Experimentation And Modeling Of Intracellular Calcium Responses During The Cellular Deformation Processes Of Single Cells Passing Through Narrow Microfluidic-channels

The cellular mechanical properties denote the cellular deformation characteristics under mechanical stimuli.The cellular mechanobiological properties mean the cellular characteristics of biological responses to mechanical stimuli.In many studies,the cellular mechanical and mechanobiological properties have been regarded as convenient and direct markers,by which the occurrences of diseases and the changes of cellular states could be indicated.The cellular deformation processes of single cells passing through narrow microfluidic-channels and the intracellular calcium response processes induced by it contain abundant information about the cellular biomechanical and mechanobiological properties.In recent years,many studies have pointed out that the abnormal intracellular calcium responses to mechanical stimuli,play important roles in the occurrences and developments of certain diseases.Therefore,it is of significance in revealing pathogenesis and developing new diagnosis methods to measure the cellular deformation processes of single cells passing through narrow microfluidic-channels and the intracellular calcium response processes induced by the deformation processes.With the advantages of manipulating micrometer-scale or nanometer-scale liquid,fine microstructure and easy operation,the microfluidics provides powerful tools for measuring the cellular deformation processes and the intracellular calcium response processes of single cells passing through narrow microfluidic-channels.However,the existing studies on measuring cellular deformation in microfluidic-channels did not observe the intracellular calcium response processes of single cells under mechanical stimuli,so they cannot offer more abundant biomechanical and mechanobiological information for the analyses of single cells.To address these issues,this dissertation designed and fabricated a high-throughput microfluidic chip for trapping single cells.By using the chip,the deformation and intracellular calcium response processes of single cells were sufficiently monitored,and the cellular mechanical and mechanobiological properties were measured and analyzed.A mathematical model was built to quantitatively analyze and explain the experimental phenomena.The main research contents and results of the present dissertation are:First,the present dissertation optimized the structure of single cell trapping unit and parallelized it based upon the principle of fluid mechanics,designed and fabricated a chip for trapping single cells in a high-throughput manner.Experiments validated that the proposed chip has the ability to trap single cells and measure their apparent viscosities in a high-throughput manner.According to the cellular deformation in the trapping channel monitored in experiments,this dissertation relaxed the assumption for the fixed cellular apparent viscosity and built a drop model,then derived an equation for calculating apparent viscosity,which contain the full viscosity information throughout the deformation process.Experiments validated that the apparent viscosities of Hela cells(Human cervical cancer cells)and human umbilical vein endothelial cells can be effectively measured by using this equation.Second,by using the chip,the intracellular calcium experiments were carried out and the intracellular calcium responses of single cells while squeezing through trapping channel were measured.In the experiments for the Hela cells and human umbilical vein endothelial cells,the present dissertation discovered three patterns of intracellular calcium responses,namely the short,plateau-like and two-peak calcium response patterns.This dissertation took the two-peak calcium response curve apart into six characteristic parameters and acquired these parameter values of Hela cells and human umbilical vein endothelial cells.As the statistical results demonstrated,there is one characteristic parameter where Hela cells exhibited significant difference from human umbilical vein endothelial cells.The present dissertation further probed the effects of the cytoskeleton on this calcium response process.Third,according to the changes of cellular morphological and mechanical states while the cell squeezed through the trapping channel,the present dissertation developed a mechanobiological model for the intracellular calcium responses of single cells,quantitatively analyzed and explained the mechanisms of the three patterns of intracellular calcium responses.Firstly,the present dissertation applied the Young-Laplace equation to the cellular surface tension,computed the three-dimensional cellular surface area throughout the cellular deformation process based on the numerical simulation and solved the cellular surface tension.Then,the dissertation built the cellular surface tension activating intracellular calcium response model and integrated well with the existing model in literature for the intracellular calcium homeostasis.The numerical simulation results showed that the three patterns of intracellular calcium responses simulated by the model agreed with the experimental data well.By using the model,the dissertation probed the mechanisms of the three patterns of intracellular calcium responses and made some theoretical explanations.To summarize,this dissertation designed and fabricated a high-throughput microfluidic chip for trapping single cells,by using the chip,this dissertation fully monitored the deformation and intracellular calcium response processes of single cells,extracted the cellular apparent viscosities and calcium response characteristic parameters,and built a dynamic model for analyzing the new patterns of intracellular calcium responses.The proposed microfluidic chip and methods for analyzing the mechanical and mechanobiological properties of single cells provide new ideas for in vitro assaying single cells in a high-throughput manner.