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INTRODUCTION More than a century ago cell culture was invented to study neuron outgrowth1. The demonstration that single cells could be grown in vitro, combined with the development of specific growth media and the establishment of the first cell lines, marks the birth of cell culture has a critical research tool. Investigators have developed a myriad of in vitro cellular models to better understand various normal and pathological cellular processes, as well as to screen and characterize potential therapeutic modalities. These advancements happened in concert with the development of many technologies impacting the 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 biological research laboratories. During the past decade, cell-based assays have become fundamental and irreplaceable tools in drug discovery and development. This trend was driven by two main factors: first, the completion of the human genome combined with advances in functional genomics and proteomics led to the need to evaluate thousands of potential new targets, and second, the high attrition rate of therapeutic candidates at the preclinical and clinical stages. This high attrition rate generated the need for more biologically relevant, high-throughput screening approaches placing cell-based assay technologies at the forefront of the drug discovery strategies. As a result, investigators ability to develop rapid, flexible, robust and cost-effective high-throughput cell-based assays became of paramount importance. Despite significant technological progresses in enabling technologies such as molecular labeling and the advent of high content screening approaches, cell-based assays still have major limitations due in part to their well plate format and associated costs. Although wells are an efficient and simple strategy to segregate experimental conditions in various formats from 6 to 1536 wells per plate, they have major restrictions when using suspension, loosely adherent and in some cases, fully adherent cells 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 could be implemented for high-throughput cell-based screenings efforts. Since the addition and removal of reagents to the wells through vacuum aspiration can compromise the retention of the cells, various technologies have been developed to circumvent these limitations. This is the case of the homogeneous assays that have been developed in recent years. These assays provide a convenient “add, mix and read” approach for a number of assays including luminescent cell viability assays, that use cellular ATP as a surrogate for cell number2 and caspase assays that use fluorescent or luminescent substrates to measure the activation of specific caspases within the cells3-6. Homogeneous approaches also include the cell-based reporter assays that take advantage of reporter genes such as luciferase, b-galactosidase and b-glucuronidase7-9. In these cases cells contain the reporter gene under the control of a specific promoter containing multiple response elements. In the presence of activating or repressing conditions, the amount of reporter enzyme translated is modulated impacting the signal generated in presence of the specific substrate.

ABSTRACT Cellular assays represent a great opportunity for researchers to test various molecules in a more biologically relevant context than biochemical assays. Many of these cell-based assays could be adapted for high-throughput and high content screening assays, providing investigators the opportunity to interrogate large number of samples and conditions. Despite significant technical advances made during the last few years, high-throughput/high content cellular assays still suffer from key limitations in working with a large number and variety of cell lines. A key limitation is the use of suspension cell lines, especially when multi-step staining procedures are required. Here, we demonstrate that the use of a well-less plate system which significantly improves the flexibility of our high content screening platform. The well-less format utilizes surface tension to maintain the cell population on the glass surface of plate in 2.5mm diameter drops, while a gentle buffer exchange allows the cells to remain on the plate surface throughout a variety of experimental procedures. We performed cell viability experiments in the presence of the antimitotic agent monomethyl auristatin E (MMAE) on the suspension cell line U-937 in immunofluorescence and chemiluminescence viabi l i ty assays. We also performed immunofluorescence-based, protein-protein interaction assays using a large library of single transmembrane, multi-transmembrane and secreted proteins expressed in COS7 cells. The expressed library was incubated with tagged bait proteins to interrogate novel protein-protein interactions at the cell surface. This resulted in the successful expression and binding of several known ligands to their respective receptors, i.e. PD-1 binding to PD-L1, NGF to NGFR, PVRL2 to PVRIG, and HVEM binding to BTLA. We also examined changes in cell morphology using both established and primary human cell lines infected with fluorescent organelle-specific baculovirus constructs. Live imaging of the infected cells revealed alterations in the trafficking patterns of GFP-labeled early endosomes in the presence of the dynamin inhibitor Dynasore. We also observed changes in the actin cytoskeletal structure in the presence of Cytochalasin D and Blebbistatin. The added flexibility in terms of cell lines and readouts enables the screening platform to truly perform high-throughput and high content assays to examine a wide variety of cellular processes.

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SUMMARY AND CONCLUSIONS The well-less DropArray plate technology represents a revolutionary approach to overcome the limitations in developing rapid, flexible, robust, cost-effective, and most importantly, scientifically relevant cell-based assays. The developed platform enables the use of adherent, semi-adherent and suspension cells with almost any cell-based assay modalities. We have also combined the DropArray technology within automation platforms that can be easily deployed for a broad spectrum of high-throughput applications. This technology can be used for protein/antibody, small molecule or siRNA screens using, homogeneous or reporter gene fluorescent and luminescent readouts. The DropArray technology can also be used for more complicated assays and readouts such as multi-step immunofluorescence high-content analyses. Future investigations will explore additional cell-based applications using multiple cell types in 3D models and more physiologically relevant readouts for high-throughput, high-content in vitro assays.

ACKNOWLEDGEMENTS AND REFERENCES 1.  Harrison, R. (1907) Anat. Rec. 1:116-128 2.  Crouch, S.P.M. et al. (1993) J. Immunol. Meth. 160, 81–88 3.  Karvinen, J. et al. (2002) J. Biomol. Screen. 7, 223–231 4.  Préaudat, M. et al. (2002) J. Biomol. Screen. 7, 267–274 5.  Liu, D. et al. (2004) J. Biol. Chem. 279, 48434–48442 6.  Ren, Y.G. et al. (2004) Mol. Biol. Cell 15, 5064–5074 7.  Brasier, A.R., Ron, D. (1992) Methods Enzymol. 216, 386–397. 8.  Nielsen, D. A. et al. (1983) PNAS USA 80, 5198-5202. 9.  Marathe SV, McEwen JE (1995). Gene 154, 105–107.

We would like to thank Wai Lee Wong (Genentech) for her help and introduction to Curiox Biosystems. We would also like to thank Travis More, Hyunjae Lee, and Noo Li Jeon (Seoul National University) for their help and guidance in conducting cell retention assays as well as fluid velocity computer modeling and testing.

Figure 1: DropArray Plate And Washing Procedure A B Beginning of Oil Addition

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Figure 1A Photographs of the DropArray 384 well-less plate at the start and end on the cover oil addition. The oil prevents evaporation of the 2µl drops on the plate surface. Figure 2B The plate washing procedure. (1) The plates start off with 2µl drops covered by incubation oil. (2) The plate is inverted and gravity assisted by a vacuum pump is used to aspirate the oil from the plate (3). (4) Wash buffer is added, the plate is leveled and then gently rocked back and forth (5). The plate is inverted once again and the pump removes the wash buffer before adding fresh rinsing oil (6).

Figure 2: Adherent And Suspension Cells Withstand Extensive Washing

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A B Figure 2A Images of suspension cells (U-937) and weakly adherent cells (293S) after one to four 4X wash cycles, the equivalent of 16 washes by hand. The cells were labeled with CMFDA and scanned using an ImageXpress Velos laser scanning cytometer. Scale bar = 100µm. Figure 2B The percent of cells retained on the glass surface after each round of wash cycles. Greater than 85% of the suspension cells remain on the plate surface after four 4X washes.

Figure 3: Extracellular Protein-Protein Interactions

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Figure 3A COS7 cells transfected with the indicated receptor construct (black) were incubated with hFc tagged bait protein (red) corresponding to the ligand for the receptors. The cells were then fixed and stained with and anti-hFc antibody conjugated to AlexaFluor 488. The images were acquired using a 20X objective on a GE IN Cell Analyzer 2000. Scale bar = 70µm. Figure 3B COS7 transfected with either an HNT or OPCML expression construct and incubated with NEGR1-hFc bait protein. The cells were stained with anti-human AF488 (green) and scanned on the IN Cell Analyzer 2000. The same reagents for transfection and staining were used on a standard 384-well microtiter plate (Aurora) and a DropArray plate for a side-by-side comparison of the two formats. Hoechst staining (blue) of the nuclei shows the same cell density on both plate formats. Scale bar = 70µm. Figure 3C A graph of the mean intensity of the green channel from the images acquired. Using three different cell types, equivalent intensity values are seen when NEGR1-hFc binds to cells expressing three different expression constructs of known NEGR1 binding partners. The same values are generated from the standard 384-well format, but only for the adherent cell line (COS7), while the weakly adherent (293T) and suspension adapted (293S) cell lines were not retained during the washing procedure on the standard 384-well plate format.

Figure 4: Fluorescence And Luminescence Cell Viability Assays A C B D

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Figure 4A Two concentrations of U-937 cells were treated with increasing concentrations of the antimitotic agent MMAE on DropArray plates. After treatment, the cells were then lysed and incubated with CellTiter Glo to measure the amount of ATP in the cells. This is a correlative readout for cell viability and proliferation. The plate was read on a microplate luminometer after placing a custom-made plastic grid over the plate to prevent light contamination from well-to-well. Figure 4B U-937 cells were labeled with CMFDA 24 hours prior to the addition of MMAE. After MMAE treatment, the DropArray plate was washed to remove dead cells and cellular debris. The plate was scanned on a laser scanner and the number of cells remaining was calculated. Figure 4C and Figure 4D U-937 cells treated with increasing concentrations of MMAE were dual stained with ethidium bromide and acridine orange (EB/AO) to measure live, apoptotic, and necrotic cells. This dual stain causes live cells to fluoresce green while apoptotic cells retain a red-orange fluorescence.

Figure 5: Drug/Chemical-Induced Morphological Changes Figure 5 COS7 cells were treated with 50µM -(-)Blebbistatin, control medium or 0.4µM Cyto. D overnight. The cells were then fixed with 4% PFA and stained with phalloidin-FITC to visualize the actin cytoskeleton and Hoechst for nuclei. Both Blebbistatin and Cyto. D caused drastic changes to the actin cytoskeleton as was expected. Scale bar = 70µm

Figure 6: Live Cell Imaging And Multicolor Staining In A 2µL Drop A

Figure 6A COS7 cells infected with a GFP-labeled, Rab5a targeting baculovirus construct were imaged every 60 seconds over the course of 30 minutes on the GE IN Cell Analyzer 2000 before and after the addition of 100µM Dynasore. Green arrowheads indicate the starting position of a single endosome and red arrowheads indicate the ending position of that same endosome. Scale bar = 10µm Figure 6B and Figure 6C hRPE cells co-stained with: Hoechst, ZO-1 and phalloidin. Scale bars = 70µm

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High-Throughput Cellular Assays Using A Well-Less Plate Format G. Quiñones1, K. Nicholes1, M. Lye2, N. Kim2 and J-P Stephan1

1Genentech, South San Francisco, CA, USA, 2Curiox Biosystems, Singapore