MEMS-based Massively-parallelized Mechanoporation Instrumentation for Ultrahigh Throughput Cellular Manipulation

Doctor of Philosophy, Graduate Program in Mechanical Engineering
University of California, Riverside, December 2012
Dr. Masaru P. Rao, Chairperson

Many applications in cell biology, genetic engineering, cell-based therapeutics, and drug discovery require precise and safe methods for introducing membrane-impermeable molecules into cells. This can be implemented satisfactorily by  microinjection. However, disadvantages of traditional  manual  microinjection include  high degree of operator skill, low throughput and labor-intensiveness. Many studies have focused on developing automated and high-throughput  systems  for  microinjection  to  address  these  limitations.  However,  none  have  provided  sufficient throughput for applications such as ex vivo cell therapy, where manipulation of many millions of cells is required. Herein,  we  propose  an  ultrahigh  throughput  (UHT)  mechanoporation  concept  that  seeks  to  address  these  limitations.  The  mechanoporation  device  is  a  massively-parallelized  MEMS-based  platform  for  passively  delivering molecules into living cells via mechanical cell membrane penetration. Studies focusing on device design, fabrication and validation at the proof-of-concept level are presented in this dissertation.

Detailed system concept and design is introduced, which integrates functions of cell transfer, capture, penetration and release into a single piece of instrumentation using a  microfluidic approach. System operating parameters are analytically analyzed and numerically simulated. Results from these studies agree with previous studies by others in related applications, and suggest reasonable operation feasibility without detrimental effect on cells. Those estimated operating parameters also provide basis to develop test models in practical cell studies. The device fabrication utilized conventional silicon MEMS technologies, and we successfully produced millimeter-scale device chips containing an array of ten thousand hemispherical capture wells with monolithically integrated solid penetrators. A flow circuit system involving a syringe pump, pressure transducer, and fixture set supporting the device chip was developed,  and  preliminary  functional  testing  was  carried  out.  Device  validation  tests  using  K562  cells  obtained about  15%  average  penetration  efficiency  of  live  cells  after  manipulation.  Subsequent  testing  with  fluorescent beads  and  Mouse  Embryonic  Fibroblast  (MEF)  cell  identified  several  key  issues  responsible  for  the  lower-than-expected efficiency, thus suggesting that improved performance may be possible with further system and operation optimization.

The  UHT  mechanoporation  device  developed  in  this  effort  shows  promise  for  providing  an  efficient  and  safe method  for  introducing  membrane  impermeable  molecules  into  cells  with  ultrahigh  throughput.  Moreover,  these studies also represent key steps towards our long-term goal of developing instrumentation capable of UHT cellular manipulation  via  active  microinjection.  This  new  instrumentation  will  have  broad  potential  for  advancing  understanding of fundamental cellular processes, as well as facilitating clinical translation of cell-based therapies.