| The capability of cells for micro-chemical environment recognition and response in cell signal transduction,plays an important role in cell development,tissue repair and immunity.As the development of microfluidics technology,in vitro simulation or reconstruction of the extracellular complex environment in microfluidic chip for cellular chemical stimulation have gradually become one of the most effective way to investigate cell dynamic signaling,such as signal transduction and cell-cell communication.Existing chemical perturbations were implemented via perfusion systems.However,their limitations are impeding further development,especially the drawbacks in lack of precision for spatial and temporal control of chemical stimuli.In this thesis,combining innovative microchannel design with external precision control,we have proposed five unique microfluidic methods for different applications: single cell high spatial and temporal local stimulation,high-precision single-cell stimulation,multiple waveform stimulation,high-throughput chemical stimulation and so on.(1)A new microfluidic strategy was developed for investigating the intercellular propagation of Ca2+ waves based on flexible hydrodynamic gating technique with temporally and spatially controlled noninvasive laminar flow.Clusters of target cells were seeded at stimulation microchannel with the hydrostatic pressures and selective digestion.Localized stimulation of the target cells with ATP successfully induced the propagation of intercellular Ca2+ waves among a population of adjacent contacting cells.Using this microfluidic method,we further investigated inhibition of gap junction-mediated intercellular communication by octanol,which demonstrated that this method is reliable for analyzing gap junction-mediated intercellular calcium signaling in real time.(2)A new microfluidic approach,termed as gated pinched-flow(GPF),was developed based on the multiplexed positive pressure control system.Theoretical model for GPF was then established and further validated by both numerical simulations and experiments.Based on the high precision repetitive stimulation,repeated intercellular calcium waves of the same cluster cells were also successfully observed.Localized stimulation of the target single cell located in different space of the microchannel was also successfully realized with high temporal resolution(<50 ms).(3)A precision guided microfluidic chemical stimulation strategy was further reported for cell dynamic signaling with high spatial(~12 ms)and temporal resolution(infinitesimal pinched width in theory and lateral shifting resolution of 16.3±2.4 μm/0.1kPa).Theory model was established and validated by both numerical simulations and experiments.The multi-channel pressure control system was constructed to automatically implement chemical perturbations with various stimulation times,relaxation times and number of cycles.The width and the position of the chemical microflow could also be precisely controlled with high reliability and accuracy.Investigations of intracellular and intercellular calcium signaling were successfully demonstrated using the proposed method.(4)A chemical arbitrary waveform generator was further developed for dynamically investigating the cell signaling,and also further validated by both numerical simulations and experiments.This platform was able to realize impulse chemical waveform with frequency higher than 10 Hz,and continuous chemical waveform with frequency higher than 0.2 Hz.Fast generating impulse chemical waveform with different concentration of drugs provided a novel method to obtain dose response curve of same single cells rapidly.Upon the continuous chemical waveform stimulation,stimulated cells can response to different amplitude of the input signal,however,can only response to the low frequency of the input signal with high fidelity.(5)Finally,we have developed a microfluidic single-cell dynamic cytometry strategy for cell signaling analysis by creatively applied dynamic cell trapping and releasing automatically base on the novel multi-channel pressure control system.Dose-response curves and EC50 values for He La cells treated with adenosine 5′-triphosphate(ATP),histamine(HA)and acetyl choline(ACH)were successfully obtained in single-cell resolution,which is also potentially suitable for other agonists or antagonists investigation.Single-cells dynamic signaling were further implemented by sequential stimulation or simultaneous stimulation.In addition,simultaneous stimulation to two different cells,He La and NIH 3T3 cells,have also proven to be attainable or practical.These results revealed that this microfluidic method provides a versatile means for probing single cell signaling,which is potentially useful in chemical biology,cell biology and pharmacology studies.In summary,we have demonstrated five unique microfluidic methods for cellular chemical stimulation with high spatial and temporal resolution,high-precision and high-throughput,which will open up new avenues for cell dynamic signaling and the drug screening. |
