The Microfluidic BIOCHIP Advantage for Single-cell DRUG DISCOVERY

BY PAUL C.H. LI, PHD

The Purpose of Cellular Analysis
The cell-based assay has been an important in vitro test in preclinical trials in the later stages of the drug-discovery process. In preclinical trials, before the content-rich in vivo animal tests, high throughput in vitro studies must first be performed. These in vitro tests involve two different stages of high throughput screening (HTS), such as affinity-based assays and cell-based assays. The cell-based assays are considered as high-content screening (HCS) and they partially alleviate the burden in the animal studies required for drug discovery. Bearing in mind that there are cost factors and cell heterogeneities in cell-based assays, there are additional advantages to using fewer cells, or even single cells in drug testing.

What the Microfluidic Biochip Offers
Recently, biochemical studies have benefited from the microfluidic chip technique.1,2 In particular, some of these studies have been conducted on biological cells retained within a microchip.3-9
The microchip has all of the advantages of microscale analysis, including reduction in device size, less reagent consumption and simplified waste disposal. In addition, there are multi-channel capabilities to increase sample throughput. Besides, there is potential integration with various unit operations. In cell-based tests, there are important unit operations such as cell manipulation and retention, reagent delivery without flushing away cells, and background correction to account for changes due to reagents.

Why Perform Single-cell Analysis?
Cell heterogeneity may prevent us from identifying cellular changes upon treatment with reagents. Our studies have revealed the heterogeneity observed in a commonly used cell viability test: enzymatic hydrolysis of fluorescein diacetate (FDA).10 Fluorescein was generated from FDA by the intracellular carboxyesterase in viable cells.11 We found that the test only worked on dormant yeast cells (cells directly taken from a culture plate) to produce a response, but did not generally work for budding yeast cells. Nevertheless, after subjecting them to nutrient depletion, we obtained a response.5,6 This indicates that the test applied to a batch of cells may only work for cells under certain conditions, and this may obscure the observation and data interpretation if the cells that work are small in number. Since the search for medicinal components of clinically successful Chinese herbal medicine by HTS has been elusive, we believe that cell heterogeneity could be an important factor that results in missing the hits.

Challenges and Breakthroughs in Single-cell Analysis
Most recent microfluidic studies were performed on groups of single cells, with only a few performed on an individual cell.3-9 Even so, given the technical challenges in cell retention during cell selection and during reagent delivery, microchip single-cell experiments were generally limited to only one type of stimulus or with only one repetition, or over a short period of time. Even though the problem of cell retention is solved, another challenge arises: cellular measurements are usually obscured by background changes upon switching between different reagents.
These challenges have been alleviated by the invention of a microfludic biochip that consists of a cell-retention structure (Fig. 1). The structure consists of a V-shaped barrier — with a stretch in its middle — opposite to a reagent flow channel.5,6 In addition, the 3-D fluid flow naturally present in the cell retention structure was ingeniously employed to select and retain a single cell (Fig. 2). Here, a rabbit cardiac muscle cell was selected, following a method superior to those previously reported, using physical U-shaped cell barriers.3 Furthermore, the fluid flow was used to retain a single cell and to scan or shuttle it through a fixed detection window (Fig. 3).

How Does the Chip Work?
By 3-D liquid flow control, scanning the cell back and forth through a fixed detection window could generate a series of peaks representing the cell fluorescent signal (Fig. 4). It is shown that the fluorescent intensity of a yeast cell, as given by the peak height, began to rise due to increased FDA metabolism (to generate fluorescein). Background correction can also be achieved using the baseline signal of the series of peaks.
A bonus in the single-cell experiment is the possibility of observing the cell while it is growing, that is, during single-cell culture in the chip.5,6
With microfluidic cell retention, we present single-cell measurements due to multiple stimuli. This provides sufficient information regarding single-cell biochemistry unattainable by measurements performed on an ensemble of cells. Specifically, we initiated the influx of a model substrate (FDA) in a single yeast cell, and observed the formation and efflux of the metabolite (fluorescein) in response to multiple stimuli over a long period of time.5,6 Dynamic studies of FDA metabolism in yeast have been performed by flow cytometry,12 but these studies could not completely reveal the complexity of this complex influx- hydrolysis (by esterase) -efflux process.

Applications of the Microfluidic Single-cell Biochip
The single-cell biochip can be used to test drugs that will elicit calcium responses in mammalian cells. The RAW 264.7 cell (a mouse macrophage cell line) was used and an anti-tumour compound present in the herb licorice isoliquiritigenin (IQ) was tested.13 We found that IQ was able to elicit calcium responses in the RAW cell retained in the microchip.14
Other applications involve testing of drugs that target various cell signalling pathways (e.g., NF-κB).4 In addition, the microchip method can be applied for the study of drug metabolism, efflux-mediated multiple drug resistance or cell toxicity. The single-cell assay is a novel method in drug discovery, which not only saves on cost and reduces the number of animal tests, but also addresses the issues of cell heterogeneity and single-cell biochemistry.

References
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(14)     Li, P.C.H. et al. “Cell selection, retention, culture and scanning on a microfluidic chip for single-cell biochemical studies pertai