Cell-based screens are widely used across biology and biotechnology to associate genetic programs with outputs of those programs (i.e., phenotypes). As diverse and large-scale molecular libraries become available, the need increases for instruments capable of screening large populations of cells and separating those with phenotypic differences. Methods for screening large populations are currently limited primarily to growth, or require robotics and other expensive instrumentation. There is increasing evidence that the electrical properties of cells confer useful phenotypic information, as judged by ~20 yrs of separations based upon dielectrophoresis. However, existing electrical separation devices are not suitable for screening due to a variety of issues, including batch operation that limits throughput, sensitivity to particle size that limits specificity, and deleterious particle-particle interactions that limit both throughput and specificity. The research supported by this grant will enable the development of a microfluidic cell separation technology that separates cells based upon their electrical properties, specifically their polarizability, and is ideally positioned for large-scale screens. The instrument consists of a microfluidic device that flows cells through a micrometer- width channel with a liquid conductivity gradient, and separates them along that gradient by applying an electric field from embedded electrodes, resulting in the cells attaining a position that is dictated by their electrical properties. The first step in the development of the instrument is to engineer a device that integrates valves for recovery of cells and conductivity generators for automatic control of the liquid conductivity. Then instrument will be optimized to increase resolution, throughput, and specificity to make it suitable for large-scale screens, after which the team will undertake a screen of a yeast library to validate the instrument's performance. By disseminating this instrument through collaborations, publications, and via providing microfluidic devices to the community, this instrument will dramatically enhance the ability to undertake high-throughput screens. The project will train undergraduate and graduate students, as well as interact with the Women's Technology program which brings high-school girls to MIT for a four-week program, and a research program involving local Boston high-school students in a week-long project.
Genetic or phenotypic screens require the ability to select a small fraction of targeted cells from a large, heterogeneous background. One of the largest challenges in applied biology is to perform these screens in a way that possesses both high throughput and high purity. Our approach to this problem is to develop a new equilibrium method, called iso-dielectric separation (IDS), for sorting cells based upon electrically distinguishable phenotypes. Equilibrium methods sort cells according to their intrinsic properties, and thus do not require that any labels be developed and applied to the targeted cells. Furthermore, they have the potential to be both preparative and analytic, meaning that they are able to provide both separation as well as quantitative information about the population of cells. The IDS device developed in our lab exhibits all of these characteristics, and offers the additional advantage of operating under continuous-flow. This enables high throughput, label-free, analytic and preparative separations capable of resolving multiple sub-populations of cells from heterogeneous backgrounds in a microfluidic format. In terms of outcomes, over the course of the project we re-engineered and improved the IDS device in order to develop an automated system that allowed cell characterization with minimal user intervention. The major biological outcome was that we performed a genetic screen of the yeast deletion library using the device, providing the first genome-wide map of how genotype maps to electrical phenotype. In terms of human personnel, we trained graduates students and undergraduate researchers in how to perform research at the interface between engineering and biology.
