Three-Dimensional Co-culture Microfluidic Model And Its Application On Cancer Research

Cell microenvironment plays a crucial role in cell morphology,functional development and tissue repair.The interaction of the processes in tumorigenesis,progression,metastasis and its microenvironment has become a hot topic in current oncology.It is difficult to recreate the tumor microenvironment in traditional vitro two-dimensional(2D)culture conditions.Its inherent defects cannot effectively correspond to the factors in environment of in vivo cancer cells,such as the dimension of cell growth,the interactions between cells,the biochemical factor group,the physicochemical property of extracellular matrix(ECM),and the stress stimulus.Recently,microfluidic technology shows its unique advantages in highly efficient drug screening and delivery,physiology and disease model building,and the simulation of tissue repair.It has been used to build functional “Lab on a chip”.Aiming at cancer and tumor problems,microchips could provide controllable condition to correspond to certain physical or chemical factors,and independently studied the physiological characteristics and functional response of tumor cells with its effect.The configurations of these devices are usually too simple or too complex,examine single cell type in the stimulus conditions of single factor.It has become an important development direction of microfluidic model to be able to effectively deal with the complicated process and necessary link of tumor progression,and integrate the interaction between cancer cells and tumor microenvironment.Therefore,in order to cope with researchers' urgent need of the comprehensive conditions and the integral vision,there are enormous challenges in constructing integrated multi-factor in vitro model by virtue of microfluidic technology for cancer research.In order to analyze tumor cell migration under more integrated condition,this study abstracted and extracted tumor microenvironment,designed kinds of microfluidic configurations and fabricated the chip,built a simple and effective in vitro three-dimensional(3D)co-culture model of tumor cells.Then we applied the model to investigate kinds of tumor cells,and analyzed its biological efficacy in tumor progression and metastasis,hoping to expand new vision and help to clarify the interaction of cells and tumor microenvironment.The main content of this study is summarized as follows:(1)Analyzing the steps of stromal infiltration and angiogenesis in tumor progression and metastasis model.Abstracting blood vessels,matrix and tumor in microenvironment to the space-distributed microchannel configuration reasonably,to design a chip and modify the simple three-channel configuration.Results showed that the adjustment of microchannel sizes promoted HepG2 and human umbilical vein endothelial cells(ECs)to form a 3D structure in Matrigel.Hexagonal microspots and trapezoidal narrow microbridges can control and locate fluid more accurately.The treatment of vacuum affixed method can bond the chip non-permanently,and effectively make up the shortcomings of the chip in fabrication,cleaning and reuse.The chip can satisfy the cell co-culture and multi-factor integration in space,configuration and function,and lay the foundation for the follow-up cell experiment.(2)In order to verify the advantages of the chip in 3D culture condition and the effect of biochemical factors compare with 2D conditions,we used the chip to investigate three types of ovarian cancer cell lines.By contrasting 2D and 3D culture with biochemical factor conditions of NGF and its inhibitor,we investigated colony formation of cancer cell under the multiple factors environment.Results showed that the 3D culture could promote the formation of obvious tumor cell colony of CAOV3 and OVCAR3,and effectively affect the polarization and filopodia extension of SKOV3.Although the morphology and adhesion of all three cell types were modulated by Ro 08-2750(inhibitor of NGF)in 2D condition,NGF could significantly affect the formation of CAOV3(inhibitory)and OVCAR3(promoted)cell colony in 3D condition and both significantly antagonized by inhibitor.The results verified the advantages of the chip in cell 3D culture,conditional control,investigation of function and real-time observation,and endorsed the modeling of tumor cells 3D co-culture model.(3)We further integrated some important factors in the tumor microenvironment to construct tumor cell 3D co-culture model,such as co-culture,multi-dimension culture,the interaction between cell-cell and cell-matrix,biochemical factors and its concentration distribution,geometric distribution of space.In the constructed microenvironment,we investigated the 3D co-culture of two types of tumor cells(HepG2 and CAOV3)and ECs.Results showed that HepG2 cells formed a 3D network in Matrigel,and co-culture condition made ECs polarize and extend filopodia.Mutual induction of tumor cells and EC promoted ECs migrate into Matrigel and invasive ability of CAOV3,the invasion of tumor cell corresponding to the lowly invasion of HepG2 and highly invasion of CAOV3.Compared with the traditional 2D experiments,the model showed more behavior and response of cells and provided more data to analysize.The results validated the function of the model and can be used to investigate tumor growth and metastasis.(4)Combining with the traditional experiments,we applied the 3D co-culture model to integrally investigate EC migration which induced by liver cancer stem-like cell(SC)in chip.Western bolt and ELISA analysis showed that SCs had a higher expression level of proteins related to adhesion,degradation,migration than mature liver cancer cell(MC).Transwell migration analysis showed that SCs had stronger ability of inducing ECs migration than MCs.Results of chip experiments showed that the SCs did not form the normal cell spheres in Matrigel.ECs extended filopodia into 3D gel,which corresponded to the early sprouting behavior of angiogenesis,which was promoted by the co-culture of tumor cells.SCs were significantly stronger than MCs in inducing ECs migration.SCs seemed to be more sensitive to the effect of ECs,and had greater potential of invasion and metastasis.The multi-factor condition in the chip provided more details than the traditional migration experiment.The model provided integrated results and verified them from the multi-dimensional degree.Above all,this study designed the configuration and size of microchannel,selected the hexagonal microspot and trapezoidal microbridge,adopted the bonding method of vacuum affixed treatment,and effectively improved the abilities of chip in cell growth,fluid control,fabrication and use.The 3D culture condition and NGF had significant influence on the adhesion and aggregation of ovarian cancer cells,which indicated that it was better in investigating the formation of ovarian cancer cell colony in the chip.Based on the chip,the in vitro tumor cells 3D co-culture model could integrate some factors to simulate the tumor microenvironment to a certain extent.Cancer cells can interact with ECs and facilitate each other to migrate into ECM,and show the corresponding invasive properties.Applying model to investigate liver cancer stem-like cell could effectively evaluate the stronger ability of SCs in inducing ECs migration than MCs,and the greater potential of metastasis in invasive ability.Experimental results above showed that: the microfluidic chip and tumor cell co-culture model,which were designed and fabricated in this study,could be more intuitive and comprehensive research on cancer metastasis,analyze the problems from the angle of formation of cancer cell colony,stimulation of biochemical factors,interaction between cells,cell migration.The advantages of the model were verified by comparing 2D and 3D,the traditional experiment and the chip model,cell kind and type.The kinds of cell involved evaluated the high adaptability of application of the model in the scope.The details of cell behavior that revealed in the model embodied its development potential for investigating cancer cells aggregation and tumor angiogenesis the model in the depth.The method not only can be used to investigate related fundamental problems of cancer progression and metastasis,but also can be used for comparison and verification combined with traditional experiments.At the same time,it provides new vision and thinking of multi-dimensional,multi cell type,multi-factor.It is expected to become a new supplement between simple basic experiment and complex in vivo model.