive to BCoV, as shown by a seven-fold increase in viral RNA after 72 h. Immunostaining of differentiation markers showed a mixed population of differentiated cells. Gene expression ratios at 72 h showed that pro-inflammatory responses such as IL-8 and IL-1A remained unchanged in response to BCoV infection. Expression of other immune genes, including CXCL-3, MMP13, and TNF-α, was significantly downregulated. This study shows that the bovine enteroids had a differentiated cell population and were permissive to BCoV. Further studies are necessary for a comparative analysis to determine whether enteroids are suitable in vitro models to study host responses during BCoV infection.

A kidney organoid is a three-dimensional (3D) cellular aggregate grown from stem cells in vitro that undergoes self-organization, recapitulating aspects of normal renal development to produce nephron structures that resemble the native kidney organ. These miniature kidney-like structures can also be derived from primary patient cells and thus provide simplified context to observe how mutations in kidney-disease-associated genes affect organogenesis and physiological function. In the past several years, advances in kidney organoid technologies have achieved the formation of renal organoids with enhanced numbers of specialized cell types, less heterogeneity, and more architectural complexity. Microfluidic bioreactor culture devices, single-cell transcriptomics, and bioinformatic analyses have accelerated the development of more sophisticated renal organoids and tailored them to become increasingly amenable to high-throughput experimentation. However, many significant challenges remain in realizing the use of kidney organoids for renal replacement therapies. This review presents an overview of the renal organoid field and selected highlights of recent cutting-edge kidney organoid research with a focus on embryonic development, modeling renal disease, and personalized drug screening.

The p38 inhibitor SB202190 is a necessary component of the medium used for normal colorectal mucosa cultures. Sato et al. suggested that the primary activity of SB202190 may be EGFR signaling stabilization, causing an increased phosphorylation of Erk1-2 sustaining organoid proliferation. However, the growth of some colorectal cancer (CRC)-derived organoid cultures is inhibited by this molecule via an unknown mechanism. We biochemically investigated SB202190 activity on a collection of 25 primary human CRC organoids, evaluating EGFR, Akt and Erk1-2 activation using Western blot. We found that Erk1-2 phosphorylation was induced by SB202190 in 20 organoid cultures and inhibited in 5 organoid cultures. A next-generation sequencing (NGS) analysis revealed that the inhibition of p-Erk1-2 signaling corresponded to the cultures with BRAF mutations (with four different hits, one being undescribed), while p-Erk1-2 induction was apparently unrelated to other mutations involving the EGFR pathway (Her2, KRAS and NRAS). We found that SB202190 mirrored the biochemical activity of the BRAF inhibitor Dabrafenib, known to induce the paradoxical activation of p-Erk1-2 signaling in BRAF wild-type cells. SB202190 was a more effective inhibitor of BRAF-mutated organoid growth in the long term than the specific BRAF inhibitors Dabrafenib and PLX8394. Overall, SB202190 can predict BRAF-activating mutations in patient-derived organoids, as well as allowing for the identification of new BRAF variants, preceding and enforcing NGS data.

Bone defects have caused immense healthcare concerns and economic burdens throughout the world. Traditional autologous allogeneic bone grafts have many drawbacks, so the emergence of bone tissue engineering brings new hope. Bone tissue engineering is an interdisciplinary biomedical engineering method that involves scaffold materials, seed cells, and “growth factors”. However, the traditional construction approach is not flexible and is unable to adapt to the specific shape of the defect, causing the cells inside the bone to be unable to receive adequate nourishment. Therefore, a simple but effective solution using the “bottom-up” method is proposed. Microspheres are structures with diameters ranging from 1 to 1000 µm that can be used as supports for cell growth, either in the form of a scaffold or in the form of a drug delivery system. Herein, we address a variety of strategies for the production of microspheres, the classification of raw materials, and drug loading, as well as analyze new strategies for the use of microspheres in bone tissue engineering. We also consider new perspectives and possible directions for future development.

Tissue organoids hold enormous potential as tools for a variety of applications, including disease modeling and drug screening. To effectively mimic the native tissue environment, it is critical to integrate a microvasculature with the parenchyma and stroma. In addition to providing a means to physiologically perfuse the organoids, the microvasculature also contributes to the cellular dynamics of the tissue model via the cells of the perivascular niche, thereby further modulating tissue function. In this review, we discuss current and developing strategies for vascularizing organoids, consider tissue-specific vascularization approaches, discuss the importance of perfusion, and provide perspectives on the state of the field.

Spheroids are expected to aid the establishment of an in vitro-based cell culture system that can realistically reproduce cellular dynamics in vivo. We developed a fluoropolymer scaffold with an extracellular matrix (ECM) dot array and confirmed the possibility of mass-producing spheroids with uniform dimensions. Controlling the quality of ECM dots is important as it ensures spheroid uniformity, but issues such as pattern deviation and ECM drying persist in the conventional microstamping method. In this study, these problems were overcome via ECM dot printing using a resin mask with dot-patterned holes. For dot diameters of φ 300 μm, 400 μm, and 600 μm, the average spheroid diameters of human iPS cells (hiPSCs) were φ 260.8 μm, 292.4 μm, and 330.7 μm, respectively. The standard deviation when each average was normalized to 100 was 14.1%. A high throughput of 89.9% for colony formation rate to the number of dots and 89.3% for spheroid collection rate was achieved. The cells proliferated on ECM dots, and the colonies could be naturally detached from the scaffold without the use of enzymes, so there was almost no stimulation of the cells. Thus, the undifferentiated nature of hiPSCs was maintained until day 4. Therefore, this method is expected to be useful in drug discovery and regenerative medicine.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Organoid models have gained importance in recent years in determining the toxic effects of drugs in cancer studies. Organoid designs with the same standardized size and cellular structures are desired for drug tests. The field of microfluidics offers numerous advantages to enable well-controlled and contamination-free biomedical research. In this study, simple and low-cost microfluidic devices were designed and fabricated to develop an organoid model for drug testing for renal cancers. Caki human renal cancer cells and mesenchymal stem cells isolated from human umbilical cord were placed into alginate hydrogels. The microfluidic system was implemented to form size-controllable organoids within alginate hydrogels. Alginate capsules of uniform sizes formed in the microfluidic system were kept in cell culture for 21 days, and their organoid development was studied with calcein staining. Cisplatin was used as a standard chemotherapeutic, and organoid sphere structures were examined as a function of time with an MTT assay. HIF-1α, CXCR4 and CXCL-12 chemokine protein, and CXCR4 and CXCL-12 gene levels were tested in organoids and cisplatin responses. In conclusion, it was found that the standard renal cancer organoids made on a lab-on-a-chip system can be used to measure drug effects and tumor microenvironment responses.

The liver produces and stores various nutrients that are necessary for the body and serves as a chemical plant, metabolizing carbohydrates, fats, hormones, vitamins, and minerals. It is also a vital organ for detoxifying drugs and exogenous harmful substances. Culturing liver cells in vitro under three-dimensional (3D) conditions is considered a primary mechanism for liver tissue engineering. The 3D cell culture system is designed to allow cells to interact in an artificially created environment and has the advantage of mimicking the physiological characteristics of cells in vivo. This system facilitates contact between the cells and the extracellular matrix. Several technically different approaches have been proposed, including bioreactors, chips, and plate-based systems in fluid or static media composed of chemically diverse materials. Compared to conventional two-dimensional monolayer culture in vitro models, the ability to predict the function of the tissues, including the drug metabolism and chemical toxicity, has been enhanced by developing three-dimensional liver culture models. This review discussed the methodology of 3D cell cultures and summarized the advantages of an in vitro liver platform using 3D culture technology.

Biophysical factors play a fundamental role in human embryonic development. Traditional in vitro models of organogenesis focused on the biochemical environment and did not consider the effects of mechanical forces on developing tissue. While most human tissue has a Young's modulus in the low kilopascal range, the standard cell culture substrate, plasma-treated polystyrene, has a Young's modulus of 3 gigapascals, making it 10,000-100,000 times stiffer than native tissues. Modern in vitro approaches attempt to recapitulate the biophysical niche of native organs and have yielded more clinically relevant models of human tissues. Since Clevers' conception of intestinal organoids in 2009, the field has expanded rapidly, generating stem-cell derived structures, which are transcriptionally similar to fetal tissues, for nearly every organ system in the human body. For this reason, we conjecture that organoids will make their first clinical impact in fetal regenerative medicine as the structures generated ex vivo will better match native fetal tissues. Moreover, autologously sourced transplanted tissues would be able to grow with the developing embryo in a dynamic, fetal environment. As organoid technologies evolve, the resultant tissues will approach the structure and function of adult human organs and may help bridge the gap between preclinical drug candidates and clinically approved therapeutics. In this review, we discuss roles of tissue stiffness, viscoelasticity, and shear forces in organ formation and disease development, suggesting that these physical parameters should be further integrated into organoid models to improve their physiological relevance and therapeutic applicability. It also points to the mechanotransductive Hippo-YAP/TAZ signaling pathway as a key player in the interplay between extracellular matrix stiffness, cellular mechanics, and biochemical pathways. We conclude by highlighting how frontiers in physics can be applied to biology, for example, how quantum entanglement may be applied to better predict spontaneous DNA mutations. In the future, contemporary physical theories may be leveraged to better understand seemingly stochastic events during organogenesis.

A craniopharyngioma (CP) is a rare epithelial tumor of the sellar and parasellar region. CPs are difficult to treat due to their anatomical proximity to critical nervous structures, which limits the ability of the surgeon to completely resect the lesion, exposing patients to a high risk of recurrence. The treatment of craniopharyngiomas is primarily surgery and radiotherapy. So far, neither a cell line nor an animal model has been established, and thus data on other treatment options, such as chemotherapy and immunotherapy, are limited. Here, the expression profile of the pan-cancer antigen B7-H3 in various cancer types including CP was examined by immunohistochemistry. An in vitro organoid model was established by using fresh tissue biospecimens of CP. Based on the organoid model, we evaluated the antitumor efficacy of B7-H3-targeted immunotherapy on CP. As a result, the highest expression of B7-H3 was observed in CP tissues across various cancer types. Although B7-H3-targeted chimeric antigen-receptor T cells show obvious tumor-killing effects in the traditional 2D cell culture model, limited antitumor effects were observed in the 3D organoid model. The B7-H3-targeted antibody-DM1 conjugate exhibited a potent tumor suppression function both in 2D and 3D models. In conclusion, for the first time, we established an organoid model for CP and our results support that B7-H3 might serve as a promising target for antibody-drug conjugate therapy against craniopharyngioma.

Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition.

3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal and multiphoton microscopy, have been used to elucidate data from 3-D cell cultures and whole organs, but their reach inside the 3-D tissues is restrained by the light scattering of the tissues, limiting their effective reach to 100–200 µm, which is simply not enough. Tissue clearing protocols, developed mostly for larger specimens usually involve multiple steps of viscous liquid submersion, and are not easily adaptable for much smaller spheroids and organoids. In this work, we have developed a novel tissue clearing solution tailored for small spheroids and organoids. Our tissue clearing protocol, called HyClear, uses a mixture of DMSO, HPG and urea to allow for one-step tissue clearing of spheroids and organoids, and is compatible with high-throughput screening studies due to its speed and simplicity. We have shown that our tissue clearing agent is superior to many of the commonly used tissue clearing agents and allows for elucidating better quality data from drug screening experiments.

As one of the leading causes of death from disease, cancer continues to pose a serious threat to human health globally. Despite the development of novel therapeutic regimens and drugs, the long-term survival of cancer patients is still very low, especially for those whose diagnosis is not caught early enough. Meanwhile, our understanding of tumorigenesis is still limited. Suitable research models are essential tools for exploring cancer mechanisms and treatments. Herein we review and compare several widely used in vitro and in vivo murine cancer models, including syngeneic tumor models, genetically engineered mouse models (GEMM), cell line-derived xenografts (CDX), patient-derived xenografts (PDX), conditionally reprogrammed (CR) cells, organoids, and MiniPDX. We will summarize the methodology and feasibility of various models in terms of their advantages and limitations in the application prospects for drug discovery and development and precision medicine.

Organ-on-a-chip (OOC) provides microphysiological conditions on a microfluidic chip, which makes up for the shortcomings of traditional in vitro cellular culture models and animal models. It has broad application prospects in drug development and screening, toxicological mechanism research, and precision medicine. A large amount of data could be generated through its applications, including image data, measurement data from sensors, ~omics data, etc. A database with proper architecture is required to help scholars in this field design experiments, organize inputted data, perform analysis, and promote the future development of novel OOC systems. In this review, we overview existing OOC databases that have been developed, including the BioSystics Analytics Platform (BAP) developed by the University of Pittsburgh, which supports study design as well as data uploading, storage, visualization, analysis, etc., and the organ-on-a-chip database (Ocdb) developed by Southeast University, which has collected a large amount of literature and patents as well as relevant toxicological and pharmaceutical data and provides other major functions. We used examples to overview how the BAP database has contributed to the development and applications of OOC technology in the United States for the MPS consortium and how the Ocdb has supported researchers in the Chinese Organoid and Organs-On-A-Chip society. Lastly, the characteristics, advantages, and limitations of these two databases were discussed.

The diverse therapeutic feasibility of the sea urchin-derived naphthoquinone pigment, Echinochrome A (Ech A), has been studied. Simple and noninvasive administration routes should be explored, to obtain the feasibility. Although the therapeutic potential has been proven through several preclinical studies, the biosafety of orally administered Ech A and its direct influence on intestinal cells have not been evaluated. To estimate the bioavailability of Ech A as an oral administration drug, small intestinal and colonic epithelial organoids were developed from mice and humans. The morphology and cellular composition of intestinal organoids were evaluated after Ech A treatment. Ech A treatment significantly increased the expression of LGR5 (~2.38-fold change, p = 0.009) and MUC2 (~1.85-fold change, p = 0.08). Notably, in the presence of oxidative stress, Ech A attenuated oxidative stress up to 1.8-fold (p = 0.04), with a restored gene expression of LGR5 (~4.11-fold change, p = 0.0004), as well as an increased expression of Ly6a (~3.51-fold change, p = 0.005) and CLU (~2.5-fold change, p = 0.01), markers of revival stem cells. In conclusion, Ech A is harmless to intestinal tissues; rather, it promotes the maintenance and regeneration of the intestinal epithelium, suggesting possible beneficial effects on the intestine when used as an oral medication.

Alpha-synuclein’s role in diseases termed “synucleinopathies”, including Parkinson’s disease, has been well-documented. However, after over 25 years of research, we still do not fully understand the alpha-synuclein protein and its role in disease. In vitro cellular models are some of the most powerful tools that researchers have at their disposal to understand protein function. Advantages include good control over experimental conditions, the possibility for high throughput, and fewer ethical issues when compared to animal models or the attainment of human samples. On the flip side, their major disadvantages are their questionable relevance and lack of a “whole-brain” environment when it comes to modeling human diseases, such as is the case of neurodegenerative disorders. Although now, with the advent of pluripotent stem cells and the ability to create minibrains in a dish, this is changing. With this review, we aim to wade through the recent alpha-synuclein literature to discuss how different cell culture setups (immortalized cell lines, primary neurons, human induced pluripotent stem cells (hiPSCs), blood–brain barrier models, and brain organoids) can help us understand aggregation pathology in Parkinson’s and other synucleinopathies.

The cerebral cortex forms early in development according to a series of heritable neurodevelopmental instructions. Despite deep evolutionary conservation of the cerebral cortex and its foundational six-layered architecture, significant variations in cortical size and folding can be found across mammals, including a disproportionate expansion of the prefrontal cortex in humans. Yet our mechanistic understanding of neurodevelopmental processes is derived overwhelmingly from rodent models, which fail to capture many human-enriched features of cortical development. With the advent of pluripotent stem cells and technologies for differentiating three-dimensional cultures of neural tissue in vitro, cerebral organoids have emerged as an experimental platform that recapitulates several hallmarks of human brain development. In this review, we discuss the merits and limitations of cerebral organoids as experimental models of the developing human brain. We highlight innovations in technology development that seek to increase its fidelity to brain development in vivo and discuss recent efforts to use cerebral organoids to study regeneration and brain evolution as well as to develop neurological and neuropsychiatric disease models.

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has severely affected socio-economic conditions and people’s life. The lung is the major target organ infected and (seriously) damaged by SARS-CoV-2, so a comprehensive understanding of the virus and the mechanism of infection are the first choices to overcome COVID-19. Recent studies have demonstrated the enormous value of human organoids as platforms for virological research, making them an ideal tool for researching host–pathogen interactions. In this study, the various existing lung organoids and their identification biomarkers and applications are summarized. At the same time, the seven coronaviruses currently capable of infecting humans are outlined. Finally, a detailed summary of existing studies on SARS-CoV-2 using lung organoids is provided and includes pathogenesis, drug development, and precision treatment. This review highlights the value of lung organoids in studying SARS-CoV-2 infection, bringing hope that research will alleviate COVID-19-associated lung infections.

Compared with traditional two-dimensional culture, a three-dimensional (3D) culture platform can not only provide more reliable prediction results, but also provide a simple, inexpensive and less time-consuming method compared with animal models. A direct in vitro model of the patient’s tumor can help to achieve individualized and precise treatment. However, the existing 3D culture system based on microwell arrays has disadvantages, such as poor controllability, an uneven spheroid size, a long spheroid formation time, low-throughput and complicated operation, resulting in the need for considerable labor, etc. Here, we developed a new type of microdevice based on a 384-well plate/96-well plate microarray design. With our design, cells can quickly aggregate into clusters to form cell spheroids with better roundness. This design has the advantage of high throughput; the throughput is 33 times that of a 384-well plate. This novel microdevice is simple to process and convenient to detect without transferring the cell spheroid. The results show that the new microdevice can aggregate cells into spheroids within 24 h and can support drug and radiation sensitivity analyses in situ in approximately one week. In summary, our microdevices are fast, efficient, high-throughput, simple to process and easy to detect, providing a feasible tool for the clinical validation of individualized drug/radiation responses in patients.

The transplantation of pluripotent stem cell (PSC)-derived liver organoids has been studied to solve the current donor shortage. However, the differentiation of unintended cell populations, difficulty in generating multi-lineage organoids, and tumorigenicity of PSC-derived organoids are challenges. However, direct conversion technology has allowed for the generation lineage-restricted induced stem cells from somatic cells bypassing the pluripotent state, thereby eliminating tumorigenic risks. Here, liver assembloids (iHEAs) were generated by integrating induced endothelial cells (iECs) into the liver organoids (iHLOs) generated with induced hepatic stem cells (iHepSCs). Liver assembloids showed enhanced functional maturity compared to iHLOs in vitro and improved therapeutic effects on cholestatic liver fibrosis animals in vivo. Mechanistically, FN1 expressed from iECs led to the upregulation of Itgα5/β1 and Hnf4α in iHEAs and were correlated to the decreased expression of genes related to hepatic stellate cell activation such as Lox and Spp1 in the cholestatic liver fibrosis animals. In conclusion, our study demonstrates the possibility of generating transplantable iHEAs with directly converted cells, and our results evidence that integrating iECs allows iHEAs to have enhanced hepatic maturation compared to iHLOs.

Renewal of the intestinal epithelium is orchestrated by regenerative epithelial proliferation within crypts. Recent studies have shown that lysophosphatidic acid (LPA) can maintain intestinal epithelial renewal in vitro and conditional deletion of Lpar5 (Lpar5iKO) in mice ablates the intestinal epithelium and increases morbidity. In contrast, constitutive Lpar5 deletion (Lpar5cKO) does not cause a defect in intestinal crypt regeneration. In this study, we investigated whether another LPA receptor (LPAR) compensates for constitutive loss of LPA5 function to allow regeneration of intestinal epithelium. In Lpar5cKO intestinal epithelial cells (IECs), Lpar2 was upregulated and blocking LPA2 function reduced proliferation and increased apoptosis of Lpar5cKO IECs. Similar to Lpar5cKO mice, the absence of Lpar2 (Lpar2−/−) resulted in upregulation of Lpar5 in IECs, indicating that LPA2 and LPA5 reciprocally compensate for the loss of each other. Blocking LPA2 in Lpar5cKO enteroids reduced phosphorylation of Akt, indicating that LPA2 maintains the growth of Lpar5cKO enteroids through activation of the PI3K-Akt pathway. The present study provides evidence that loss of an LPAR can be compensated by another LPAR. This ability to compensate needs to be considered in studies aimed to define receptor functions or test the efficacy of a LPAR-targeting drug using genetically engineered animal models.

Despite tremendous advancements in technologies and resources, drug discovery still remains a tedious and expensive process. Though most cells are cultured using 2D monolayer cultures, due to lack of specificity, biochemical incompatibility, and cell-to-cell/matrix communications, they often lag behind in the race of modern drug discovery. There exists compelling evidence that 3D cell culture models are quite promising and advantageous in mimicking in vivo conditions. It is anticipated that these 3D cell culture methods will bridge the translation of data from 2D cell culture to animal models. Although 3D technologies have been adopted widely these days, they still have certain challenges associated with them, such as the maintenance of a micro-tissue environment similar to in vivo models and a lack of reproducibility. However, newer 3D cell culture models are able to bypass these issues to a maximum extent. This review summarizes the basic principles of 3D cell culture approaches and emphasizes different 3D techniques such as hydrogels, spheroids, microfluidic devices, organoids, and 3D bioprinting methods. Besides the progress made so far in 3D cell culture systems, the article emphasizes the various challenges associated with these models and their potential role in drug repositioning, including perspectives from the COVID-19 pandemic.

The COVID-19 pandemic has driven the scientific community to adopt an efficient and reliable model that could keep up with the infectious disease arms race. Coinciding with the pandemic, three dimensional (3D) human organoids technology has also gained traction in the field of infectious disease. An in vitro construct that can closely resemble the in vivo organ, organoid technology could bridge the gap between the traditional two-dimensional (2D) cell culture and animal models. By harnessing the multi-lineage characteristic of the organoid that allows for the recapitulation of the organotypic structure and functions, 3D human organoids have emerged as an essential tool in the field of infectious disease research. In this review, we will be providing a comparison between conventional systems and organoid models. We will also be highlighting how organoids played a role in modelling common infectious diseases and molecular mechanisms behind the pathogenesis of causative agents. Additionally, we present the limitations associated with the current organoid models and innovative strategies that could resolve these shortcomings.

Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze–thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids.

The biological taste system has the unique ability to detect taste substances. Biomaterials originating from a biological taste system have been recognized as ideal candidates to serve as sensitive elements in the development of taste-based biosensors. In this study, we developed a taste bud organoid-based biosensor for the research of taste sensation. Taste bud organoids prepared from newborn mice were cultured and loaded onto the surface of a 64-channel microelectrode array (MEA) chip to explore the electrophysiological changes upon taste; an MEA chip was used to simultaneously record multiple-neuron firing activities from taste bud organoids under different taste stimuli, which helped to reveal the role of taste buds in taste sensing. The obtained results show that taste cells separated from the taste epithelium grew well into spherical structures under 3D culture conditions. These structures were composed of multiple cells with obvious budding structures. Moreover, the multicellular spheres were seeded on a 64-channel microelectrode array and processed with different taste stimuli. It was indicated that the MEA chip could efficiently monitor the electrophysiological signals from taste bud organoids in response to various taste stimuli. This biosensor provides a new method for the study of taste sensations and taste bud functions.

Organoids are novel in vitro cell culture models that enable stem cells (including pluripotent stem cells and adult stem cells) to grow and undergo self-organization within a three-dimensional microenvironment during the process of differentiation into target tissues. Such miniature structures not only recapitulate the histological and genetic characteristics of organs in vivo, but also form tissues with the capacity for self-renewal and further differentiation. Recent advances in biomaterial technology, particularly hydrogels, have provided opportunities to improve organoid cultures; by closely integrating the mechanical and chemical properties of the extracellular matrix microenvironment, with novel synthetic materials and stem cell biology. This systematic review critically examines recent advances in various strategies and techniques utilized for stem-cell-derived organoid culture, with particular emphasis on the application potential of hydrogel technology in organoid culture. We hope this will give a better understanding of organoid cultures for modelling diseases and tissue engineering applications.

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder and the leading cause of death among older individuals. Available treatment strategies only temporarily mitigate symptoms without modifying disease progression. Recent studies revealed the multifaceted neurobiology of AD and shifted the target of drug development. Established animal models of AD are mostly tailored to yield a subset of disease phenotypes, which do not recapitulate the complexity of sporadic late-onset AD, the most common form of the disease. The use of human induced pluripotent stem cells (HiPSCs) offers unique opportunities to fill these gaps. Emerging technology allows the development of disease models that recapitulate a brain-like microenvironment using patient-derived cells. These models retain the individual’s unraveled genetic background, yielding clinically relevant disease phenotypes and enabling cost-effective, high-throughput studies for drug discovery. Here, we review the development of various HiPSC-based models to study AD mechanisms and their application in drug discovery.

The emergence of human-induced Pluripotent Stem Cells (hiPSCs) has dramatically improved our understanding of human developmental processes under normal and diseased conditions. The hiPSCs have been differentiated into various tissue-specific cells in vitro, and the advancement in three-dimensional (3D) culture has provided a possibility to generate those cells in an in vivo-like environment. Tissues with 3D structures can be generated using different approaches such as self-assembled organoids and tissue-engineering methods, such as bioprinting. We are interested in studying the self-assembled organoids differentiated from hiPSCs, as they have the potential to recapitulate the in vivo developmental process and be used to model human development and congenital defects. Organoids of tissues such as those of the intestine and brain were developed many years ago, but heart organoids were not reported until recently. In this review, we will compare the heart organoids with the in vivo hearts to understand the anatomical structures we still lack in the organoids. Specifically, we will compare the development of main heart structures, focusing on their marker genes and regulatory signaling pathways.

The achievement of an HIV cure is dependent on the eradication or permanent silencing of HIV-latent viral reservoirs, including the understudied central nervous system (CNS) reservoir. This requires a deep understanding of the molecular mechanisms of HIV’s entry into the CNS, latency establishment, persistence, and reversal. Therefore, representative CNS culture models that reflect the intercellular dynamics and pathophysiology of the human brain are urgently needed in order to study the CNS viral reservoir and HIV-induced neuropathogenesis. In this study, we characterized a human cerebral organoid model in which microglia grow intrinsically as a CNS culture model to study HIV infection in the CNS. We demonstrated that both cerebral organoids and isolated organoid-derived microglia (oMG), infected with replication-competent HIVbal reporter viruses, support productive HIV infection via the CCR5 co-receptor. Productive HIV infection was only observed in microglial cells. Fluorescence analysis revealed microglia as the only HIV target cell. Susceptibility to HIV infection was dependent on the co-expression of microglia-specific markers and the CD4 and CCR5 HIV receptors. Altogether, this model will be a valuable tool within the HIV research community to study HIV–CNS interactions, the underlying mechanisms of HIV-associated neurological disorders (HAND), and the efficacy of new therapeutic and curative strategies on the CNS viral reservoir.

Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover relevant disease mechanisms and enhance the translation of therapeutic findings to strengthen neuromuscular disease precision medicine. By concentrating on idiopathic inflammatory muscle disorders, we summarize the recent evolution of the novel in vitro models to study disease mechanisms and therapeutic strategies. A particular focus is laid on the integration and simulation of multicellular interactions of muscle tissue in disease phenotypes in vitro. Finally, the requirements of a neuromuscular disease drug development workflow are discussed with a particular emphasis on cell sources, co-culture systems (including organoids), functionality, and throughput.

An organoid is a miniaturized and simplified in vitro model with a similar structure and function to a real organ. In recent years, the use of organoids has increased explosively in the field of growth and development, disease simulation, drug screening, cell therapy, etc. In order to obtain necessary information, such as morphological structure, cell function and dynamic signals, it is necessary and important to directly monitor the culture process of organoids. Among different detection technologies, imaging technology is a simple and convenient choice and can realize direct observation and quantitative research. In this review, the principle, advantages and disadvantages of imaging technologies that have been applied in organoids research are introduced. We also offer an overview of prospective technologies for organoid imaging. This review aims to help biologists find appropriate imaging techniques for different areas of organoid research, and also contribute to the development of organoid imaging systems.

A treatment with direct healing effects on the gastrointestinal epithelial barrier is desirable for inflammatory bowel disease (IBD). Interleukin-27 (IL-27) is an immunoregulatory cytokine, and oral delivery is an effective treatment in murine models of IBD. We aimed to define IL-27 effects on the human gastrointestinal epithelial barrier. We characterised gene and protein expression of permeability mediators in a human colon-derived organoid model. Functional permeability was determined in an organoid-derived 2D monolayer by transepithelial electrical resistance. IL-27 effects on epithelial innate immune responses were assessed through expression of cytokines, anti-microbial peptides and MUC genes. IL-27 effects on wound healing and proliferation were determined in human colon epithelial cell lines. IL-27 led to restoration of permeability regulation following inflammatory cytokine insult (p = 0.001), associated with differential expression of tight junction mediators with decrease in claudin 2 (p = 0.024) and increase in claudin 4 (p < 0.001), E-cadherin (p < 0.001) and zona occludens (p = 0.0014). IL-27 evoked differential gene expression of epithelial-derived innate immune responses (reduced IL1B and IL18, and increased IL33, HBD1, MUC1 and MUC2; p < 0.012). IL-27 induced epithelial barrier wound healing through restitution (p < 0.001), and increased proliferation (p < 0.001) following injury. Overall, IL-27 provokes mucosal healing of the human gastrointestinal epithelial barrier.

Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell–cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids’ crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt-associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level.