| At present, the drug screening techniques for glucagon-like peptide-1 (GLP-1) receptor agonist are mainly based on detecting the intracellular cyclic adenosine monophosphate (cAMP) levels of the activated signal pathway. As many factors influence cAMP levels, the specificity is too bad to meet the request of drug screening. Therefore, it is urgently to establish a high-efficiency and sensitive high-throughput drug screening cellular model. To meet the requirements of high-throughput screening, the conditions for large scale cultivating GLP-1R/U-2 OS tagged with green fluorescent protein (GFP) were optimize in this study.Firstly, the cell seeding density was optimized by using a 96-well culture plate containing 10% fetal bovine serum in DMEM medium (100μl/well). By using 0.2× 106 cells/ml seeding density may make cell confluence more than 80% after 48h cultivation, while the confluence of 0.5×106 cells/ml seeding density can reach over 80% only in 24h. All cultivated cells can be used in the subsequent studies and experiments immediately. The paper also studied the state of cells under different concentrations of G418. The result showed that no significant effect on the proliferation number and the proportion of fluorescent cells with different concentrations of G418 in same culture time. The proportion of fluorescent cells shares a growing trend when the cultivation time was extended. The proportion of fluorescent cells can finally make over 50% under different concentrations of G418. The serum optimization experiments showed that the cell proliferation speed is the most rapid when using fetal bovine serum at 20% volume ratio, while by using the fetal calf serum, the serum volume ratio of 20% can reach to the maximum number of cell proliferation. So the fetal bovine serum should be used in the large-scale cultivation. Then the significance of initial concentration of glucose and glutamine added concentrations for cell proliferation were also analyzed. The results showed that the initial concentration of glucose had significant impact on cell growth, while of the added concentration of glutamine had not significant effect on cell growth. Finally, orthogonal design study on three factors related with cell growth:serum, glucose and glutamine concentration was performed. The results showed that serum concentrations are significant factor fort cell growth, whereas glucose and glutamine concentration are only minor factors, the best medium parameters studied were:the concentration of glucose, fetal bovine serum and glutamine was 3.5g/L,20% and 2mM respectively. The optimal seeding density is 1×104 cells/hole. Using the optimized culture medium, GLP-1R/U-2 OS cell line may reach 80% confluence after 24 hours cultivation, and may be in good condition. At the end, the function of cultivated cells was proved. A significant receptor ligand binding reaction can be observed after the addition of the positive control Exendin-4, which is in accordance with a dose-dependent curve. After cost estimates, the production of an experimental 96 cells plate costs about ten RMB in total, and do not need to add G418.In this paper, seeding density, serum type and concentration, G418 screening concentration, initial glucose and glutamine concentration in terms of cell lines cultivation were optimized. Through orthogonal experiment, serum, glucose and glutamine added concentration three factors were optimized, and the optimal culture conditions were obtained, which laid the foundation for the large scale cultivation of GLP-1R/U-2 OS cell lines. |
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