The structure of intestinal tissue is complex.In vitro simulation of intestinal structure and function is important for studying intestinal development and diseases.Recently,organoids have been successfully constructe...The structure of intestinal tissue is complex.In vitro simulation of intestinal structure and function is important for studying intestinal development and diseases.Recently,organoids have been successfully constructed and they have come to play an important role in biomedical research.Organoids are miniaturized three-dimensional(3D)organs,derived from stem cells,which mimic the structure,cell types,and physiological functions of an organ,making them robust models for biomedical research.Intestinal organoids are 3D micro-organs derived from intestinal stem cells or pluripotent stem cells that can successfully simulate the complex structure and function of the intestine,thereby providing a valuable platform for intestinal development and disease research.In this article,we review the latest progress in the construction and application of intestinal organoids.展开更多
Given the severe toxicity and widespread presence of cadmium(Cd)in staple foods such as rice,accurate dietary exposure assessments are imperative for public health.In vitro bioavailability is commonly used to adjust d...Given the severe toxicity and widespread presence of cadmium(Cd)in staple foods such as rice,accurate dietary exposure assessments are imperative for public health.In vitro bioavailability is commonly used to adjust dietary exposure levels of risk factors;however,traditional planar Transwell models have limitations,such as cell dedifferentiation and lack of key intestinal components,necessitating a more physiologically relevant in vitro platform.This study introduces an innovative three-dimensional(3D)intestinal organoid model using a microfluidic chip to evaluate Cd bioavailability in food.Caco-2 cells were cultured on the chip to mimic small intestinal villi's 3D structure,mucus production,and absorption functions.The model's physiological relevance was thoroughly characterized,demonstrating the formation of a confluent epithelial monolayer with well-developed tight junctions(ZO-1),high microvilli density(F-actin),and significant mucus secretion(Alcian blue staining),closely resembling the physiological intestinal epithelium.Fluorescent particle tracking confirmed its ability to simulate intestinal transport and diffusion.The Cd bioavailability in rice measured by the 3D intestinal organoid model((9.07±0.21)%)was comparable to the mouse model((12.82±3.42)%)but significantly lower than the Caco-2 monolayer model((26.97±1.11)%).This 3D intestinal organoid model provides a novel and reliable strategy for in vitro assessment of heavy metal bioavailability in food,with important implications for food safety and risk assessment.展开更多
Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechani...Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechanically dissociated 3 D organoids of mouse duodenum were used to generate a polarized epithelium containing all cell types found in the tissue of origin. The organoid-derived cell monolayers were exposed to 4%(w/v) of ‘undigested(non-hydrolysed)-soluble' fraction of protein sources used as feed ingredients [soybean meal(SBM) and casein], or alternative protein sources(spray dried plasma protein, and yellow meal worm), or controls for 6 h prior to RNA isolation and transcriptomics. All protein sources altered expression of unique biological processes in the epithelial cells. Exposure of intestinal organoids to SBM downregulated expression of retinol and retinoid metabolic processes as well as cholesterol and lipid biosynthetic pathways, consistent with the reported hypotriglyceridaemic effect of soy protein in vivo. These findings support the use of intestinal organoids as models to evaluate complex interactions between dietary ingredients and the intestinal epithelium and highlights some unique host effects of alternative protein sources in animal feed and potentially human food.展开更多
To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts were isolated from jejunum of ...To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.RESULTSWe established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5<sup>+</sup> cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5<sup>+</sup> cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSIONThe maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.展开更多
Increased micro-and nanoplastic(MNP)pollution poses significant health risks,yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood.Addressing this knowledge gap requires t...Increased micro-and nanoplastic(MNP)pollution poses significant health risks,yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood.Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods,enabling multi-parameter single cell analysis.We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy(FLIM)to study MNP interactions with gut epithelium.To facilitate this,we optimized live imaging of porcine and mouse small intestinal organoids with an‘apical-out’topology.Subsequently,we produced a set of pristine MNPs based on PMMA and PS(<200 nm,doped with deep-red fluorescent dye)and evaluated their interaction with organoids displaying controlled epithelial polarity.We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake.Using a phasor analysis approach,we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy.The resulting‘fluorescence lifetime barcoding’enabled distinguishing of different types of MNP and their interaction sites within organoids.Finally,we studied short(1 day)-and long(3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function,total cell energy budget and epithelial inflammation.We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells.The presented FLIM approach will advance the study of MNP toxicity,their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems.展开更多
Intestinal organoids,derived from intestinal stem cell self-organization,recapitulate the tissue structures and behav-iors of the intestinal epithelium,which hold great potential for the study of developmental biology...Intestinal organoids,derived from intestinal stem cell self-organization,recapitulate the tissue structures and behav-iors of the intestinal epithelium,which hold great potential for the study of developmental biology,disease modeling,and regenerative medicine.The intestinal epithelium is exposed to dynamic mechanical forces which exert profound effects on gut development.However,the conventional intestinal organoid culture system neglects the key role of mechanical microenvironments but relies solely on biological factors.Here,we show that adding cyclic stretch to intestinal organoid cultures remarkably up-regulates the signature gene expression and proliferation of intestinal stem cells.Furthermore,mechanical stretching stimulates the expansion of SOX9+progenitors by activating the Wnt/β-Catenin signaling.These data demonstrate that the incorporation of mechanical stretch boosts the stemness of intestinal stem cells,thus benefiting organoid growth.Our findings have provided a way to optimize an organoid generation system through understanding cross-talk between biological and mechanical factors,paving the way for the application of mechanical forces in organoid-based models.展开更多
With research burgeoning in nanoscience and nanotechnology,there is an urgent need to develop new biological models that can simulate native structure,function,and genetic properties of tissues to evaluate the adverse...With research burgeoning in nanoscience and nanotechnology,there is an urgent need to develop new biological models that can simulate native structure,function,and genetic properties of tissues to evaluate the adverse or beneficial effects of nanomaterials on a host.Among the current biological models,three-dimensional(3D)organoids have developed as powerful tools in the study of nanomaterial-biology(nano-bio)interactions,since these models can overcome many of the limitations of cell and animal models.A deep understanding of organoid techniques will facilitate the development of more efficient nanomedicines and further the fields of tissue engineering and personalized medicine.Herein,we summarize the recent progress in intestinal organoids culture systems with a focus on our understanding of the nature and influencing factors of intestinal organoid growth.We also discuss biomimetic extracellular matrices(ECMs)coupled with nanotechnology.In particular,we analyze the application prospects for intestinal organoids in investigating nano-intestine interactions.By integrating nanotechnology and organoid technology,this recently developed model will fill the gaps left due to the deficiencies of traditional cell and animal models,thus accelerating both our understanding of intestine-related nanotoxicity and the development of nanomedicines.展开更多
Diet and nutrition significantly influence health, largely by regulating intestinalnutrient absorption. The intestinal epithelium, as the primary site for nutrientuptake, undergoes continuous renewal driven by precise...Diet and nutrition significantly influence health, largely by regulating intestinalnutrient absorption. The intestinal epithelium, as the primary site for nutrientuptake, undergoes continuous renewal driven by precise regulation of intestinalstem cells (ISCs). Nutrient sensing and metabolism are key determinants of ISCfate, making ISCs a central link between nutrient metabolism and the regulationof intestinal tissue renewal and homeostasis. Understanding how ISCs respond ormake adaptations to nutritional signals is therefore vital for maintaining intestinalhomeostasis. Recent studies have spotlighted the origin and identity of ISCs andbroadened our insight into the plasticity and function of ISCs under differentconditions. Mitochondria, the central hubs of energy production and metabolicsignals provided by dietary components and metabolic substrates, such as glucose,amino acids, and lipids, govern the intricate balance between self-renewal anddifferentiation of ISCs. This review highlights the importance of nutrient sensing,metabolic regulation, and mitochondrial function in the specification of ISC fate.A thorough understanding of these mechanisms paves the way for the developmentof stem cell-based therapy for the mucosal healing of gastrointestinaldiseases and diet intervention to foster body health.展开更多
The development of“mini-guts”organoid originates from the identification of Lgr5+intestinal stem cells(ISCs)and circumambient signalings within their specific niche at the crypt bottom.These in vitro self-renewing“...The development of“mini-guts”organoid originates from the identification of Lgr5+intestinal stem cells(ISCs)and circumambient signalings within their specific niche at the crypt bottom.These in vitro self-renewing“mini-guts”,also named enteroids or colonoids,undergo perpetual proliferation and regulated differentiation,which results in a high-performance,self-assembling and physiological organoid platform in diverse areas of intestinal research and therapy.The triumphant reconstitution of ISC niche in vitro also relies on Matrigel,a heterogeneous sarcoma extract.Despite the promising prospect of organoids research,their expanding applications are hampered by the canonical culture pattern,which reveals limitations such as inaccessible lumen,confine scale,batch to batch variation and low reproducibility.The tumor-origin of Matrigel also raises biosafety concerns in clinical treatment.However,the convergence of breakthroughs in cellular biology and bioengineering contribute to multiform reconstitution of the ISC niche.Herein,we review the recent advances in the microfabrication of intestinal organoids on hydrogel systems.展开更多
BACKGROUND Intestinal ischemia-reperfusion(I/R)injury(II/RI)is a critical condition that results in oxidative stress,inflammation,and damage to multiple organs.Zinc,an essential trace element,offers protective benefit...BACKGROUND Intestinal ischemia-reperfusion(I/R)injury(II/RI)is a critical condition that results in oxidative stress,inflammation,and damage to multiple organs.Zinc,an essential trace element,offers protective benefits in several tissues during I/R injury,but its effects on intestinal II/RI remain unclear.METHODS C57BL/6 mice were pretreated with zinc sulfate(ZnSO4,10 mg/kg)daily for three days before I/R injury was induced via superior mesenteric artery occlusion(SMAO)and abdominal aortic occlusion(AAO)models.Tissue and serum samples were collected to evaluate intestinal,liver,and kidney damage using Chiu’s score,Suzuki score,and histopathological analysis.Caco-2 cells and intestinal organoids were used for in vitro hypoxia-reoxygenation injury models to measure reactive oxygen species(ROS)and superoxide dismutase(SOD)levels.RESULTS Zinc pretreatment significantly reduced intestinal damage in the SMAO and AAO models(P<0.001).The serum levels of liver enzymes(alanine aminotransferase,aspartate aminotransferase)and kidney markers(creatinine and urea)were lower in the zinc-treated mice than in the control mice,indicating reduced hepatic and renal injury.In vitro,zinc decreased ROS levels and increased SOD activity in Caco-2 cells subject to hypoxia-reoxygenation injury.Intestinal organoids pretreated with zinc exhibited enhanced resilience to hypoxic injury compared to controls.CONCLUSION Zinc pretreatment mitigates II/RI and reduces associated multiorgan damage.These findings suggest that zinc has potential clinical applications in protecting against I/R injuries.展开更多
Methionine and its hydroxy analogue(MHA)have been shown to benefit mouse intestinal regeneration.The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestina...Methionine and its hydroxy analogue(MHA)have been shown to benefit mouse intestinal regeneration.The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development.Here,we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development.The results showed that 125-mm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting.We found that transforming growth factor-b inhibitor(A8301)supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells(ISC).The mitogen-activated protein kinase p38 inhibitor(SB202190)promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells,goblet cells and Paneth cells.However,the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301.The glycogen synthase kinase 3 inhibitor(CHIR99021),valproic acid(VPA)alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells.Chicken serum significantly improved organoid formation,especially in the presence of A8301,SB202190,CHIR99021,and VPA,but inhibited the differentiation of Paneth cells and enteroendocrine cells.Chicken serum at a concentration of 0.25%meets the requirement of chicken intestinal organoid development,and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1.Moreover,commercial mouse organoid culture medium supplemented with A8301,SB202190,CHIR99021,VPA,and chicken serum promotes chicken organoid budding.Based on the chicken intestinal organoid model,we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size,and this effect was reinforced with increased cycloleucine concentrations.Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine.In conclusion,our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.展开更多
A healthy intestine plays an important role in the growth and development of farm animals.In small intestine,Paneth cells are well known for their regulation of intestinal microbiota and intestinal stem cells(ISCs).Al...A healthy intestine plays an important role in the growth and development of farm animals.In small intestine,Paneth cells are well known for their regulation of intestinal microbiota and intestinal stem cells(ISCs).Although there has been a lot of studies and reviews on human and murine Paneth cells under intestinal homeostasis or disorders,little is known about Paneth cells in farm animals.Most farm animals possess Paneth cells in their small intestine,as identified by various staining methods,and Paneth cells of various livestock species exhibit noticeable differences in cell shape,granule number,and intestinal distribution.Paneth cells in farm animals and their antimicrobial peptides(AMPs)are susceptible to multiple factors such as dietary nutrients and intestinal infection.Thus,the comprehensive understanding of Paneth cells in different livestock species will contribute to the improvement of intestinal health.This review first summarizes the current status of Paneth cells in pig,cattle,sheep,horse,chicken and rabbit,and points out future directions for the investigation of Paneth cells in the reviewed animals.展开更多
Perfluorooctanoic acid(PFOA)and perfluorooctanesulfonate(PFOS)continue to be extensively present in the natural environment and seriously threaten human health.The intestinal tract is the primary organ of PFOA/PFOS ex...Perfluorooctanoic acid(PFOA)and perfluorooctanesulfonate(PFOS)continue to be extensively present in the natural environment and seriously threaten human health.The intestinal tract is the primary organ of PFOA/PFOS exposure due to the consumption of contaminated food and drinking water.However,it remains unclear how PFOA/PFOS affects intestinal function and overall health.The aim of this study was to investigate the influence of PFOA/PFOS on the absorption of fatty acids in the intestine and the underlying mechanisms using three-dimensional(3D)intestinal organoids.Our results showed that PFOS,but not PFOA,could significantly enhance the fatty acid uptake capacity without obvious damage to the organoids.Furthermore,PFOS markedly reduced the protein levels of ChgA in enteroendocrine cells,but with no observed impact on aldolase B+enterocytes.Mechanistically,exposure to PFOS induced the activation of the peroxisome proliferator-activated receptor(PPAR)αpathway in intestinal organoids,with enhanced expression of PPARαtarget genes associated with fatty acid metabolism,such as Fabp1 and Cd36(fatty acid transporter genes),Acox1 and Pdk4(fatty acid oxidation genes),and Plin2 and Plin3(lipid droplet synthesis genes).These data suggest that PFOS have the potential to affect the absorption function of the intestinal epithelium through the PPARαpathway,and its effect is much stronger than that of PFOA.Our findings also highlight that organoids can be used as a valuable model for conducting toxicological research on environmental chemicals.展开更多
Understanding the regulatory mechanisms of intestinal organoid morphogenesis remains a fundamental chal-lenge in organoid biology.Emerging evidence highlights mechanical bistability as a critical regulator,mediated by...Understanding the regulatory mechanisms of intestinal organoid morphogenesis remains a fundamental chal-lenge in organoid biology.Emerging evidence highlights mechanical bistability as a critical regulator,mediated by dynamic lumen-actomyosin feedback.The recently developed 3D vertex model demonstrates that crypt curvature modulates actomyosin localization via mechanosensitive pathways,creating two stable morphological states-bulged or budded-depending on mechanical history.This model advances beyond static vertex models by incorporating epithelial thickness variations and lumen pressure effects,explaining previously unresolved phenomena like irreversible crypt budding and snap-through transitions.The findings establish a new framework for understanding mechanical decision-making in epithelial tissues,with implications for organoid engineering and developmental biology.展开更多
Dietary factors play a crucial role in irritable bowel syndrome(IBS)pathogenesis.Therefore,the dietary contraindications for patients with IBS require further supplementation.Recent investigations have revealed that g...Dietary factors play a crucial role in irritable bowel syndrome(IBS)pathogenesis.Therefore,the dietary contraindications for patients with IBS require further supplementation.Recent investigations have revealed that ginger consumption may pose a risk of aggravating the symptoms and incidence of IBS;however,the specific mechanism remains unknown.In this study,we developed experimental IBS and intestinal organoid differentiation screening models to elucidate the mechanisms underlying the gingermediated exacerbation of IBS symptoms.Subsequently,we used a knockout approach combined with click chemistry as well as virus infection to identify the toxic components of ginger and the target mechanism.Our results showed that a daily intake of 90 to 300 mg/kg ginger(equivalent to a human daily dose of O.6 to 2 g per person)may pose a risk of exacerbating IBS symptoms.Furthermore,a component derived from 6-gingerol(ginger's main ingredient)through in vivo gastric acid and heat processing inhibited the formation of the elF3 transcription initiation complex by covalently binding to the Cys^(58)site of elF3A,a key factor regulating intestinal crypt stem celldifferentiation,further reducing the goblet cellnumber and related mucus layer thickness and increasing lipopolysaccharide infiltration and low-grade inflammation in the ileum crypts,thereby exacerbating the symptoms of IBS in mice.Our study suggests that dietary ginger aggravates IBS and provides safety evaluation methods for the proper use of foods in specific populations.展开更多
Early weaning in piglets can cause a series of negative effects.This causes serious losses to the livestock industry.N-Acetyl-D-glucosamine(D-GlcNAc)plays an important role in regulating the homeostasis of the intesti...Early weaning in piglets can cause a series of negative effects.This causes serious losses to the livestock industry.N-Acetyl-D-glucosamine(D-GlcNAc)plays an important role in regulating the homeostasis of the intestine.This study aimed to investigate the effects of D-GlcNAc on the growth performance and intestinal function of weaned piglets.Twenty-four weaned piglets([Yorkshire×Landrace]Duroc,6.58±0.15 kg,n=8)at 21 d old were fed 3 diets supplemented with 0(control),1 and 3 g/kg D-GlcNAc.The intestinal organoid model was used to verify the regulatory mechanism of D-GlcNAc on intestinal epithelial cells.On the whole,supplementation of D-GlcNAc in the piglet diet has no significant effect on the growth performance and diarrhoea of weaned piglets(P>0.05).The apparent digestibility of nutrients and mRNA abundance of nutrient transporters in the 1 g/kg D-GlcNAc group were increased significantly(P<0.05).D-GlcNAc did not affect villus height(VH)and crypt depth(CD)but resulted in a numerically shorter VH and shallower CD,which lead to an increase in ileal VH:CD ratio(P<0.05).Cell shedding rates in the ileum villi increased(P<0.05).The relative length and weight of the small intestine of weaned piglets increased(P<0.05).In vitro studies found that the budding rates of organoids treated with 0.1 mmol/L D-GlcNAc increased on the d 3 and 5(P<0.05).The average budding numbers per budding organoid treated with 0.1 and 10 mmol/L D-GlcNAc increased on d 3(P<0.05).D-GlcNAc upregulated leucine rich repeat containing G protein-coupled receptor 5(Lgr5^(+))and Chromogranin A mRNA abundance in organoids(P<0.05).Mucin 2(Muc2)expression increased when treated with 1 and 10 mmol/L D-GlcNAc(P<0.05).In conclusion,dietary D-GlcNAc cannot improve the growth performance of weaned piglets.However,it can promote the growth and development of the intestinal tract and improve the digestion and absorption capacity of the intestine,which is achieved by affecting the activity of intestinal stem cells.展开更多
基金funded by the National Natural Science Foundation of China(No.32300659)Shenzhen Science and Technology Innovation Commission Project(No.JCYJ20230807143302004)+6 种基金Zhangjiakou City Key R&D Plan Project(No.2421118D),Zhangjiakou City Key R&D Plan Project(No.2322088D)The Natural Science Project of Hebei North University(No.XJ2024034 and XJ2024035)Medical Science Research Subject Plan Project of Hebei Provincial Health Commission(No.20240782)Project of Administration of Traditional Chinese Medicine of Hebei Province(No.2025392)The 2025 Government-funded Training Project for Outstanding Clinical Medicine Talents(No.ZF2025264)Research Project of Medical Innovation for Chinese Youth(Project Leader:Jun Xue).g Project for Outstanding Clinical Medicine Talents(No.ZF2025264)Research Project of Medical Innovation for Chinese Youth(Project Leader:Jun Xue)。
文摘The structure of intestinal tissue is complex.In vitro simulation of intestinal structure and function is important for studying intestinal development and diseases.Recently,organoids have been successfully constructed and they have come to play an important role in biomedical research.Organoids are miniaturized three-dimensional(3D)organs,derived from stem cells,which mimic the structure,cell types,and physiological functions of an organ,making them robust models for biomedical research.Intestinal organoids are 3D micro-organs derived from intestinal stem cells or pluripotent stem cells that can successfully simulate the complex structure and function of the intestine,thereby providing a valuable platform for intestinal development and disease research.In this article,we review the latest progress in the construction and application of intestinal organoids.
基金supported by National key research and development program of China(2022YFF1102500)。
文摘Given the severe toxicity and widespread presence of cadmium(Cd)in staple foods such as rice,accurate dietary exposure assessments are imperative for public health.In vitro bioavailability is commonly used to adjust dietary exposure levels of risk factors;however,traditional planar Transwell models have limitations,such as cell dedifferentiation and lack of key intestinal components,necessitating a more physiologically relevant in vitro platform.This study introduces an innovative three-dimensional(3D)intestinal organoid model using a microfluidic chip to evaluate Cd bioavailability in food.Caco-2 cells were cultured on the chip to mimic small intestinal villi's 3D structure,mucus production,and absorption functions.The model's physiological relevance was thoroughly characterized,demonstrating the formation of a confluent epithelial monolayer with well-developed tight junctions(ZO-1),high microvilli density(F-actin),and significant mucus secretion(Alcian blue staining),closely resembling the physiological intestinal epithelium.Fluorescent particle tracking confirmed its ability to simulate intestinal transport and diffusion.The Cd bioavailability in rice measured by the 3D intestinal organoid model((9.07±0.21)%)was comparable to the mouse model((12.82±3.42)%)but significantly lower than the Caco-2 monolayer model((26.97±1.11)%).This 3D intestinal organoid model provides a novel and reliable strategy for in vitro assessment of heavy metal bioavailability in food,with important implications for food safety and risk assessment.
基金the financial support from the Wageningen University&Research “IPOP Customized Nutrition” program(grant no.4417801270)financed by Wageningen University&Research,the Dutch Ministry of Economic Affairs, Agriculture&Innovation, the graduate school Wageningen Institute of Animal Science(WIAS)+1 种基金industrial partners Trouw Nutrition, The Netherlands and Darling Ingredient International, The Netherlandssupported by the applied and engineering sciences division of The Netherlands Organisation for Scientific Research(NWO project 14935)and DSM Nutritional Products。
文摘Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechanically dissociated 3 D organoids of mouse duodenum were used to generate a polarized epithelium containing all cell types found in the tissue of origin. The organoid-derived cell monolayers were exposed to 4%(w/v) of ‘undigested(non-hydrolysed)-soluble' fraction of protein sources used as feed ingredients [soybean meal(SBM) and casein], or alternative protein sources(spray dried plasma protein, and yellow meal worm), or controls for 6 h prior to RNA isolation and transcriptomics. All protein sources altered expression of unique biological processes in the epithelial cells. Exposure of intestinal organoids to SBM downregulated expression of retinol and retinoid metabolic processes as well as cholesterol and lipid biosynthetic pathways, consistent with the reported hypotriglyceridaemic effect of soy protein in vivo. These findings support the use of intestinal organoids as models to evaluate complex interactions between dietary ingredients and the intestinal epithelium and highlights some unique host effects of alternative protein sources in animal feed and potentially human food.
基金a grant of the Korea Institute of Radiological and Medical Sciences,funded by Ministry of Science,ICT and Future Planning,South Korea,No.1711031810/50586-2016 and No.1711031808/50581-2016
文摘To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.RESULTSWe established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5<sup>+</sup> cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5<sup>+</sup> cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSIONThe maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.
基金supported by the Special Research Fund(BOF)grants(BOF/STA/202009/003,BOF/BAF/1 y/25/1/004)Research Foundation Flanders(FWO,I001922N,I004124N)the European Union,fliMAGIN3D-DN Horizon Europe-MSCA-DN No.101073507 grants.
文摘Increased micro-and nanoplastic(MNP)pollution poses significant health risks,yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood.Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods,enabling multi-parameter single cell analysis.We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy(FLIM)to study MNP interactions with gut epithelium.To facilitate this,we optimized live imaging of porcine and mouse small intestinal organoids with an‘apical-out’topology.Subsequently,we produced a set of pristine MNPs based on PMMA and PS(<200 nm,doped with deep-red fluorescent dye)and evaluated their interaction with organoids displaying controlled epithelial polarity.We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake.Using a phasor analysis approach,we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy.The resulting‘fluorescence lifetime barcoding’enabled distinguishing of different types of MNP and their interaction sites within organoids.Finally,we studied short(1 day)-and long(3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function,total cell energy budget and epithelial inflammation.We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells.The presented FLIM approach will advance the study of MNP toxicity,their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems.
基金This work was supported from the National Key Research and Development Program of China(2018YFA0109400)the National Natural Science Foundation of China(32022022,11972002,11972001,62071085)+2 种基金Beijing Natural Science Foundation(Z200017)Natural Science Foundation of Shandong Province(ZR2019PC026)the Open Research Fund of Shandong Key Laboratory of Biophysics of Dezhou College(SD2018BP005).
文摘Intestinal organoids,derived from intestinal stem cell self-organization,recapitulate the tissue structures and behav-iors of the intestinal epithelium,which hold great potential for the study of developmental biology,disease modeling,and regenerative medicine.The intestinal epithelium is exposed to dynamic mechanical forces which exert profound effects on gut development.However,the conventional intestinal organoid culture system neglects the key role of mechanical microenvironments but relies solely on biological factors.Here,we show that adding cyclic stretch to intestinal organoid cultures remarkably up-regulates the signature gene expression and proliferation of intestinal stem cells.Furthermore,mechanical stretching stimulates the expansion of SOX9+progenitors by activating the Wnt/β-Catenin signaling.These data demonstrate that the incorporation of mechanical stretch boosts the stemness of intestinal stem cells,thus benefiting organoid growth.Our findings have provided a way to optimize an organoid generation system through understanding cross-talk between biological and mechanical factors,paving the way for the application of mechanical forces in organoid-based models.
基金supported by the National Key Research and Development Program of China(No.2021YFA1200900)the National Natural Science Foundation of China(NSFC,No.32271460)+7 种基金the Major instrument project of NSFC(No.22027810)NSFC Major Research Plan-Integrated Program(No.92143301)the Innovative Research Group Project of NSFC(No.11621505)the CAS international cooperative project(No.GJHZ201949)the CAS Interdisciplinary Innovation Team,the CAS Key Research Program for Frontier Sciences(No.QYZDJ-SSSLH022)the Research and Development Project in Key Areas of Guangdong Province(No.2019B090917011)CAMS Innovation Fund for Medical Sciences(No.CIFMS 2019-I2M-5-018)the Strategic Priority Research Program of Chinese Academy of Sciences(No.XDB36000000).
文摘With research burgeoning in nanoscience and nanotechnology,there is an urgent need to develop new biological models that can simulate native structure,function,and genetic properties of tissues to evaluate the adverse or beneficial effects of nanomaterials on a host.Among the current biological models,three-dimensional(3D)organoids have developed as powerful tools in the study of nanomaterial-biology(nano-bio)interactions,since these models can overcome many of the limitations of cell and animal models.A deep understanding of organoid techniques will facilitate the development of more efficient nanomedicines and further the fields of tissue engineering and personalized medicine.Herein,we summarize the recent progress in intestinal organoids culture systems with a focus on our understanding of the nature and influencing factors of intestinal organoid growth.We also discuss biomimetic extracellular matrices(ECMs)coupled with nanotechnology.In particular,we analyze the application prospects for intestinal organoids in investigating nano-intestine interactions.By integrating nanotechnology and organoid technology,this recently developed model will fill the gaps left due to the deficiencies of traditional cell and animal models,thus accelerating both our understanding of intestine-related nanotoxicity and the development of nanomedicines.
基金Supported by the Ministry of Science and Technology of China,No.2022YFF1203300National Key Research and Development Program,No.2023YFC2307001China Postdoctoral Science Foundation,No.2023TQ0125 and No.2024M751031.
文摘Diet and nutrition significantly influence health, largely by regulating intestinalnutrient absorption. The intestinal epithelium, as the primary site for nutrientuptake, undergoes continuous renewal driven by precise regulation of intestinalstem cells (ISCs). Nutrient sensing and metabolism are key determinants of ISCfate, making ISCs a central link between nutrient metabolism and the regulationof intestinal tissue renewal and homeostasis. Understanding how ISCs respond ormake adaptations to nutritional signals is therefore vital for maintaining intestinalhomeostasis. Recent studies have spotlighted the origin and identity of ISCs andbroadened our insight into the plasticity and function of ISCs under differentconditions. Mitochondria, the central hubs of energy production and metabolicsignals provided by dietary components and metabolic substrates, such as glucose,amino acids, and lipids, govern the intricate balance between self-renewal anddifferentiation of ISCs. This review highlights the importance of nutrient sensing,metabolic regulation, and mitochondrial function in the specification of ISC fate.A thorough understanding of these mechanisms paves the way for the developmentof stem cell-based therapy for the mucosal healing of gastrointestinaldiseases and diet intervention to foster body health.
基金Supported by the 333 High Level Talents Training Project of Jiangsu Province,No.BRA2019011and Project of Military Research,No.CLB19J025 and No.TGKS2019002。
文摘The development of“mini-guts”organoid originates from the identification of Lgr5+intestinal stem cells(ISCs)and circumambient signalings within their specific niche at the crypt bottom.These in vitro self-renewing“mini-guts”,also named enteroids or colonoids,undergo perpetual proliferation and regulated differentiation,which results in a high-performance,self-assembling and physiological organoid platform in diverse areas of intestinal research and therapy.The triumphant reconstitution of ISC niche in vitro also relies on Matrigel,a heterogeneous sarcoma extract.Despite the promising prospect of organoids research,their expanding applications are hampered by the canonical culture pattern,which reveals limitations such as inaccessible lumen,confine scale,batch to batch variation and low reproducibility.The tumor-origin of Matrigel also raises biosafety concerns in clinical treatment.However,the convergence of breakthroughs in cellular biology and bioengineering contribute to multiform reconstitution of the ISC niche.Herein,we review the recent advances in the microfabrication of intestinal organoids on hydrogel systems.
文摘BACKGROUND Intestinal ischemia-reperfusion(I/R)injury(II/RI)is a critical condition that results in oxidative stress,inflammation,and damage to multiple organs.Zinc,an essential trace element,offers protective benefits in several tissues during I/R injury,but its effects on intestinal II/RI remain unclear.METHODS C57BL/6 mice were pretreated with zinc sulfate(ZnSO4,10 mg/kg)daily for three days before I/R injury was induced via superior mesenteric artery occlusion(SMAO)and abdominal aortic occlusion(AAO)models.Tissue and serum samples were collected to evaluate intestinal,liver,and kidney damage using Chiu’s score,Suzuki score,and histopathological analysis.Caco-2 cells and intestinal organoids were used for in vitro hypoxia-reoxygenation injury models to measure reactive oxygen species(ROS)and superoxide dismutase(SOD)levels.RESULTS Zinc pretreatment significantly reduced intestinal damage in the SMAO and AAO models(P<0.001).The serum levels of liver enzymes(alanine aminotransferase,aspartate aminotransferase)and kidney markers(creatinine and urea)were lower in the zinc-treated mice than in the control mice,indicating reduced hepatic and renal injury.In vitro,zinc decreased ROS levels and increased SOD activity in Caco-2 cells subject to hypoxia-reoxygenation injury.Intestinal organoids pretreated with zinc exhibited enhanced resilience to hypoxic injury compared to controls.CONCLUSION Zinc pretreatment mitigates II/RI and reduces associated multiorgan damage.These findings suggest that zinc has potential clinical applications in protecting against I/R injuries.
基金This work was supported by the founding of the National Natural Science Foundation of China(No.32072752,No.31772620,No.31970814)the System for Poultry Production Technology,Beijing Agriculture Innovation Consortium(Project Number:BAIC04-2020)Natural Key R&D Project of China(2020YFA0113200).
文摘Methionine and its hydroxy analogue(MHA)have been shown to benefit mouse intestinal regeneration.The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development.Here,we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development.The results showed that 125-mm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting.We found that transforming growth factor-b inhibitor(A8301)supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells(ISC).The mitogen-activated protein kinase p38 inhibitor(SB202190)promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells,goblet cells and Paneth cells.However,the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301.The glycogen synthase kinase 3 inhibitor(CHIR99021),valproic acid(VPA)alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells.Chicken serum significantly improved organoid formation,especially in the presence of A8301,SB202190,CHIR99021,and VPA,but inhibited the differentiation of Paneth cells and enteroendocrine cells.Chicken serum at a concentration of 0.25%meets the requirement of chicken intestinal organoid development,and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1.Moreover,commercial mouse organoid culture medium supplemented with A8301,SB202190,CHIR99021,VPA,and chicken serum promotes chicken organoid budding.Based on the chicken intestinal organoid model,we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size,and this effect was reinforced with increased cycloleucine concentrations.Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine.In conclusion,our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.
基金the Joint Funds of the National Natural Science Foundation of China(U22A20511)China Agriculture Research System(CARS-36)Hubei Provincial Key R&D Program(2021BBA083).
文摘A healthy intestine plays an important role in the growth and development of farm animals.In small intestine,Paneth cells are well known for their regulation of intestinal microbiota and intestinal stem cells(ISCs).Although there has been a lot of studies and reviews on human and murine Paneth cells under intestinal homeostasis or disorders,little is known about Paneth cells in farm animals.Most farm animals possess Paneth cells in their small intestine,as identified by various staining methods,and Paneth cells of various livestock species exhibit noticeable differences in cell shape,granule number,and intestinal distribution.Paneth cells in farm animals and their antimicrobial peptides(AMPs)are susceptible to multiple factors such as dietary nutrients and intestinal infection.Thus,the comprehensive understanding of Paneth cells in different livestock species will contribute to the improvement of intestinal health.This review first summarizes the current status of Paneth cells in pig,cattle,sheep,horse,chicken and rabbit,and points out future directions for the investigation of Paneth cells in the reviewed animals.
基金supported by the National Key Research and Development Project of China(2020YFC1808204)the Youth Talent Lifting Project of China National Tobacco Quality Supervision&Test Center(552021CR0030)the State Key Laboratory of Environmental Chemistry and Ecotoxicology,Chinese Academy of Sciences(KF2021-17).
文摘Perfluorooctanoic acid(PFOA)and perfluorooctanesulfonate(PFOS)continue to be extensively present in the natural environment and seriously threaten human health.The intestinal tract is the primary organ of PFOA/PFOS exposure due to the consumption of contaminated food and drinking water.However,it remains unclear how PFOA/PFOS affects intestinal function and overall health.The aim of this study was to investigate the influence of PFOA/PFOS on the absorption of fatty acids in the intestine and the underlying mechanisms using three-dimensional(3D)intestinal organoids.Our results showed that PFOS,but not PFOA,could significantly enhance the fatty acid uptake capacity without obvious damage to the organoids.Furthermore,PFOS markedly reduced the protein levels of ChgA in enteroendocrine cells,but with no observed impact on aldolase B+enterocytes.Mechanistically,exposure to PFOS induced the activation of the peroxisome proliferator-activated receptor(PPAR)αpathway in intestinal organoids,with enhanced expression of PPARαtarget genes associated with fatty acid metabolism,such as Fabp1 and Cd36(fatty acid transporter genes),Acox1 and Pdk4(fatty acid oxidation genes),and Plin2 and Plin3(lipid droplet synthesis genes).These data suggest that PFOS have the potential to affect the absorption function of the intestinal epithelium through the PPARαpathway,and its effect is much stronger than that of PFOA.Our findings also highlight that organoids can be used as a valuable model for conducting toxicological research on environmental chemicals.
基金supported by the National Natural Science Foundation of China(Grant Nos.12025207,12372319)USTC Research Funds of the Double First-Class Initiative(YD2090002024,YD2090092204).
文摘Understanding the regulatory mechanisms of intestinal organoid morphogenesis remains a fundamental chal-lenge in organoid biology.Emerging evidence highlights mechanical bistability as a critical regulator,mediated by dynamic lumen-actomyosin feedback.The recently developed 3D vertex model demonstrates that crypt curvature modulates actomyosin localization via mechanosensitive pathways,creating two stable morphological states-bulged or budded-depending on mechanical history.This model advances beyond static vertex models by incorporating epithelial thickness variations and lumen pressure effects,explaining previously unresolved phenomena like irreversible crypt budding and snap-through transitions.The findings establish a new framework for understanding mechanical decision-making in epithelial tissues,with implications for organoid engineering and developmental biology.
基金supported by the National Natural Science Foundation of China(grant numbers 82125037,8197-3498,82104508,and 81774283)Science Foundation for Distinguished Young Scholars of Jiangsu Province(BK20231528)+5 种基金the National Key Research and Development Program of China(2023YFC2308200)Key R&D Program of Jiangsu Province(grant number BE2022815)Jiangsu Provincial Medical Innovation Center(grant number CXZX202225)Young Elite Scientists Sponsorship Program of the China Association for Science and Technology(grant number 2021-QNRC2-B17)333 high-level talents project of Jiangsu Province for Y.Y.(BRA202202)Suzhou GuSu Medical Talent Project(GSWS2022100).
文摘Dietary factors play a crucial role in irritable bowel syndrome(IBS)pathogenesis.Therefore,the dietary contraindications for patients with IBS require further supplementation.Recent investigations have revealed that ginger consumption may pose a risk of aggravating the symptoms and incidence of IBS;however,the specific mechanism remains unknown.In this study,we developed experimental IBS and intestinal organoid differentiation screening models to elucidate the mechanisms underlying the gingermediated exacerbation of IBS symptoms.Subsequently,we used a knockout approach combined with click chemistry as well as virus infection to identify the toxic components of ginger and the target mechanism.Our results showed that a daily intake of 90 to 300 mg/kg ginger(equivalent to a human daily dose of O.6 to 2 g per person)may pose a risk of exacerbating IBS symptoms.Furthermore,a component derived from 6-gingerol(ginger's main ingredient)through in vivo gastric acid and heat processing inhibited the formation of the elF3 transcription initiation complex by covalently binding to the Cys^(58)site of elF3A,a key factor regulating intestinal crypt stem celldifferentiation,further reducing the goblet cellnumber and related mucus layer thickness and increasing lipopolysaccharide infiltration and low-grade inflammation in the ileum crypts,thereby exacerbating the symptoms of IBS in mice.Our study suggests that dietary ginger aggravates IBS and provides safety evaluation methods for the proper use of foods in specific populations.
基金This work was supported by Hunan Province Key Field R&D Program(2019NK2193)Key Programs of Frontier Scientific Research of the Chinese Academy of Sciences(QYZDY-SSWSMC008)+1 种基金Natural Science Foundation of Hunan Province(2017JJ1020)Young Elite Scientists Sponsorship Program by CAST(YESS20160086).
文摘Early weaning in piglets can cause a series of negative effects.This causes serious losses to the livestock industry.N-Acetyl-D-glucosamine(D-GlcNAc)plays an important role in regulating the homeostasis of the intestine.This study aimed to investigate the effects of D-GlcNAc on the growth performance and intestinal function of weaned piglets.Twenty-four weaned piglets([Yorkshire×Landrace]Duroc,6.58±0.15 kg,n=8)at 21 d old were fed 3 diets supplemented with 0(control),1 and 3 g/kg D-GlcNAc.The intestinal organoid model was used to verify the regulatory mechanism of D-GlcNAc on intestinal epithelial cells.On the whole,supplementation of D-GlcNAc in the piglet diet has no significant effect on the growth performance and diarrhoea of weaned piglets(P>0.05).The apparent digestibility of nutrients and mRNA abundance of nutrient transporters in the 1 g/kg D-GlcNAc group were increased significantly(P<0.05).D-GlcNAc did not affect villus height(VH)and crypt depth(CD)but resulted in a numerically shorter VH and shallower CD,which lead to an increase in ileal VH:CD ratio(P<0.05).Cell shedding rates in the ileum villi increased(P<0.05).The relative length and weight of the small intestine of weaned piglets increased(P<0.05).In vitro studies found that the budding rates of organoids treated with 0.1 mmol/L D-GlcNAc increased on the d 3 and 5(P<0.05).The average budding numbers per budding organoid treated with 0.1 and 10 mmol/L D-GlcNAc increased on d 3(P<0.05).D-GlcNAc upregulated leucine rich repeat containing G protein-coupled receptor 5(Lgr5^(+))and Chromogranin A mRNA abundance in organoids(P<0.05).Mucin 2(Muc2)expression increased when treated with 1 and 10 mmol/L D-GlcNAc(P<0.05).In conclusion,dietary D-GlcNAc cannot improve the growth performance of weaned piglets.However,it can promote the growth and development of the intestinal tract and improve the digestion and absorption capacity of the intestine,which is achieved by affecting the activity of intestinal stem cells.
