| Objective: This work aimed at setting a micro fluidic system which can mimic microcirculation in vivo. Long time cell culture, protein detection and chemotherapy analysis can be finished through flow medium in the system.The integration of various chemical devices and complex operations onto a microchip, also known as micro total analysis systems (-TAS) or labs-on-a-chip, is currently a major interest for researchers due to their desirable characteristics, including reductions in reagent consumption, space requirements and analysis time. Especially, the mechanical micro- fluidic pump can be utilized in a micro-fluidic system having a simplified network of tubes and pumps, and used as liquid delivery systems to create vivo-like microenvironments. However, till now little study focuses on long time cell culture, drug analysis and cell functional detection under the condition of vivo-like micro fluidic system. In addition, compare with common research platform, it will be more significant for the study on chemotherapy through continuous flow medium.Lung cancer is the leading cause of cancer death in human beings all over the world. Chemotherapy is the major clinical strategy for adminis- tration of lung cancer with the advanced stages. However, the success of the treatment is limited by the intrinsic or acquired resistance of cancer cells to chemotherapy.Human cancer cells can exhibit a cross-resistant phenotype against several unrelated antineoplastic drugs that differ widely with respect to molecular structure and target specificity. This phenomenon has been termed as multi drug resistance (MDR). Pg-p is the main protein associated with MDR and capable of decreasing the intracellular concentration of a broad range of cytotoxic agents in an energy-dependant mediated efflux. But little study focuses on the drug resistance caused by pg-p in human lung cancer cell lines.Methods: A human carcinoma SPCA cell line has been used as a model in this work. A connection between micro-fluidic pump and micro chip has been conducted as to build the micro fluidic system, which can be used for cell culture. In order to explore the suitable flow rate,cells were cultured under the flow rate of 1mm/24h,5mm/24h,10mm/24h or 15mm/24h and the morphology of cells was observed after 24h culture. About the culture time, with the flow rate around 15mm/24h, morphology of cells were observed at 24h,48h,72h and 96h after injection into the micro channel, then cells viability was measured with staining of trypan. In order to study the availability of the system, expression of pg-p levels under the condition with (experiment) or without (control) inhibition by calcium ionophore verapamil was detected by immunofluorescence through a fluoresce micro- scope. Further study focused on the correlation between expression of pg-p and resistance to apoptotic induced by VP16. Apoptotic rate was detected by PI staining. To confirm the results of the micro fluidic systems, we also did apoptotic detection by flow cytometry with a traditional method with Annexin V and PI.Results: The SPCA cells can be cultured successfully in the micro fluidic system. It was shown if the flow rate achieved 15mm/24h, because of the strong shearing force, morphology of cells turned round and cells were flowed away. So the flow rate should be set under 15mm/24h. Under the flow rate around 15mm/24h, it can be seen cells grew and propagate well in the system under observation at 24h,48h,72h and 96h as well as cells viability was less than 1% with staining of trypan. Pg-p expression detection with immunofluorescence, cellular membrane presented green fluorescence (excitation at 488nm) and the intensity of the fluorescence was clearly decreased with verapamil inhibition in experiment group. PI staining indicated that the cells of experiment group was more stronger than that in control. Flow cytometry measurement showed that the percentage of apoptotic cells was more than 2 fold in experiment group as compared with control group: experiment (19.94%) versus control (8.63%). Our results demonstrate there was a good correlation between micro fluidic system and traditional method.Conclusion: We successfully set a vivo microcirculation like micro fluidic system which can be used for at least 4 days cell culture and analysis in medicine. In addition, it also includes many advantages such as lower quantity of chemical reagents, simple handling course, high level of integration and so on. We also find that pg-p inhibition enhances VP16-induced apoptosis in lung cancer cells. |
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