Ning Bao

Electroporation of Cells in Microfluidic Droplets

Droplet-based microfluidics has raised a lot of interest recently due to its wide applications to screening biological/chemical assays with high throughput. Despite the advances on droplet-based assays involving cells, gene delivery methods that are compatible with the droplet platform have been lacking. In this report, we demonstrate a simple microfluidic device that encapsulates cells into aqueous droplets and then electroporates the encapsulated cells. The electroporation occurs when the cell-containing droplets (in oil) flow through a pair of microelectrodes with a constant voltage established in between. We investigate the parameters and characteristics of the electroporation. We demonstrate delivering enhanced green fluorescent protein (EGFP) plasmid into Chinese hamster ovary (CHO) cells. We envision the application of this technique to high-throughput functional genomics studies based on droplet microfluidics.

Recent advances in electric analysis of cells in microfluidic systems.

AbstractMicrofluidics offers an ideal platform to integrate cell-based assays with electric measurements. The technological advances in microfluidics, microelectronics, electrochemistry, and electrophysiology have greatly inspired the development of microfluidic/electric devices that work with a low number of cells or single cells. The applications of these microfluidic systems range from the detecting of cell culture density to the probing of cellular functions at the single-cell level. In this review, we introduce the recent advances in the electric analysis of cells on a microfluidic platform, specifically related to the quantification and monitoring of cells in static solution, on-chip patch-clamp measurement, and examination of flowing cells. We also point out future directions and challenges in this field.n FigureDifferent microfluidic devices applied to electrical analysis of cells

Microfluidic Electroporative Flow Cytometry for Studying Single-Cell Biomechanics

Biomechanical properties of cells yield important information on the disease state of cells such as transformation and metastasis. Screening of cells based on their biomechanical properties provides rapid tools for label-free diagnosis and staging of cancers. However, existent single-cell techniques for measuring biomechanical properties suffer from low throughput (<1 cell/min). This prevents the application of these assays to a large cell population, which produces information with statistical significance. In this study, we applied microfluidics-based electroporative flow cytometry (EFC) that combined electroporation with flow cytometry to study deformability of cells at the single-cell level with a throughput of approximately 5 cells/s. The cell swelling during flow-through electroporation was recorded in real time. We believe that the degree of such swelling was indicative of the cell deformability and the cytoskeleton mechanics. Three cell types (MCF-10A, MCF-7, and 12- O-tetradecanoylphorbol-13-acetate-treated MCF-7) with different malignancy and metastatic potential were tested using our approach. We found that the more malignant and metastatic cell types exhibited more swelling due to higher cell deformability. Furthermore, the disruption of microtubules by colchicine caused substantial change in the EFC results, which confirmed that EFC data strongly reflected the cytoskeletal mechanics. Finally, the cell type with the highest metastatic potential also suffered the most cell death due to the flow-through electroporation treatment, presumably due to the most substantial cell swelling, which could irreversibly rupture the membrane. EFC provides a new method for examining single-cell biomechanics with high throughput. We believe that this technique will be useful for mechanistic studies of cytoskeleton dynamics and clinical applications such as diagnosis and staging of cancers in general.

Microfluidic CARS cytometry

Coherent anti-stokes Raman scattering (CARS) flow cytometry was demonstrated by combining a laser-scanning CARS microscope with a polydimethylsiloxane (PDMS) based microfluidic device. Line-scanning across the hydrodynamically focused core stream was performed for detection of flowing objects. Parameters were optimized by utilizing polystyrene beads as flowing particles. Population measurements of adipocytes isolated from mouse fat tissues demonstrated the viability of microfluidic CARS cytometry for quantitation of adipocyte size distribution. CARS cytometry could be a new modality for quantitative analysis with vibrational selectivity.

Microfluidic electroporation of tumor and blood cells: observation of nucleus expansion and implications on selective analysis and purging of circulating tumor cells

Circulating tumor cells (CTCs) refer to cells that detach from a primary tumor, circulate in the blood stream, and may settle down at a secondary site and form metastases. The detection and characterization of CTCs are clinically useful for diagnosis and prognosis purposes. However, there has been very little work on purging CTCs from the blood. In this study, we systematically studied electroporation of tumor and blood cells in the context of selective purging and analysis of CTCs, using M109 and mouse blood cells as models. Electroporation is a simple and effective method for disruption of the cell membrane by applying an external electric field. We applied a microfluidic flow-through electroporation to process cells with various electroporation durations and field intensities. With duration of 100-300 ms, we found that the thresholds for electroporation-induced lysis started at 300-400 V cm(-1) for M109, 400-500 V cm(-1) for white blood cells and 1100-1200 V cm(-1) for red blood cells. Due to the substantial difference, we demonstrated the selective electroporation of tumor cells among blood cells and the scale-up of the flow-through electroporation devices for processing samples of millilitre volumes. Using Coherent Anti-stokes Raman Scattering (CARS) and fluorescence microscopy tools, we observed the dramatic increase in the size of the nucleus of a tumor cell in response to the applied field. We suggest that the nucleus expansion is a newly discovered mechanism responsible for rapid tumor cell death resulted from electroporation.

A microfluidic cell array with individually addressable culture chambers

Microfluidic arrays of living cells have raised a lot of interests recently due to their potential for high throughput screening of cell-based assays. This report presents a microfluidic cell array with individually addressable chambers controlled by pneumatic valves for cell culture and treatment. There are two modes for the cell array to be operated. In the first mode, different groups of cells are directed into designated chambers for culturing and observation. We demonstrate the delivery and culture of enhanced green fluorescent protein (EGFP) expressing and nonfluorescent Chinese hamster ovary (CHO) cells into specific chambers in the array. In the second mode, the chambers are first seeded with the same cell type and different reagents are delivered to specific chambers for cell treatment. We treat cells in designated chambers with Calcein AM and CellTrace calcein red-orange AM to demonstrate the principle. We envision that this microfluidic cell array technology will pave the way to automated high-throughput screening of biomolecules and drugs based on observing cellular phenotypes and responses.

Genomic DNA Extraction from Cells by Electroporation on an Integrated Microfluidic Platform

The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of the DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ∼36% for Chinese hamster ovary cells) and bacterial cells (up to ∼45% for Salmonella typhimurium) that is comparable to the widely used chemical lysis. The DNA extraction efficiency has dependence on both the electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedures.

A microfluidic device for physical trapping and electrical lysis of bacterial cells

In this letter, we report a simple microfluidic device that integrates the capture of bacterial cells using a microscale bead array and the rapid electrical lysis for release of intracellular materials. We study the retention of Escherichia coli cells with different concentrations in this type of bead array and the optimal electrical parameters for the electroporative release of intracellular proteins. Our design provides a simple solution to the extraction of intracellular materials from a bacterial cell population based entirely on physical methods without applying chemical or biological reagents.

Total Internal Reflection Fluorescence Flow Cytometry

Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells, and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of approximately 100-150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface.

Release of intracellular proteins by electroporation with preserved cell viability.

Extraction of intracellular proteins from cells is often an important first step for conducting molecular biology and proteomics studies. Although ultrasensitive detection and analytical technology at the single molecule level is becoming routine, protein extraction techniques have not followed suit and still call for complete lysis that leads to cell death. In principle, with refined extraction techniques, intracellular proteins can potentially be extracted without killing the cell. In this Letter, we demonstrate that electroporation is capable of releasing intracellular proteins from adherent Chinese hamster ovary cells while preserving the cell viability. By tuning the duration and intensity of an electric pulse, we were able to control the average amount of protein release and the percentage of viable cells after the operation. Our results indicate that a substantial fraction of the cell population was able to release proteins under electroporation and survive the procedure. Interestingly, at the single cell level, the probability for cell death does not increase with more protein release. This work paves the way to extracting and analyzing intracellular proteins while keeping cells live.