Research On Cell Manipulation And Separation In Microfluidic Systems

Accurate and high-throughput cell separation is a key technology in molecular and cell biology,biotechnology,and medicine.At present,with the development of micro-fabrication techniques,microfluidic technology has become an important research field in the advancement of novel biological analyses and the integration of cell biological functions.With the increasing cancer incidents,efficient methods for cancer cell separation play a very important role in the early diagnosis and prognosis of cancer.Microfluidic technology has many advantages for the separation of cancer cells,such as low cost,small sample volume,and high-throughput sample processing.High-throughput microfluidic cell separation usually relies on a variety of physical principles,such as dielectrophoresis,hydrodynamic forces,acoustophoresis,and magnetophoresis,etc.Among these methods,cell separation based on acoustophoresis and magnetophoresis have attracted extensive attention because of their high efficiency and simplicity in structure.Therefore,this thesis was focused on developing new cell separation methods using these two physical principles.In Chapter Two,microparticle alignment and cell separation with acoustic microfluidic chips were studied.A microfluidic chip with a piezoelectric transducer that generated ultrasonic standing waves?USWs?was fabricated to control the location of particles.In addition,three representative particles were prepared,including poly?lactic-co-glycolic acid??PLGA?microspheres,silica-coated magnetic microbeads?Si O₂ magnetic beads?,and polydimethylsiloxane?PDMS?microspheres,and investigated the responses of these particles to USWs in a microfluidic chip.Results showed that depending on the sign?positive or negative?of the acoustic contrast factors of the particles,they displayed different alignment behaviors by moving towards the pressure nodes or antinodes of an USW,respectively.Specifically,PLGA microspheres and Si O₂ magnetic beads?positive acoustic contrast factors?are able to align in the middle of the microfluidic channel,while PDMS microspheres?negative acoustic contrast factor?aggregated along the side walls of the channel,which is beneficial for cell capture.Then the surface of PDMS microspheres was modified with collagen by physical adsorption.The collagen was utilized to attach MC3T3-E1 cells to the elastomeric microspheres so as to separate cell-microsphere complexes from non-adherent cells.Further results demonstrated that the functional PDMS microspheres with a negative acoustic contrast factor can be used to capture and transport cells to the pressure antinodes via acoustic radiation forces in a microfluidic chip.Cell viability tests revealed that the ultrasonic manipulation didn't not exert any harmful effect to the cells,and can be safely used for cell separation and detection.In Chapter Three,magnetic beads were functionalized with hyaluronic acid?HA?for capture and separate cancer cells in microfluidic chip.The surface of aminated Si O₂ beads were modified with hyaluronic acid?HA?,then characterized by TGA and Zeta potential measurements.He La cells over-expressing CD44 receptors were incubated with HA-modified Si O₂ microbeads,drawn into the microfluidic channel,and separated from the cells not binding with the microbeads under an external magnetic field.Results showed that using HA modified Si O₂ magnetic beads to enrich cancer cells is of high specificity and feasible.By exerting magnetic field around the microfluidic chip,the magnetic separation technique does not cause damage to the cells.And the strong binding between the cells and the modified magnetic beads significantly enhances the separation efficiency.The whole magnetic separation process is high throughput and fast.In conclusion,we studied cell separation methods based on different physical principles in microfluidic chips.The first method is to use acoustic radiation force from USWs on an elastomeric particle carrier to achieve cell separation.The second method is to use functional magnetic beads to separation cells in magnetic field.The research in this thesis holds a great potential for the development of cell separation carriers of high efficiency and low cost.