Application of a new wall-less plate technology to complex multistep cell-based investigations using suspension cells

Abstract

• The DropArray technology is compatible with the retention of suspension cells in multistep procedures thus enabling novel assay methods.• This technology enabled visualization and quantification of specific killing events triggered by bispecific antibodies engaging T cells. IntroductionThe demonstration that single cells could be grown in vitro 1 combined with the development of specific growth media 2 and later the establishment of the first cell lines [3][4][5][6] definitively marks the birth of cell culture as a critical research tool. Since then, investigators have developed a myriad of in vitro cellular models to further the understanding of various normal and pathologic cellular processes as well as to screen and characterize potential therapeutic modalities. All this progress occurred in concert with the development of many technologies that have impacted the investigator's ability to establish specific culture conditions and measure or visualize different cellular signals. As a result, cell-based assays are today almost universally used in biology research laboratories.During the past decade, cell-based assays have become fundamental and irreplaceable tools in the drug discovery and development industry. This trend was driven mainly by 2 rationales. First, the completion of the human genome, 7 combined with advances in functional genomics and proteomics, 8 has led to the need to evaluate thousand of potential new targets. Second, the high attrition rate of therapeutic candidates at the preclinical and clinical stages generated the need for more biologically relevant highthroughput screening approaches, bringing cell-based assay technologies to the forefront of the drug-discovery strategy. As a result, investigators' ability to develop rapid, flexible, robust, and costeffective high-throughput cell-based assays became of paramount importance.Despite significant technological progress in enabling technologies such as molecular labeling and the advent of high content screening approaches, cell-based assays, in part because of their well-plate format, continue to have major limitations. Although wells are an efficient and simple strategy to segregate experimental conditions in formats from 6-to 1536-well plates, they have major restrictions when suspension, loosely adherent, and in some cases fully adherent cells are used, especially with high-throughput formats such as 96-, 384-, and 1536-well plates. Microwell plates not only limit the use of certain cells but also significantly reduce the spectrum of experimental procedures that can be implemented in high-throughput cell-based screenings. Because the addition and removal of reagents to the wells could definitively compromise the cells, various technologies have been developed to circumvent these limitations. The homogeneous assay, developed in recent years, provides a convenient "add, mix, and read" approach to explore a broad spectrum of biologic events from cell viability/ Submitted July 31, 2012; accepted November 24, 2012. Prepublished online as Bl...

Miniaturization of Mitotic Index Cell-Based Assay Using “Wall-Less” Plate Technology

“…13 Cells in mitosis are typically characterized by round-shape features, which make them highly susceptible to shear force disturbance when washing buffer is introduced into a typical microtiter plate. 15,16 As a consequence, cell-based assays used with conventional microtiter plates could limit the user's ability to quantify cells precisely in the mitotic phase with high-content imaging.…”

“…Such a low shear force has been previously established to show high retention of suspension cells on a DropArray plate. 13 Mitotic cells display similar properties as suspension cells and offer challenges in being handled on conventional microtiter plates due to the general mechanism of the direct nozzle wash dispenser, which can greatly disturb nonadherent cells. The absence of walls between wells in the DropArray plate allows the washing buffer to be dispensed from the side of the plate with a laminar flow, reducing the movement of nonadherent (mitotic) cells and helping cell retention.…”

“…A more detailed description about fabrication of DropArray can be found elsewere. 12,13 Each circular plastic area is rendered hydrophilic with a plasma treatment. Each circular well can then accommodate a drop of 2-4 mL in volume (Fig.…”

“…A more detailed description about DropArray washing station properties and theory are available elsewhere. [12][13][14] Cell Seeding and Compound Treatment on the DropArray Plate HeLa (ATCC) were cultured in Nunc tissue culture flask in D10 growth media (Dulbecco's modified Eagle's medium [DMEM] high glucose [4.5 g/L] supplemented with 10% fetal calf serum, 5% penicillin-streptomycin). The cells were maintained in a humidified 37°C incubator with an air to 5% CO 2 atmosphere.…”

“…DropArray also offers a different washing method using laminar flow, which circumvents the classic microwell-based washing limitations by allowing gentle but efficient washing with high cell retention. 12,13 In this study, the use of the DropArray technology enabled the development of a mitotic index assay, which was validated with the antimitotic drug docetaxel.…”