Preparation Of Hydrogel Microtubes Based On Microfluidic To Study The Migration Behavior Of Tongue Squamous Carcinoma Cells

Tongue squamous cell carcinoma（TSCC）is one of the most common types of head and neck cancer with a high morbidity and mortality rate.Due to the abundance of blood vessels and nerves around the tongue,as well as its flexibility of movement,TSCC is susceptible to early spread and lymph node metastases,even distant metastases.The 5-year survival rate of advanced TSCC is approximately 50%.Non-steroidal antiinflammatory drugs（NSAIDs）are a group of medication with diverse structures and similar effects,clinically treating rheumatic diseases and reducing inflammation.NSAIDs are widely used for antipyretics,analgesics,platelet aggregation inhibitors,and the prevention of strokes.Clinical and epidemiological studies have revealed that frequent use of aspirin and other NSAIDs could reduce the risk of several cancers（colon cancer,stomach cancer,breast cancer,and lung cancer）,and improve survival rate after diagnosis.It has been confirmed that aspirin could reduce migration and invasion of TSCC.There is a wide variety of NSAIDs,and whether all of them have anti-tumor migration has not been studied yet.Objective: In order to screen NSAIDs with anti-tongue squamous cell migration in vitro,it is necessary to construct a new 3D dynamic in vitro screening model to make up for the defects of the traditional static 2D cell migration model.The successful construction of an in vitro drug screening model has screened out NSAIDs with excellent anti-migration ability of TSCC,which provides a theoretical basis for preclinical experiments.Methods:1.Design a microinjection device to prepare hollow calcium alginate microtubes,explore the influence of preparation parameters on the microtube structure,and select the best preparation parameters.Optical microscopy,scanning electron microscopy（SEM）and confocal laser microscopy（CLSM）to characterize the microtube structure.The vertical orientation of CAL-27 cell suspension was introduced on the basis of the microtubule structure,the optimal preparation parameters were selected,and the cell microtubes were characterized by fluorescence microscopy and CLSM.2.The alginate hydrogel was biomodified by adding natural extracellular matrix component（fibrin）,cultured for 72 h in vitro,and the biocompatibility of the hydrogel was verified by Calcein-AM/PI live/dead cell staining and cytoskeleton staining.3.Three NSAIDs（aspirin,indomethacin,nimesulide）were selected,the cytotoxicity of the drugs was verified by CCK-8,and the appropriate drug concentrations were screened for cell migration experiments.The effects of three NSAIDs on the migration of CAL-27 cells were evaluated by scratch assay and cell microtubes assay,respectively.Results:1.The structural characterization proved the hollow structure of the microtubes.The measurement results of the inner,outer diameters and the thickness of microtubes prepared under different parameters showed that there is a linear relationship between the preparation parameters and the microtube structure.Based on the principle of hydrodynamics,the orderly arrangement of cells is realized.2.The observation of in vitro cultured cells,live/dead cell staining and cytoskeleton staining showed that adding fibrin can improve the biological activity of calcium alginate hydrogel.CAL-27 cells maintained spherical and showed a clear spreading.It indicated that the cell 3D culture model was successfully established.3.The results of cytotoxicity experiments showed that the cytotoxicity of three NSAIDs（aspirin,indomethacin,nimesulide）was positively correlated with drug concentration and duration of action.The scratch assay results showed that all three drugs had the ability to resist the migration of CAL-27 cells in vitro.The cell microtube assay results were the same as the scratch assay,and the effect of nimesulide was more significant.Conclusion: Using the designed microfluidic device,the in vitro 3D culture model of CAL-27 cells was successfully constructed and three NSAIDs（aspirin,indomethacin,nimesulide）with anti-migration effect of CAL-27 cells were successfully screened by the cell microtube model.