Reconstitution Of Human 3D Liver And Brain Organoids Models For Preliminary Applications Using Microfluidic Organs-on-chips Technology

The construction of in vitro biomimetic 3D tissue and organ models holds great appeal for biomedical applications in disease modeling,drug development,regenerative medicine and clinical therapy.Current model systems mostly rely on animal models and traditional twodimensional(2D)cell cultures,which are difficult to reflect human physiology,as well as faithfully predict human responses to drugs or external stimuli.Recently,the emerging organson-chips provide a promising platform to establish in vitro human organ microphysiology based on microfluidic technology.Organs-on-chips are a frontier and interdisciplinary field by combining with biology,physics,chemistry,materials science,bioengineering and so on.According to the key structural and fundtional characteristics of human specific tissues or organs,we build physiologically relevant in vitro 3D tissues and organ models by integrating stem cell biology,microfluidic organs-on-chips with bioengineering technologies,which provide proof-of-concepts and new strategies to engineer human tissues or organs.These established organ models offer powerful platform for applications in developmental biology,disease studies and drug testing.The detailed work in this thesis includes:First,we present a new strategy to generate hiPSCs-derived 3D brain organoids using an organ-on-a-chip system in a controlled manner.This perfusable chip system was designed with multiple parallel channels structures that enabled 3D culture,in situ neural differentiation,and organization of brain organoids.The brain organoids on chip display neural differentiation,regionalization and cortical organization,which recapitulate the key features of early human brain development.Moreover,the brain organoids exhibited enhanced neural differentiation and cortical organization under fluid flow conditions compared to that in conventional static cultures,suggesting the role of mechanical fluid flow in promoting brain organogenesis.The brain organoids-on-a-chip system is simple and flexible,which may open new avenues for brain developmental studies,disease modeling and drug development.Second,we established an in vitro liver organoids-on-a-chip model for hepatotoxic drugs assessment.A perfusable microfluidic chip that contained micropillar array was established to enable the construction of functional 3D liver organoids derived from hiPSCs in a controlled manner,which recapitulates the key features of human liver formation.The results indicated the role of mechanical fluid flow in promoting the functions of the liver organoids.Moreover,this model can be used for hepatotoxic drug testing,which exhibited hepatotoxic responses to acetaminophen in a dose-and time-dependent manner.The in vitro liver models provide a new approach and platform for drug testing and toxicology research.Third,we presented a stem cell-based human brain organoids-on-a-chip to probe the effects of prenatal nicotine exposure on brain development in vitro.With nicotine exposure,brain organoids showed increased apoptosis,premature differentiation of neurons and impaired differentiation of brain regions and cortical organization.These findings indicated that nicotine exposure is closely related to impaired neurogenesis in developmental fetal brain during early gestation.The created brain organoids on chip system may provide a versatile and valuable platform to study brain development,neurological disorders and toxicity predictions at early stages.Forth,we studied human nonalcoholic fatty liver disease(NAFLD)using a liver organoids-on-a-chip system.Upon free fatty acids(FFAs)exposure,this system allowed to characterize the pathological features of NAFLD in liver organoids under perfused culture conditions,including lipid droplet formation and triglyceride accumulation,abnormal expressions of lipid metabolism-associated genes,elevated levels of reactive oxygen species and inflammatory cytokines.These results suggested that liver organoids on chip can recapitulate the typical pathological features of NAFLD progression,which may provide a promising platform for studying the pathogenesis of NAFLD and drug development.