(1271-D) Developments in Quadruplexing Microplate Assays: From 1536 to 6444 Assays in a Standard Microplate

Abstract: The ability to perform multiple assays within a single microtiter plate provides novel opportunities in high throughput screening, extending its utility in early-stage drug discovery. The technology development presented aims to maximize the use of standard microtiter plates and HTS plate reader instrumentation through controlled, simultaneous dispensing and culturing of physically separated cell-lines that share a common microwell for drug dosing. Applications for such technology include, but are not limited to:
Co-Culture Drug Screening: Co-culturing separate but adjacent cell-lines can enable the detection of cross-communications which is especially critical in cancer research. Anti-tumor drug effects can be better ascertained by not only observing primary cancer cell activity, but its associative activation of cancer associated fibroblasts, angiogenesis, and immune cell activation. High content analysis of the wells can reveal the effect of the drug compound on each cell line in a single read. This approach affords the opportunity to run co-culture assays without the need for specialized labware.
Multiplex Primary Drug Discovery: The ability to execute primary screening campaigns using multiple cell lines can improve throughput, data variance, and costs. Assay development typically takes place in multiple stages, with hit compounds being tested against a variety of different cell lines. One disadvantage of this is that it requires multiple rounds of testing for any single candidate drug. Cell responses may vary between tests, affecting the overall quality of the results. By testing multiple cell lines at once, dosing and readout variances are greatly reduced, improving overall analysis. Drug screening would not only include the primary cellular target but could also include counterscreens against control cell-lines to test for promiscuity and/or cytotoxicity.
Natural Product (NP) Discovery: A critical bottleneck in natural product research is to ascertain the potential utility of any NP entity against different disease models. The ability to test each NP against four different disease models could significantly improve the speed and costs that arise with NP evaluation. Such testing could have a singular focus: 1) infectious diseases against gram-positive and gram-negative pathogen lines, 2) multiple resistant cancer cell lines or across cell models of unrelated diseases.

Currently the work presented here is proof of concept that integrates the development of a specialized dispenser with formulation of an appropriate hydrogel and the development of screening assays for testing the quadruplexing approach. In this work a BioRaptr FRD dispenser had been modified with a custom dispensing tip solution and alterations to the electronic wiring to enable 4-tip simultaneous dispensing. Initial testing has shown successful quantitative quadrant dispensing with a standard 1536w microplate. Presented are the operational capabilities of the quadruplexing dispenser and its ability to dispense 4 different cell lines into each corner of a 1536-well plate along with the future work aimed at evaluating its assay performance for early-stage drug screening.