We have developed new tools to interrogate the effects of small molecules on biological function (Figure 1). These include cell- and protein-based high-throughput screening, on chip immunoassays and high-throughput biocatalysis on a microarray platform (Lee et al. Proc. Natl. Acad. Sci. USA 102, 983-987 (2005); Sukumaran et al. J. Biomol. Screen. 14, 668-678 (2009)]. With respect to the cell-based screening platform, we developed (in collaboration with Doug Clark’s group at UC Berkeley) a miniaturized three-dimensional (3D) cellular array chip (the DataChip, Figure 2) that has been used for in vitro toxicological assessment of drug candidates and other chemicals in high-throughput [Lee et al. Proc. Natl. Acad. Sci. USA 105, 59-63 (2008)]. Although recent emphasis has been placed on understanding cellular metabolism in 3D, relatively little effort has been spent using 3D cultures to study cytotoxicity, particularly at very small volumes consistent with high-throughput screening. Furthermore, we have recently begun to investigate the toxicity of compounds on human stem and progenitor cells in comparison to their differentiated progeny.
Figure 1. New high-throughput tools being developed. Upper left is the DataChip (Data Analysis Toxicology Assay Chip) for high throughput 3D cell culture screens. Upper right is the MetaChip (Metabolizing Enzyme Assay Chip for high-throughput interrogation of enzyme function. In this case, CYP450 inhibition assays7. Lower left is a modification of the DataChip to enable on-chip, in-cell immunofluorescence assays and quantitative analysis of protein expression levels within cells6. Lower right is a schematic of an "Artificial Golgi", which includes an image of a digital microfluidic device.
![Illustration of the DataChip [Lee et al. Proc. Natl. Acad. Sci. USA 105, 59-63 (2008)]](https://enzymes.che.rpi.edu/sites/default/files/styles/wide/public/2022-03/Figure-DD-2.png?itok=mYlxEFVA)
Figure 2. The DataChip[Lee et al. Proc. Natl. Acad. Sci. USA 105, 59-63 (2008)]. Upper left represents the hemispherical spots generated by microarray spotting of cells in alginate solution, which upon contact with poly-L-lysine and BaCl2 results in near immediate gelation. The cells grow well up to 5 days in culture (upper right). Lower left shows an actual image of a stained DataChip (live-dead assay) and representative dose response curves for drug candidates and chemicals in the presence or absence of metabolizing enzymes contained within a MetaChip and stamped on top of the DataChip. Lower right is the output of the on-chip, in-cell immunofluorescence assay [Fernandes et al. Anal. Chem. 80, 6633-6639 (2008)]. The dose response to an activator of HIF-1α in a pancreatic tumor cell line (triangles represent standard Western blotting analysis on mL scale and open squares represent the on-chip immunofluorescence assay in volumes as low as 30-nL).
We also developed a novel chip design that enables interrogation of differential expression of various drug-metabolizing enzymes (DMEs) in the human liver. Such information is relevant to inter-individual variability in drug pharmacokinetic profiles, drug efficacy and toxicity. Specifically, we developed the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity [Kwon et al. Nat. Commun. 5, 3739 (2014)]. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip (Figure 3). The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray (Figure 4). Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.
Figure 3. Illustration of TeamChip schematics and photographs. (a) Micropillar/microwell chip components in relation to a standard glass microscope slide. (b) The micropillar chip containing THLE-2 cells encapsulated in matrigel droplets. (c) The microwell chip containing recombinant adenoviruses carrying genes for drug-metabolizing enzymes (the red color indicates the no-virus control and the two colors represent different viruses). (d) Stamping of the micropillar/microwell chips for drug-metabolizing gene expression. (e) Experimental procedure for use of the TeamChip. [Kwon et al. Nat. Commun. 5, 3739 (2014)].
Figure 4. Illustration depicting the effects of combinatorial expression of human drug-metabolizing enzymes on the toxicity of tamoxifen. (a) Layout of the microwell chip containing 84 combinations of multiple recombinant adenoviruses (three sets of recombinant adenoviruses dispensed sequentially) to prepare the TeamChip for high-throughput gene transduction, and an additional microwell chip containing 200 mM tamoxifen for metabolism-induced toxicity screening. (b) Scanned image of THLE-2 cells expressing 84 combinations of multiple drug-metabolizing enzymes on the chip exposed to 200 mM tamoxifen for 48 h (top) and normalized THLE-2 cell viability at different drug-metabolizing enzyme expression levels (bottom). The viability of multiple drug-metabolizing enzyme-expressing THLE-2 cells exposed to tamoxifen was normalized by the fluorescent intensity of THLE-2 cells incubated in the absence of compound. The least toxic region in the scanned image is highlighted in a yellow box, and the red circle in the graph designates normalized THLE-2 cell viability calculated from the least toxic region that involved the combination of CYP1A2 and UGT1A4.
