(439f) Uniform In Vitro Gene Transfection Via High-Throughput Nano-Electroporation Chip | AIChE

(439f) Uniform In Vitro Gene Transfection Via High-Throughput Nano-Electroporation Chip


Zhao, X. - Presenter, The Ohio State University
Wu, Y. - Presenter, The Ohio State University
Gao, K. - Presenter, The Ohio State University
Boukany, P. E. - Presenter, The Ohio State University
Lee, L. J. - Presenter, The Ohio State University

A major disadvantage of most current drug/gene delivery systems is that dosage uniformity at cellular level cannot be achieved, with micro injection being the only exception. The standard deviation of gene copies transfected into individual cells through bulk electroporation, for example, can be as large as 250%. Therefore, the cellular responses to a single transfection will be different from each other and the overall result is an average, making it hard to draw precise and comprehensive conclusion.

We have developed a novel in vitro transfection method, nanoelectroporation (NEP), which provides controlled dosage delivery and thus can achieve uniform transfection at cellular-level. The NEP device has two arrays of micro channels facing each other, with their converging ends connected by nano tubes, which are created through etching gold-coated DNA wire away. Cells are placed at the end of micro channels and the unique dosage control characteristic is attributed to the close contact of cell membrane to nano channel, which condenses electric field and facilitates convection flow rather than diffusion. Being critical for NEP process, a precise cell placement is provided by optical trapping for small amount of cells, yet it becomes increasingly inefficient when cell population increases. Therefore, it is necessary to develop high-throughput NEP to transfect large amount of cells and extend the frontiers of NEP.

Our first design inherits the micro-nano-micro channel setup with greatly reduced cell side channel length allowing only one cell per micro channel, and replaces optical trapping with centrifugal force loading. By placing the chip at the periphery of a rotating disc, cells will spontaneously flow from a remote reservoir to the micro channel inlets and fill the channels with a solid contact. Excessive amount of cells will flow to a drain reservoir. After electroporation, de-loading can be achieved simply by turning the chip around for another centrifuge. Micro fluidic design on loading side and the operation conditions are optimized to ensure the best performance. For example, tethered antibody cell trapping spots can be deposited in front of micro channels to help capture one cell for each micro channel or even achieve selectivity on mixed cell sample. The overall design can be densely integrated so that one chip is capable of handling thousands of cells in several minutes.

Another new design replaces horizontal nanochannels with vertical nanonozzle array on a thin film produced by e-beam lithography or femtosecond laser fabrication. Such nanonozzle array provides similar electric field condensing function as aforementioned nanochannels. With this design, NEP can be performed on Z direction with cells sitting on top of the nanonozzle array and genes delivered from the bottom. The arrangement and distance between nozzles can be modified for optimal transfection efficiency. The nanonozzle array based NEP can transfect millions of cells at same time with controlled dosage delivery.

In this study, plasmid that encodes green fluorescence protein (pGFP) is used as the model gene and its delivery efficiency is characterized in mouse embryonic fibroblasts. With high throughput NEP, we are able to better understand the relationship between delivered dosage and transient/stable expression.