Construction And Application Of Microfluidic Based Multiplex Single-cell Array

In recent years, along with the intensive study of the cell heterogeneity, single-cell analysis technology becomes one hot topic in the life science research. Conventional bulk experiments normally ignore the differences among individual cells within the same cell population due to the average of results. Whereas single-cell analysis could observe and study these ignored cell heterogeneity and be helpful for reveal the critical functional information from individual or several cells. In order to realize rapid, convenient and automatic parallel analysis of cell function, heterogeneity and intercellular interaction at single-cell level, simple and reliable single-cell analysis technology becomes one issue which is urgently needed to be study and addressed. Microfluidics, merges of physics, biology, chemistry and computer science, shows its particular advantages such as precise manipulation of micro-scale fluids and particles, easy accessibility, integration and high-throughput for single-cell analysis. And it has already been widely applied in the area of single-cell analysis. In this study, we combined the pneumatic microvalve arrays(P?VAs) and controllable air plugs in the microchannels with the microfluidic foundation to develop and demonstrate two methods for generation of multiplex single-cell array with multiple cell types, respectively. These methods are convenient, fast, simple, reliable, universal and scalable. They could be utilized for study of high-throughput single-cell drug screening, tissue engineering, neural networks and intercellular interaction at single-cell level. The results obtained in the present work are as follows:1. In this study, by combining pneumatic microvalve arrays(P?VAs) and hydrodynamic single-cell trapping sites, we developed a method for the generation of multiplex single-cell array with multiple cell types. Precisely controlled by actuated pressure, the P?VAs could form reversible microbarriers in the fluidic channel to guide different types of cells being stepwise trapped into the corresponding trapping sites to form multiplex single-cell array. According this method, we designed a microfluidic device which was intergrated with two sets of P?VAs. A multiplex single-cell array with two cell types was successfully generated in this device. The general protocol also was optimized in this device. The application of non-adherent K562 cells and adherent cultured A549 cells in the multiplex single-cell array proved the universality of this method for the usage of different cell types.2. Based on theoretical analysis and computational simulation results, we designed another microfluidic chip which was integrated with four individual sets of P?VAs for generation of multiplex single-cell array with three cell types. Using the general protocol optimized in the prototype chip, a multiplex single-cell array with three cell types was successfully generated. The scalability of this method, which means that multiplex single-cell array with more cell types could be generated by designing more multiplex P?VAs and operation steps, was proved. Finally, the generated multiplex single-cell array was utilized for the analysis of cellular esterase heterogeneity of three cell types at single-cell level. The results indicated that the esterase heterogeneity was not only exited among different cell types, but also exited among individual cells within the same population.3. In this study, we utilized the hydrophobicity and gas-permeability of PDMS(the common material for microfluidic device preparation) to developed another microfluidic method for convenient and fast generation of multiplex single-cell array. Precisely controlled by injected flow rate, air plugs would be controllably created and eliminated in different bypass channels to function as microvalves. Combining the bypass channels which could function as single-cell trapping sites, the multiplex single-cell array with A549 cells and K562 cells was rapidly generated. The universality of this method for the usage of different cell types was then proved. In the meantime, according to the cell spacing analysis in the array, the stability, controllability and predictability of cell positions in this multiplex single-cell array was also confirmed. It would be helpful for development of automatic data processing method.4. Using the air plugs assisted multiplex single-cell array generation method, we redesigned the main channel width to make sure the distance between every two adjacent bypass channels was the same. And the dimensional distribution of two types of bypass channels was reorganized to generate multiplex single cell patterning arrays with various patterns. The results indicated that this method was scalable for application of tissue engineering, intercellular paracrine communication and neuron networks generation with particular permutation and combination of different bypass channels.5. In this study, we also designed a microfluidic chip for convenient and fast generation of heterotypic single-cell pairing array. Based on the air plugs assisted multiplex single-cell array generation method, we designed the single-cell pairing site which organized two different bypass channels into one cell trapping site. Using different injected flow rates, cells of different types was stepwise trapped into the same site to form the heterotypic single-cell pairing array. It would be promising for intercellular interaction study at single-cell level in the future.