Scalable 3D Assays for Drug Screening

VIAFLO_96_384_VIAFILL_CORNING_V00 1/8 Application Note High-Throughput, 3D Assay Development on Spheroids in 1536 Well Plates 3D cell models are often a better representation of the in vivo environment than traditional 2D methodologies. Accordingly, spheroid and other 3D models are increasingly being used for drug discovery. A high throughput system is essential to meet the 3D cell screening and 3D cell culture demands of most largescale drug discovery programs. Introduction Key benefits: • The Corning 1536-well spheroid microplate can be used to generate 1536 uniform, single spheroids that can be assayed by imaging, fluorescence or luminescence measurements directly in the microplate. • The VIAFILL is designed for rapid bulk liquid dispensing of volumes as low as 0.5 µl. It can precisely seed cells in a 1536 well microplate in less than 1 minute using the 16 channel dispensing cassette. • Pairing the VIAFILL with a plate stacker increases the system capacity to up to 50 plates in a row. Step-by-step procedure: Experimental set-up DU 145 (ATCC® HTB-81™) and PANC-1 (ATCC CRL-1469™) cells were routinely cultured in DMEM containing 10 % FBS. Cells were seeded into Corning 1536-well spheroid microplates at 1000 cells per well, in a volume of 5 µl per well, using the VIAFILL with the 16 channel dispensing cassette. The pipetting steps were performed using a VIAFLO 384 together with a 384 pipetting head (0.5-12.5 µl), an adjustable spring loaded plate holder with 1536 offset, and 12.5 µl SHORT, Sterile, Filter GripTips. An automated method has been established for seeding, dosing and assaying spheroids in Corning 1536-well spheroid microplates using INTEGRA Biosciences’ VIAFILL reagent dispenser and VIAFLO 384 handheld electronic pipette. With a maximum volume of 14 µl per well, accurate liquid handling is essential for the generation of quality data. The method was proven to generate robust, high quality data for assay development and high throughput screenings. Corning 1536-well Spheroid Microplate • The adjustable spring loaded plate holders with slide function and 1536 well offset offer optimal alignment of the VIAFLO 384 pipetting head with the target 1536 well plate. • The 12.5 µl SHORT GripTip pipette tips are specifically designed for 1536 well pipetting, and enable precise targeting of small wells. • Setting the Tip Align strength to 3 and using the VIAFLO 384’s z height function makes it easy and accurate to pipette into a 1536 well plate.VIAFLO_96_384_VIAFILL_CORNING_V00 2/8 1. Spheroid formation STEP: Cell seeding into the Corning 1536-well spheroid microplates using the VIAFILL reagent dispenser. HOW TO: Accurate handling of the cell suspension during seeding is critical to ensure that the spheroids are of uniform size across the entire microplate. For rapid cell seeding, use the VIAFILL reagent dispenser. Install the 16 channel dispensing cassette, and place the Corning 1536-well Spheroid Microplate on the plate sledge of the VIAFILL reagent dispenser. If using the VIAFILL with a 16 channel dispensing cassette and a 1536 well plate for the first time, adjust the stage alignment and save the settings. The stage alignment only has to be done once. Use the Repeat Dispense function of the VIAFILL to quickly fill the wells at 1000 cells per well, in a volume of 5 µl per well, with a touch off. Incubate the plates overnight at 37 °C in a humidified CO2 incubator to allow for spheroid formation. Experimental results (Corning) showed the formation of uniform, single spheroids in each well across an entire microplate (Figure 1). Application Note The experiment is divided into three steps. Overview of the steps: Step 1: Spheroid formation Step 2: Homogenous cell-based assay Step 3: High-content imaging assay VIAFILL VIAFLO 384VIAFLO_96_384_VIAFILL_CORNING_V00 3/8 Application Note Tips: • Remove the dispensing cassette crash guards to dispense the cells into the 1536 well plate with a touch off. • Using the VIAFILL reagent dispenser allows accurate, automatic seeding in less than 1 minute per plate. • The VIAFILL can be paired with the plate stacker for an increased capacity of up to 50 plates at a time. 2. Homogenous cell-based assay STEP: Spheroid treatment with staurosporine to assess the uniformity of signal, signalto-background ratio (S/B) and calculated Z' values, and demonstrate the utility of this experimental design for 3D cell screening. HOW TO: Uniformity of signal was demonstrated by treating sections of the microplate with a single concentration of buffer (control) or staurosporine to induce cell death. Spheroid viability was assessed with a homogenous luminescence cell viability assay. After DU 145 spheroid formation, dispense 1 µl of 6 µM staurosporine or buffer into each well. Use the VIAFLO 384 with a 384 channel pipetting head (0.5-12.5 µl) and 12.5 µl SHORT Sterile, Filter GripTips. Insert the Corning 1536-well Spheroid Microplate into an adjustable spring loaded plate holder B with 1536 offset placed on Position B. If using for the first time, adjust the plate holder to the Corning 1536-well Spheroid Microplate for a perfect tip alignment. Set the Tip Align strength to 3 to easily and rapidly access the center of the tiny wells of the 1536 well microplate. Use the slide function of the plate holder to move the 1536 well plate and access all four quadrants with the 384 channel pipetting head. Incubate the plates overnight at 37 °C in a humidified CO2 incubator. Figure 1: Uniform, single spheroids formed in each well across the entire spheroid microplate. Representative image of single uniform DU 145 spheroids formed in the Corning 1536-well spheroid microplate with one well digitally zoomed in. Images were taken with Thermo Fisher CellInsight CX7 high-content screening platform using a 4X objective.VIAFLO_96_384_VIAFILL_CORNING_V00 4/8 Application Note The next day, transfer 6 µl of CellTiter-Glo® 3D reagent from a 150 ml automation friendly reagent reservoir placed on Position A to each well, and incubate at room temperature for 1 hour. The luminescence signal is then read using a microplate reader (Tecan Infinite® M200). Corning’s experimental results showed good signal uniformity regardless of a well’s location in the microplate (Figure 2). The mean S/B ratio from this assay was greater than 20 for 3 independent studies (Figure 3 A). Additionally, Z' values were consistently above 0.7, indicating the robustness of the viability assay in the Corning 1536-well Spheroid Microplate (Figure 3 B). Figure 2: Uniform signal across Corning spheroid microplate. Representative image of color-scaled luminescence signal generated from an assay where DU 145 spheroids were exposed to a final concentration of buffer (green) or 1 µM staurosporine (red). Green corresponds to high luminescence values, high viability. Red corresponds to low luminescence values, low viability. Figure 3: Large, robust luminescence assay window. (A) Mean ± SD luminescence of DU 145 spheroids exposed to buffer or 1 µM staurosporine, resulting in S/B of 20.2. p<0.0001 with unpaired t-test. (B) Excellent Z' values above 0.7 in 3 independent studies. N = 2304 wells in 3 independent studies. (A) Mean ± SD luminescence of DU 145 spheroids exposed to buffer or 1 µM staurosporine, resulting in S/B of 20.2. p<0.0001 with unpaired t-test. (B) Excellent Z' values above 0.7 in three independent studies, N = 2304 wells. A BVIAFLO_96_384_VIAFILL_CORNING_V00 5/8 Application Note Tips: • For optimal alignment of the pipetting head with the target 1536 well plate, the spring loaded plate holder with 1536 offset is adjustable forwards and backwards. The easyto-use slide function of the plate holder shifts the 1536 well plate in order to access all four quadrants with a 384 channel pipetting head. • 12.5 µl SHORT GripTips have been developed to enhance 1536 well pipetting performance. These tips are specifically designed for 1536 well pipetting, and enable precise targeting of small wells. They are available in both standard and Low Retention Sterile, Filter versions. • By setting the Tip Align strength of the VIAFLO 384 to 3, pipetting into the center of the tiny wells of a 1536 well plate is as easy and accurate as pipetting into a reagent reservoir. • By defining the z heights of the VIAFLO 384, you can ensure optimal tip immersion that prevents accidental aspiration, scratching of the pipette tips on the bottom of the reservoir or damage to the spheroids. 3. High-content imaging assay STEP: Proof of concept assay. Addition of cisplatin to assess the cytotoxicity. HOW TO: Use Position A for the 150 ml automation friendly reagent reservoirs needed for this step and Position B for the Corning 1536-well spheroid microplates placed in the adjustable spring loaded plate holder B with 1536 offset. After formation, both DU 145 and PANC-1 spheroids were exposed to various concentrations of cisplatin by adding 1 µl of solution to each well using the VIAFLO 384 with 12.5 µl SHORT Sterile, Filter GripTips, followed by overnight incubation. To assess cytotoxicity, 6 µl per well of 40 µg/ml Hoechst 34580 and 16 µg/ml propidium iodide (PI) in PBS were added to each well for 1 hour. Microplates were than imaged on the Thermo Fisher CellInsight high-content screening platform. Representative images were captured directly without the need to transfer the spheroids to another microplate, demonstrating the utility of culture and subsequent imaging of spheroids in the spheroid microplate (Figure 4). Image analysis of propidium iodide staining revealed typical concentration-response curves for cisplatin cytotoxicity (Figure 5).VIAFLO_96_384_VIAFILL_CORNING_V00 6/8 Application Note Figure 4: Confocal imaging of spheroids within Corning 1536-well Spheroid Microplates. Representative z-stack images of DU 145 (top) and PANC-1 (bottom) spheroids exposed to 0, 0.025 and 83 mM cisplatin (left, middle, right, respectively) for 24 hours. Spheroids were stained with Hoechst (blue) and PI (red) to assess cell viability. Spheroids were imaged directly in the spheroid microplate with the Thermo Fisher CellInsight CX7 high-content screening platform using a 10x objective. Scale bar = 100 µm. 0 mM 0.025 mM 83 mM DU 145 PANC-1 Figure 5: Concentration-dependent cisplatin cytotoxicity. Mean ± SD PI fluorescence of DU 145 and PANC-1 spheroids following 24 hour exposure to varying concentrations of cisplatin. N = 96 wells.VIAFLO_96_384_VIAFILL_CORNING_V00 7/8 Application Note Conclusion • Using the VIAFILL and VIAFLO 384 instruments for liquid handling in Corning 1536-well spheroid microplates generated robust quality data for assay development and high throughput screens. • Corning 1536-well spheroid microplates allow for the formation of uniform, single spheroids in each well that are ideally suited for high throughput 3D drug screening. The black sidewalls and clear bottom design of these microplates are ideal for imaging, luminescence and fluorescence based cellular assays. • The combination of the VIAFILL and VIAFLO 384 instruments with the Corning 1536-well Spheroid Microplate resulted in good signal-to-background ratios, with excellent and consistent z’ values. • The VIAFILL reagent dispenser with the 16 channel dispensing cassette allows accurate, automatic plate seeding in less than 1 minute per plate. For high throughput, it can be paired with a plate stacker to automatically fill up to 50 plates. • The VIAFLO 384 handheld electronic pipette, together with the 384 channel pipetting head (0.5-12.5 µl), adjustable spring loaded plate holder with 1536 offset, and 12.5 µl SHORT GripTips allows easy, accurate and rapid pipetting into 1536 well microplates, even those containing spheroids. Tips: • When using the Repeat Dispense mode of the VIAFLO 384, define a pre- and post-dispense volume to be discarded. These two dispenses contain the largest volume errors. It is especially important to discard the last dispense as it contains the accumulated error of all previous dispenses. • The SureFlo™ array located on the flat bottom of the automation friendly reagent reservoir prevents pipette tip seal off. This allows users to rest the pipette tips directly on the bottom of the reservoir, offering an incredibly low dead volume of less than 3 ml. Materials Manufacturer Part Number Description Link INTEGRA Biosciences 5600 VIAFILL reagent dispenser https://www.integra-biosciences.com/global/en/ microplate-dispensers/viafill#parts-and-numbers INTEGRA Biosciences 5742 16 channel dispensing cassette, small bore https://www.integra-biosciences.com/global/en/ microplate-dispensers/viafill INTEGRA Biosciences 6031 VIAFLO 384 handheld electronic pipette (base unit) https://www.integra-biosciences.com/global/en/ electronic-pipettes/viaflo-96384#parts-and-numbers INTEGRA Biosciences 6131 384 channel pipetting head 12.5 µl https://www.integra-biosciences.com/global/en/ electronic-pipettes/viaflo-96384 INTEGRA Biosciences 6225, 6226 Adjustable spring loaded plate holder A/B for 24, 96, 384 and 1536 well plates, with slide function (1536 offset) https://www.integra-biosciences.com/global/en/ electronic-pipettes/viaflo-96384#parts-and-numbersVIAFLO_96_384_VIAFILL_CORNING_V00 8/8 Application Note INTEGRA Biosciences AG 7205 Zizers, Switzerland T +41 81 286 95 30 F +41 81 286 95 33 info@integra-biosciences.com INTEGRA Biosciences Corp. Hudson, NH 03051, USA T +1 603 578 5800 F +1 603 577 5529 info-us@integra-biosciences.com INTEGRA Biosciences Ltd. Egham, Surrey TW20 9EY, UK info-uk@integra-biosciences.com INTEGRA Biosciences SAS 95062 Cergy-Pontoise Cedex 1, France T +33 (0)1 34 30 76 76 F +33 (0)1 34 30 76 79 info-fr@integra-biosciences.com INTEGRA Biosciences Deutschland GmbH 35444 Biebertal, Deutschland T +49 6409 81 999 15 F +49 6409 81 999 68 info-de@integra-biosciences.com Manufacturer Part Number Description Link INTEGRA Biosciences 6475 12.5 µl SHORT, Sterile, Filter GripTips https://www.integra-biosciences.com/global/en/ griptip-selector-guide INTEGRA Biosciences 6303, 6317, 6318 150 ml automation friendly reagent reservoir https://www.integra-biosciences.com/global/en/ reagent-reservoirs/automation-friendly-reagentreservoirs Corning 4527, 4637 Corning 1536-well Black/ Clear Round Bottom Ultra-low Attachment Spheroid Microplate, with Lid, Sterile https://ecatalog.corning.com/life-sciences/b2c/ US/en/Microplates/Assay-Microplates/1536-WellMicroplates/Corning%C2%AE-1536-well-SpheroidMicroplates/p/4527 Millipore Sigma 569397 InSolution Staurosporine, Streptomyces sp. https://www.sigmaaldrich.com/catalog/product/ mm/569396?lang=en&region=US Promega G9683 CellTiter-Glo 3D Cell Viability Assay https://www.promega.com/products/cell-healthassays/cell-viability-and-cytotoxicity-assays/celltiter_ glo-3d-cell-viability-assay/?catNum=G9681&cs=y Millipore Sigma 1134357 Cisplatin https://www.sigmaaldrich.com/catalog/product/ usp/1134357?lang=en&region=US Thermo Fisher Scientific H21486 Hoechst 34580 https://www.thermofisher.com/order/catalog/product/ H21486#/H21486 AnaSpec AS-83215 Propidium iodide, 1.0 mg/ml solution in water https://www.anaspec.com/products/product. asp?id=29637 Corning 21-030-CM Corning Dulbecco’s PhosphateBuffered Saline, 1X with calcium and magnesium https://ecatalog.corning.com/life-sciences/ b2c/US/en/Media,-Sera,-and-Reagents/ Buffered-Salt-Solutions/Dulbecco's-PhosphateBuffered-Saline-(DPBS)/Corning%C2%AEDPBS-(Dulbecco%E2%80%99s-PhosphateBuffered-Saline)/p/21-030-CM