Construction And Application Of Cardiac-vascular-on-chip Based On Stem Cell And Organ-on-a-chip Technologies

At the stage of the clinical research,about one-third of drug candidates are withdrawn due to cardiotoxicity every year.Currently,animal is the most comment drug screening model,however,which is costly,inefficiently and has inherent genetic and physiological differences from the human.Therefore,it’s crucial to construct cost-effective toxicity evaluation models that can accurately simulate the physiological characteristics of the human heart.On the other hand,the in vivo heart microenvironment is maintained by blood vessel networks,which are necessary for the dynamic metabolism and information exchange between cells.Organ-on-a-chip is an emerging technology to realize in vitro dynamic physiological microenvironment.In this study,by combining stem cell technology,electrospinning technology and organ-on-a-chip technology,an in vitro cardiac-vascular-on-chip was constructed,and its application in drug cardiotoxicity evaluation was studied.The specific content is as follows:1)Human induced pluripotent stem cells(hiPSCs)were selected as the cell source.A small molecule induced differentiation method and glucose starvation purification method were used to acquire high-purity cardiomyocytes(hi PSC-CMs)in vitro.The optimization of the parameters of the differentiation process were optimized and the results showed that the optimal parameters were 75% of hiPSCs confluence,8 μM of CHIR 99021,5 μM of IWP 2and two rounds of purification.Immunofluorescence results showed that the differentiated hi PSC-CMs exhibited the expression of cardiomyocyte specific proteins,α-actinin and c Tn T.The results of flow cytometry verified that the purity of hi PSC-CMs reached to more than94.6% after purification processes.The contraction of the differentiated hi PSC-CMs was then analyzed with a custom-coded MATLAB program and demonstrated the spontaneous beating ability and maturity of the hi PSC-CMs.2)By combining soft lithography and electrospinning,a monolayer culture patch of gelatin nanofibers supported with a honeycomb PEGDA frame was fabricated.SEM images indicated that the honeycomb microstructure and the crosslinked nanofibrous net with high porosity.HUVECs were cultured on the culture patch to construct a vascular endothelial barrier.The result of live/dead assay confirmed that HUVECs on the culture patch had good cell viability.The expression of the VE-cadherin protein proved the formation and integrity of the vascular endothelial barrier.3)An openable microfluidic chip with sandwich structure was applied for the heart-vascular-on-chip construction.hi PSC-CMs were seeded in the lower chamber,and the vascular endothelial barrier patch was placed between the upper and lower layers.The expression of VE-cadherin and F-actin indicated that dynamic culture condition resulted into the morphological changes and orientation of HUVECs along the direction of flow shear force.Afterwards,two types of drugs with known cardiac effects,i.e.,ISP and E-4031 were used to evaluate the drug reaction of the in vitro cardiac-vascular model.Video signal analysis results showed that myocardial tissue without a vascular barrier could respond to drugs in time,while the drug response of myocardial models with a vascular endothelial barrier significantly was delayed.In summary,combining stem cell and organ-on-a-chip technology,we designed an in vitro cardiac-vascular model,which is promising to be provided as a new tool for the cardiotoxicity evaluation of drug candidates in drug development as well as the drug efficacy assessment in the individualized treatment.