A Cost-effective Microfluidic Cell Chip For Investigating Of Tumour Cell

Microfluidic chip technology is to integrate many basic operating units, including biological/chemical/medical sample preparation, analysis process, reaction, separation,detection etc., into a single micron-sized chip. Microfluidic technology has showed its unique advantages, and has been gradually developed into a biological, medical,chemical, electronics, materials, fluid, mechanical, new interdisciplinary research area.Due to its advantages of small in size, saving reagents and pharmaceuticals, fast response,as well as its properties in batch processing and production of disposable, etc.,great potentials are promised in both biological/medical and other areas of scientific research and practical application in the future life.The motivation of this work is to establish microfluidic cell analysis platform to investigate the effect of BRAFV600 E inhibitor PLX4032 and EGFR inhibitor gefitinib on melanoma cell growth and migration. The main results are as follows:1.Optimization of cell growth conditions on Polydimethylsiloxane(PDMS)/glass slide hydride microfluidic chipsWe firstly compared the advantages and disadvantages of the conventional liquid photoresist and photosensitive dry film, then we make a choice of the photosensitive dry film as the material for positive mold.Secondly,Achieving cell culture on chip and promise the healthy growth and proliferation of them is the basic step of using microfluidic chips to carry out a series of experiments. First, a single-channel structure was designed to optimize the conditions for on chip cell culture. The factors were evaluated include the microchannel size(height), fetal bovine serum content of the culture medium, choose of extracellular matrix protein etc. Human laryngeal epithelial(Hep-2)was studied as a model cell. Results show that: channel height of 120μm, fetal bovine serum content of10%, 10μg / ml of Collagen solution(PBS diluted) pre-treated PDMS channels can well support the cell attachment and proliferation on-chip.2.Monitoring of TGF- β 1 induced A549 cell epithelial mesenchymal transformation process by measuring cell adhesion force with a microfluidic deviceEpithelial mesenchymal transformation(EMT) is a morbid process of epithelial cells transforming into mesenchymal cells, during which the cells will gradually lose their polarity and adhesion, increasing metastasis and invasion. Studies have shown that transforming growth factor- β 1 can induce the occurrence of epithelial mesenchymal transition process.Perfusion culture apparatus used in this experiment is simple and easy to operate.Only one device can achieve cell culture under flowing media,thermostatic control, and can set multiple parameters such as velocity and time.The influence of different extracellular proteins and TGF-β1 on cells’ shear force resistance has been explored in the experiment. The results show that fibronectin can most effectively improve the cell resistance to shear force in flow medium, and it’s most appropriate and economical concentration is 10μg/ml. Moreover, transforming growth factor-β1can reduce shear force resistance ability of A549 cells, the longer application time the greater effect on cell resistance to shear force.The results demonstrate that the microfluidic device is a potential tool to characterize the epithelial-mesenchyme transition process by measuring cell adhesion force.3.The microfluidic cell chip for study of the synergist of combination Vemurafenib and Gefitinib on melanoma cell migration and the interaction between tumour cell and normal cellIn this experiment, a cost-effective and user-friendly microfluidic device was developed for studying of cell migration. A two-step photolithography procedure was conducted to reveal microchannels and micorchambers with different depth. The PDMS/glass slide hydride device was assembled between a polymethyl methacrylate(PMMA) clamp which can adjust the pressure imposed on the device to control the fluid communication between 2 main-chambers, thus identical wound(cell-free space)with clear edge can be easily formed within channel without extra chemical,mechanical force, fluidic manipulation and sophisticated microstructure.Using this device, we evaluated the synergist of combination of BRAFV600 E inhibitor vemurafenib and epidermal growth factor receptor(EGFR) inhibitor gefitinib in inhibiting of melanoma cell migration with only 20 μL cell consumption, highlighting its potential in assaying rare clinical biopsy for personalized medicine. In addition, theon-chip migration model is strictly follow the principle of conventional in vitro scratch/wound healing assay, facilitating it is translation to biologist.And then we realize the coculture of tumour cell and normal cell with the microfluidc chip,cell in the two cell lines were cultured in parallel channels in the chip,forming regular 250μm gap in the middle. In this experiment, human hepatoma cells(HepG2) and human umbilical vein endothelial cells(HUVECs)were studied as model cell lines..Different culture conditions listed as follows: basic medium, complete medium, basic medium+EGF, basic medium+VEGF, real-time monitoring of the growth and migration of the two cell lines. The results showed that: in the co-culture conditions, the cells in neighborhood area of HepG2 next to HUVECS cell began to appear apoptosis after fostering 12 h,and the HepG2 cell apoptosis are aggravated in24 h, in 36 the apoptosis phenomenon is more apparent. We can observe with the microscope that the apoptosis area present an arc-shaped pattern.