Construction And Application Of A New Generation Of Blood-brain Barrier 3D In Vitro Model

Introduction: The Blood Brain Barrier(BBB)is a highly evolved microvasculature within the Central Nervous System(CNS)and is a key barrier between circulating blood and CNS extracellular fluid.BBB can effectively prevent harmful substances in the blood from entering the CNS.However,when a disease occurs in the CNS,the BBB also restricts the entry of drugs into the CNS,making treatment of the CNS disease difficult.To this end,people continue to explore how structural cells in the blood-brain barrier are linked under various physiological or pathophysiological conditions and the expression and regulation mechanisms of some key proteins.More importantly,through these studies,it will help to find new ways to treat CNS-related diseases more rationally.Various blood-brain barrier models are used for studying the study of blood-brain barrier mechanism and new drug development.They are mainly divided into two categories,(1)in vivo models and(2)in vitro models.Although many traditional animal models have been used for the screening and research and development of BBB penetrating medicines,the purpose of real simulation of human beings cannot be achieved because of the large differences in species and poor predictive efficacy.Therefore,if human cells are used to establish a high-fidelity blood-brain barrier model in vitro,on the one hand,it can improve the accuracy of early drug efficacy(the ability to penetrate into brain tissue,etc.)of central drug candidates,and accelerate the treatment of neurotherapeutic drugs.On the other hand,it can also reduce or even replace animal experiments and guarantee animal welfare.Research purposes: In order to find new drugs and new methods for more rational treatment of diseases related to the central nervous system,people's research on the blood-brain barrier has been deepened.However,at present,due to the inadequacy of the in vitro blood-brain barrier models,they are limited in the practical application of predicting whether a drug candidate can be taken up by the central nervous system.The development of amodel that is highly compatible with the real blood-brain barrier in humans,whether it is for the study of the structural function of the blood-brain barrier,or for the development of new drugs for the treatment of central nervous system diseases,will be of great help.This experiment utilizes three major cells that make up the human blood-brain barrier,including: human primary astrocytes,human brain pericytes and human cerebral vascular endothelial cells,creating new a model in low-adhesion agarose gel plates.The 3D blood-brain barrier in vitro model of the new generation,after verifying the function of the model,further explores its practical application value.Research method: 1.Cell identification: GFAP,NG2 and CD31 were used as markers for human primary astrocytes,human brain pericytes and human cerebral vascular endothelial cells,respectively,and three cells were identified.2.Establishment and macroscopic identification of the 3D blood-brain barrier in vitro model: human cerebral pericytes,human astrocytes and human cerebral vascular endothelial cells(h CMEC/D3)in a low-adhesion agarose gel plate cultivate for 48 hours.After 48 hours,the model was formed on the surface of agarose gel,which was then visually and optically microscopically observed.3.Microstructure identification of 3D blood-brain barrier in vitro model: Cell tracer and immunofluorescence staining techniques were used to identify the cell distribution in the model and the functional proteins(such as tight junctions and P-glycoprotein)on the surface of the model.4.Functional verification of the 3D blood-brain barrier in vitro model: The VEGF-A-dependent permeability change assay and the P-glycoprotein function verification experiment were used to verify the physical barrier and bioactive barrier function of the model.5.Preliminary exploration of the application of the 3D blood-brain barrier in vitro model: the application of the model to the screening of adeno-associated viruses and peptides that can cross the blood-brain barrier,and explore the practical application value of the model.Conclusion: 1.This experiment utilizes three major cells that constitute the human blood-brain barrier,including: human primary astrocytes,human brain pericytes,and human cerebral vascular endothelial cells,in a low-adhesion agarose gel culture plate.Establish new generation of 3D blood-brain barrier in vitro model.The cell tracer technique was used to analyze the distribution of cells inside the model,and it was confirmed that the cell arrangement in the model was highly fitted to the blood-brain barrier structure in the human body.2.Immunofluorescence staining technique was used to confirm that the 3D blood-brain barrier in vitro model has a large number of blood-brain barrier-specific protein structures,such as tight junctions and P-gp efflux pumps.It was confirmed that the specific protein expression on the surface of the model was much higher than the traditional model.3.The functionality of the blood-brain barrier-specific protein on the surface of the 3D blood-brain barrier in vitro model was confirmed by using the macromolecular dextran permeability test and the efflux pump function verification experiment.The model not only has an effective physical barrier,but also restricts the transport of substances in the paracellular,and its active efflux pump also constitutes the bioactive barrier of the model.4.The application of the adeno-associated virus across the blood-brain barrier was screened by applying the 3D blood-brain barrier in vitro model for the first time,and the practical application value of the model was initially explored.After comparison with the traditional animal model screening results,the consistency of the results suggests that the 3D in vitro model of the new blood-brain barrier may replace the animal model for screening the trans-blood-brain barrier-associated virus,which not only solves the species.The problem of differences between the genera also greatly saves the experimental cycle and cost.5.Through the preliminary exploration of a new generation of blood-brain barrier 3D in vitro model screening of trans-blood-brain barrier polypeptides,it was confirmed that receptor-mediated transmembrane transport exists on the surface of the model,and that the model can be used as a predictive compound to cross the blood-brain barrier tool.6.This study has its shortcomings: First,we have not fully verified BBB-specific structure protein on the surface of this model,which makes our understanding of the model not deep enough.Secondly,the exploration of the application of the model in this study is only at the initial stage.For example,the candidate peptides of the selected polypeptides and adeno-associated viruses are somewhat single.Finally,we need to find a variety of ways to quantify the substances entering the model.In this study,only the immunofluorescence method is used to determine the amount of drug entering the model,and in the subsequent work we will introduce the method of mass spectrometry,which can screen drugs with known molecular weight but no fluorescence effect.These issues are subject to further research in the later stages.