The Drug Sensitivity Test On The Primary Non-small Cell Lung Cancer Cells For Individualized Treatment Based On Microfluidic Chip With Three-dimensional Culture Mode

Objectives: Lung cancer is one of the most common and the highest mortality ofhuman malignant tumors. Among them,80%cases of lung cancer are non-small celllung cancer (Non-small cell lung cancer, NSCLC). In recent years, with the incidencerate of lung adeno carcinoma and bronchiole-alveolar carcinoma rises in non-smokingcrowd of women， the diagnosis and treatment of NSCLC has become an importantand urgent task in clinical. Chemotherapy plays a very important role in thecomprehensive treatment of advanced NSCLC. However，it always fails in clinicbecause of lack of accurate chemotherapy drug sensitivity test. Microfluidic chip(lab-on-a-chip), with its miniaturization, integration, high-throughput, short reactiontime, less reagent consumption and other advantages has been recognized as one of themain technology platform of system biology research in this century. The micro-devicecan be used to cultivate cells in three-dimensional mode by gel perfusion inmicro-channels, and also can supply nutrients continuously through a syringe pumpwhich simulates the tumor microenvironment in vivo much more close. The purpose ofthis work lies in using the micro-fluidic chip to establish efficient and stable NSCLCprimary culture methods and to do research on Chemotherapy Drug Sensitivity Test inthree-dimensional cell culture model that can provide a basis for prediction patients’sensitivity to chemotherapeutic drugs in clinical individualized treatment of NSCLC.Methods: All8cases of fresh tumor tissue were taken from the surgicalspecimens of patients who confirmed NSCLC pathologically. Primary human NSCLCcells were cultured by using a composition which contains a variety of digestiveenzymes such as various subtypes of collagenase, the neutral protease and otherdigestive enzymes to improve digestion efficiency and purified by density gradient centrifugation. NSCLC primary cells were test by detecting the expression cytokeratin-19and carcino-embryonic antigen CEA expression in NSCLC primary cells withImmuno-fluorescence.The microfluidic chip system includes a microfluidic chip andtwo MS26micro syringes. The main components of the microchip were cell culturechambers where can be infused gel to fabricate three-dimensional cell mode andconcentration gradient generator (CGG) which can produce5:6:8drug concentrations.The cell apoptosis rate of NSCLC primary cells were detected after the treatments ofcisplatin and gefitinib in different drug combinations and concentrations.Results：The successfully cultured human primary NSCLC cell growth is ingood condition, with different forms and sizes, which can be sub-cultured stably. Theexpression of CK-19and CEA in Squamous carcinoma and adeno carcinoma primarycells were positive respectively.8cases of NSCLC patients have obvious individualsensitivity differences in the treatment of cisplatin and gefitinib in different drugcombinations and concentrations. Compared with adeno carcinoma, the drug sensitivityof squamous carcinoma is lower; the higher histological grade is, the higher the drugsensitivity; the drug sensitivity of the combination group is significantly better than thatof single group.Conclusions: In this work，efficient and stable culture methods of human primaryNSCLC cells were successfully established by using a composition which contains avariety of digestive enzymes such as various subtypes of collagenase, the neutralprotease and other digestive enzymes to improve digestion efficiency and purificationwith density gradient centrifugation. Chemotherapy Drug Sensitivity Test inthree-dimensional cell culture model using microfluidic chips can provide a basis forclinical individual therapy. Microfluidic chip, which has economic, high throughput,simple operation, easy automation, high sensitivity advantages, is a worthy simple,accurate and reliable susceptibility testing technology platform for drug susceptibilitytesting.