Study Of Key Technologies For Cell Manipulation In Microfluidic Chips

Cell manipulation technologies represented by cell separation,screening,capture,and positioned culture have great demands and significant value in biomedical applications such as in vitro diagnostic and basic biology researches.Microfluidic technologies have been widely used in cell manipulation thanks to their ability of manipulating fluids and particlesprecisely on a microscale.In this thesis,cell manipulation technologies based on microfluidic chips were studied and two key manipulation technologies,cell separation and positioned cell culture,were focused as their significance in both biomedical researches and applications.Several researches,including inertial focusing of cells in rectangular channel,inertial microfluidic-based cell separation from human blood utilizing co-flows,quantitative cell concentration based on inertial focusing,cell patterning in a microfluidic chip utilizing paired microwells and protein patterns,and microfluidic technology for separation and detection of circulating tumor cells form human blood,were carried out successively,in order to provide microfluidic tools and platforms for cell manipulation in biomedical applications and researches.The main work is summarized as follows:(1)Principles of inertial focusing of cells in rectangular channel with a low aspect ratio were studied first.The influence of particle size and flow rate on the quality of inertial focusing were investigated and high-quality focusing of 18.7?m particles and Hep G2 cells in the channel was achieved.Cell separation technologies based on inertial focusing in a low aspect ratio rectangular channel has certain problems when they are aplied in separating target cells from high-concentration suspension of various cell types,for example,separating circulating tumor cells(CTCs)form human blood.To solve these problems,a co-flow method was utilized.This method utilizes the mechanism of inertial focusing,shear-induced diffusion effect and elastic force in the blood to achieve the separation of target cells from blood cells in high concentration in a low aspect ratio rectangular micro channel.By investigating and optimizing the dilution ratio of blood and the flow rate of the co-flow,18.7 ?m particles and Hep G2 cells were successfully separated from whole blood or blood with low dilution ratio.The separation efficiency of 18.7 ?m particles from whole blood reached 82.5 ± 10%with a throughput of about 5.6 × 108 cells/min;the separation efficiency of particles from 2-fold diluted blood reached 94.1 ± 1.8%with a throughput of about 2.8 × 108 cells/min;for Hep G2 cells,the separation efficiency from 2-fold diluted blood reached 90.8%± 2.4%with a throughput of about 2.8 × 108 cells/min.This technology is competent to separate target cells directly from whole blood or blood with low dilution ratio,while maintaining a high throughput,which is very useful in the separation of CTCs from human blood.(2)Based on inertial focusing in rectangular channels,a microfluidic chip for quantitative separation and concentration of cell suspension was developed by selectively adjusting the flow resistance of the outlet channels.The influences of flow resistance of the outlet system on the trajectory and focusing quality of particles in the channel was investigated,and quantitative concentration of particle or cell suspension was achieved by adjusting the flow resistance ratio of the outlet system.Cell suspension with a wide cell concentration(up to 1.8 × 106 cells/mL)was successfully concentrated by this chip,and no obvious cell damage was induced according to the cell viability test and culture test.Compared with the traditional cell concentration method based on centrifuges,this technology shows advantages such as higher suspension quality,lower cell lost and damage,shorter processing time,higher reproducibility,lower operation requirement for the operator,and provides a convenient microfluidic alternative for cell concentration adjusting that is greatly demanded in both biology and clinical researches.(3)Utilizing paired microwells and protein patterns,a microfluidic chip for positioned cell culture at single cell level without physical nor chemical constraint was presented.By optimization of the procedure of micro contact printing(p.CP),microwells for cell capture and protein patterns for cell culture were paired precisely in an enclosed microchannel.Cells were first captured at the single cell level by microwells and then released and patterned on the protein patterns undergoing gravity.HeLa cells and SGC-996 cells were patterned and cultured successfully in the chip for up to 6 days,and their growth,migration,proliferation processes were observed and studied.Our chip is free of physical and chemical constraint which are applied to cells in the former technologies for positioned cell culture and provides cells with growth environment closer to the real condition.We envision its potential application in researches such as cell migration,neuron network formation,drug screening,single cell analysis,cell-cell interaction and so on.(4)By combining technologies of cell separation,cell capture,cell culture and immunofluorescent staining characterization,a microfluidic chip for the detection of CTCs from human blood was developed.Hela cells were used to optimize the processes of cell capture,culture and immunofluorescent staining in the chip.Based on this,CTCs were separated and captured from blood samples donated by patients with liver cancer,and CTC detection in the chip was achieved by immunofluorescent staining of CK and CD45 antibody simultaneously.This technology eliminated high fold dilution and other complex pretreatment to the blood samples,while maintaining a high throughput,which is promising to provide a high-efficiency microfluidic platform for the separation,capture and detection of human CTCs.