Novel Methods For Spatio-temporal Regulation Of Extracellular Chemical Microenviroment With Microfluidics

Cells in vivo are in a complex extracellular microenvironment.For example,many biochemical factors have concentration gradients in spatial distribution and dynamic changes in time.Therefore,the high-fidelity and high-throughput reconstruction and simulation of this dynamic microenvironment in vitro is of key scientific significance for elucidating the complexity of biological systems and revealing the dynamic mechanisms and functions of cell signaling networks.Compared with traditional methods,microfluidic chips have many advantages for studying cell dynamic signals,including miniaturization,automation,integration,high throughput,and low reagent consumption.However,the existing microfluidic methods for simulating the cellular microenvironment,such as concentration gradient generators and hydrodynamic gating technology,can only control the concentration distribution or time dynamics alone.How to simultaneously control the spatial and temporal distribution of chemical perturbation to obtain the dynamic signal response of a single cell with high throughput is still a great challenge.In this thesis,we proposed several new methods of multi-concentration/multi-drug chemical waveform generator combined with hydrodynamic gating based on the microfluidic chip,which accurately and highly controllably simulates the dynamic extracellular microenvironment,and investigates the responses of single cells to different modes of the chemical stimulation.(1)A new platform of multichannel synchronous hydrodynamic gating coupling with concentration gradient generator was developed.The platform was able to generate a series of chemical waveforms with different amplitudes(linear concentration gradients)and different frequencies(different stimulation times and buffer times)to complete multiple sets of experimental studies in parallel,thereby increasing the throughput of analysis.The platform used He La cell as the research object,applied multi-mode concentration gradient stimulation,and successfully obtained the dose-response curve of Adenosine 5’-triphosphate(ATP)and histamine,as well as the combined response curve of the two.(2)A discretionary multi-concentration synchronous chemical waveform generator combined with a single-cell trap array was proposed to study single-cell dynamic signals.Because the system contained four independent injection ports,linear concentration gradient,exponential concentration gradient or any required concentration gradient could be obtained.The cell trap array set in the cell channel was able to quickly capture single cells,making the dynamic signal acquisition of suspended cells a reality.In addition,the experimental preparation time was significantly reduced,and the operation was more convenient.Using this platform,we successfully studied the single-cell dynamic signal response of Jurkat T cells stimulated by ionomycin waveforms under linear and exponential concentration gradients.(3)A time-coded multi-concentration chemical waveform generator was designed to study the dynamic signaling of single-cell array.On the basis of generating arbitrary concentration gradients chemical waveforms,the system can also perform sequential coding of drugs,and the frequency is highly controllable.The maximum frequency of the platform was 1 Hz,and the same cell could be stimulated at multiple frequencies.He La cells was used as a research model,and pulse stimulation and continuous stimulation of different frequencies in different sequential coding modes were successfully achieved.(4)A multi-concentration chemical waveform generator with high temporal and spatial controllable,and adjustable concentration was further developed to study the dynamic response of cells.The system could not only generate multiple concentration combination waveforms with different timing codes and different frequencies,but also realize the coding of multiple concentration in the spatial distribution,which was "spacetime coding".Through this platform,not only changes in the cell response to different stimuli in the time dimension,but also the differences in the response of cells to different stimuli in the spatial dimension could be observed.We used He La cells as a research model,and successfully achieved pulse stimulation and continuous stimulation of different frequencies in different temporal and spatial coding modes.In summary,we proposed four new methods of different multi-concentration chemical stimulation microfluidic platform to simulate the dynamic extracellular microenvironment and apply them to cell signal analysis.The established method has the advantages of high throughput,high precision,high temporal and spatial resolution,and low error.We believe that it will provide new strategy for single-cell signal research and drug screening.