Improving Separation of Differentiated Embryonic Stem Cells with a Microfluidic Device

Abstract

Differences in cell cytoskeletal stiffness can be utilized to sort differentiated embryonic stem cells into distinct populations through the use of a microfluidic device. An initial microfluidic system was developed and proven by previous researchers to sort cells1. Modifications to this microfluidic system were made and aspects have been improved to increase the efficiency of moving large numbers of cells through the device for use in PCR. Preliminary data from the updated microfluidic system shows that vertical integration of cells and increasing cell count along with increasing length of experiment show the greatest promise moving forward. Polymerase Chain Reaction (PCR) is a process to analyze differences in gene expression for genes which produce proteins which may have an effect on cell stiffness. Once total cell throughput is improved to large enough numbers, PCR was then completed in a separate project on the two populations differences in levels of gene expression were compared. The genes to be tested are VIM2, ACTN13, and LMNA4, along with GapDH as a constant, which previous research suggests produce proteins which may play a role in cell stiffness. These genes would therefore have different levels of expression, as measured by PCR, in cells with different levels of cytoskeletal stiffness. Improving the microfluidic separation system will also allow for future use in research, and for commercial use in the field of artificial organ generation, by collecting larger populations of pure populations of stem cells. A system was developed to generate large quantities of cells for the graduate student advisors’ other research endeavors along with other graduate students working on similar projects. Knowing the genes that alter cytoskeletal stiffness will allow for numerous avenues of opportunity, but will greatly change the way populations of cells are isolated and purified.