Microvascularized Intestinal Organoid-on-a-chip For Intestinal Disease Modeling And Drug Development

Background Intestinal ischemia/reperfusion(I/R)injury is usually caused by vaso-occlusive disease due to thromboembolism or atherosclerosis,and acute intestinal hypoxia leads to intestinal barrier disruption and microbiota translocation,ultimately leading to multiple organ dysfunctions.Despite rapid advances in medical imaging diagnosis and clinical treatment,the mortality rate associated with intestinal I/R remains at50-80%.In the classic cascading injury model,the intestinal mucosa is most vulnerable to I/R,in which the intestinal epithelial cells are first damaged,and then immune cells in the lamina propria will activate strong inflammatory responses and in turn aggravate the damage of intestinal epithelial cells through damage-associated molecular patterns(DAMPs).Therefore,discovery of effective drug targets to alleviate epithelial damage is expected to protect intestinal I/R damage.Previous studies on molecular targets for the treatment of intestinal I/R injury mainly relied on two-dimensional(2D)cell culture models in a tri-gas incubator or animal intestinal I/R models.However,these 2D cell models are far from a spatially heterogeneous organization of native epithelial phenotypes,and the oxygen decline requires up to 24 hours,which is totally different from the in vivo I/R process that tissue hypoxia is ultrafast and epithelia start to disintegrate after around 45 minutes of ischemia.A chronic oxygen decline is likely to conceal the therapeutic targets by reshaping the molecular map as mammals have the intrinsic adaptive competence to hypoxia.Moreover,in animal I/R models,intestinal epithelia are easily affected by adjacent immune cells,thus possibly impeding the discovery of initiating molecular target.Owing to the lack of precise models integrating epithelial similarities and comparable oxygen dynamics,drug development to rescue intestinal I/R injury is facing obstacles.Method Inspired by the natural structure of intestinal villi(i.e.,intestinal epithelial cells wrap around central trophic capillaries),this study aimed to develop a biomimetic microvascularized intestinal organoid-on-a-chip.First,through microfluidic processing,a p H-responsive zinc metal-organic framework(ZIF-8)/sodium alginate(SA)hydrogel hollow tube was obtained,and human umbilical vein endothelial cells(HUVECs)were transplanted into the lumen to generate artificial blood vessels.The artificial blood vessels and intestinal organoids were then assembled on a chip in the form of organoids surrounding blood vessels.The hypoxic culture medium was continuously injected into the artificial blood vessel through a microfluidic pump for 3 hours,and the oxygen concentration in the organoids decreased rapidly through gas exchange.Then,the medium with normal oxygen concentration was injected into the artificial blood vessel to rapidly increase qxygen content for organoids.As a result,intestinal organoids were affected by rapid hypoxia-reoxygenation(HR).Subsequently,we examined the cellular damage of organoids after rapid HR.Transcriptomic sequencing was used to compare the RNA expression of intestinal organoids affected by HR with these cultured in a normal oxygen environment,thereby screening potential therapeutic targets.Based on lentivirus-transducted intestinal organoid models,cell models,and mouse intestinal I/R models,and human intestinal ischemic samples,the functions of potential molecular targets were validated.Results The microvascularized intestinal organoid-on-a-chip was successfully constructed.Through finite element analysis and oxygen meter detection,we found that the oxygen concentration in the intestinal organoid chip can be drastically reduced in a shorter time of 3hours compared with 24 hours by using the three-gas incubator to reach the comparable hypoxia state.In this way,it induced significant oxidative stress and apoptosis in the intestinal organoids of the chip.Furthermore,high-throughput RNA sequencing of harvested intestinal organoids showed that the gene of olfactomedin 4(Olfm4)was the most significantly down-regulated after hypoxia and reoxygenation(HR)of the organoid-on-a-chip,suggesting that Olfm4 may be a key signaling molecule for intestinal epithelial inflammation.In order to verify this hypothesis,we constructed lentivirus-mediated Olfm4 interference or overexpression for intestinal organoids.It was found that downregulation of Olfm4 could induce a more significant organoid inflammatory response.In addition,we up-regulated OLFM4 levels in colonic epithelial cell line(FHC)based on the OLFM4-loaded liposome delivery,which further confirmed its role in reducing cell inflammation.Moreover,in the intestinal I/R models of mice and intestinal samples of patients,we found the expression level of Olfm4 was increased in the early stage of intestinal I/R injury,indicating that bodies initiate a feedback mechanism to resist intestinal I/R injury.However,at the late stage,the Olfm4 expression was decreased and intestinal injury was further aggravated.Lentivirus-mediated Olfm4 overexpression could reverse the intestinal I/R injury at the late stage,implying that the up-regulation of intestinal Olfm4 expression is promising to reduce intestinal injury and improve therapeutic effects.Conclusion This study highlights the advantages of microvascularized intestinal organoid-on-a-chip in recapitulating oxygen dynamics during I/R injury and is promising to provide new insights for the construction of hypoxia-related disease models and drug development.Moreover,OLFM4 was found to be a potential molecular target for alleviating intestinal I/R injury.