SINGLE CELL MANIPULATION & ANALYSIS

MICROFLUIDIC PLATFORMS FOR GENE EDITING

Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A key step in such studies, is the intracellular delivery of the gene editing machinery. However, most of the current delivery methods such as bulk electroporation or chemical carriers are harsh to sensitive cell types. This often leads to poor viability and low overall efficacy, requiring the use of large starting samples.

Our group focusses on developing novel microfluidic platforms for minimally invasive and non-toxic delivery of molecular cargo into a wide variety of cell types. Two such platforms are described below:

• The Nanofountain Probe Electroporation (NFP-E) System is a nanopipette-based single-cell electroporation method that provides superior cell viability and dosage control compared to traditional methods. The automated system utilizes a convolutional neural network (CNN) to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses (see Video). We demonstrated, using a wide range of cell types and molecular cargos, that the NFP-E system minimally perturbs cells during intracellular delivery. Current work is focused on combining the automated NFP-E with micro-confinements for cell isolation, in-situ CRISPR/Cas9 gene editing, and combinatorial manipulation of single cells for the generation of isogenic cell lines.

BIOPHYSICS OF INTRACELLULAR DELIVERY

Localized electroporation has evolved as an effective technology for the non-toxic delivery of a multitude of foreign molecules into a wide variety of cell types. Although there have been numerous experimental studies on localized electroporation-based delivery, there is a lack of mechanistic understanding of the process, which makes its optimization, for different cell types and molecules, a labor-intensive task based on trial and error. To gain a mechanistic understanding of the process, we developed a multiphysics continuum scale model that predicts the transport of molecules in and out of the cell during localized electroporation. Using the model, we have identified that cell membrane tension plays a crucial role in enhancing both the amount and the uniformity of molecular transport, particularly for macromolecules. We have also identified that there exists an optimum for the electroporation pulse parameters that leads to efficient intracellular delivery.  We have experimentally validated the model predictions using the localized electroporation device (LEPD).

Our current work is focused on investigating the process of localized electroporation mediated intracellular transport at greater detail using computational and experimental approaches across multiple scales. We are using a combination of continuum and molecular scale modeling, live cell tracking on the LEPD, super resolution imaging, and machine learning based parametric calibration. We aim at gaining a mechanistic understanding of the transport process and develop quantitative models of localized electroporation mediated molecular transport.