Effects of undigested protein-rich ingredients on polarised small intestinal organoid monolayers

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.

Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

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+ 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+ 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.

Advancing intestinal organoid technology to decipher nano-intestine interactions and treat intestinal disease

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.

Mechanical stretching boosts expansion and regeneration of intestinal organoids through fueling stem cell self-renewal

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.

Current knowledge on the multiform reconstitution of intestinal stem cell niche

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.

PFOA/PFOS Facilitated Intestinal Fatty Acid Absorption by Activating the PPARα Pathway:Insights from Organoids Model

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.

Zinc pretreatment for protection against intestinal ischemiareperfusion injury

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.

Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development

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.

Paneth cells in farm animals:current status and future direction

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.

N-Acetyl-D-glucosamine improves the intestinal development and nutrient absorption of weaned piglets via regulating the activity of intestinal stem cells

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.