High-throughput, 3D assay development on spheroids in 1536 well plates

  • High-throughput, 3D assay development on spheroids in 1536 well plates

    An automated method for seeding, dosing, and assaying spheroids in a Corning 1536-well spheroid microplate using the VIAFILL and VIAFLO 384

    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 large-scale drug discovery programs. 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.

  • Table of contents

      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 large-scale drug discovery programs. 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.

    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.
    • 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.

    Overview: High-Throughput, 3D Assay Development

    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.

    The experiment is divided into three steps:

    • Step 1: Spheroid formation
    • Step 2: Homogenous cell-based assay
    • Step 3: High-content imaging assay
    Corning 1536-well Spheroid Microplate
    Corning 1536-well Spheroid Microplate

    Step-by-step procedure

    1. Spheroid formation

    Cell seeding into the Corning 1536-well spheroid microplates using the VIAFILL reagent dispenser.

    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).

    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.
    Uniform, single spheroids formed in each well across the entire spheroid microplate.
    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.

    2. Homogenous cell-based assay

    Spheroid treatment with staurosporine to assess the uniformity of signal, signal-to-background ratio (S/B) and calculated Z' values, and demonstrate the utility of this experimental design for 3D cell screening.

    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.

    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).

    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 easy-to-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.
    Uniform signal across Corning spheroid microplate.
    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).
    Figure 3: Large, robust luminescence assay window.
    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.

    3. High-content imaging assay

    Proof of concept assay. Addition of cisplatin to assess the cytotoxicity.

    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).

    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.
    Confocal imaging of spheroids within Corning 1536-well Spheroid Microplates.
    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.
    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.
    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.

    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.

    April 2020, by

    • Lydiane Saucède, PhD

      Application Scientist

      Email
    • Jan-Peter Baldin, PhD

      Product Manager

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    Any questions? I'm happy to help!

    • Tom Bentivegna

      Product Manager

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    Used instruments and accessories

    VIAFILL Reagent Dispenser Dispensing Cassette, 16 channel VIAFLO 96/384, Handheld Electronic Pipette 384 Channel Pipetting Head, 12.5 µl Adjustable spring loaded plate holders GripTip, 12.5 µl SHORT, Sterile, Filter Automation Friendly Reagent Reservoirs, 150 ml CORNING: Corning® 1536-well Spheroid Microplate MilliporeSigma: InSolution Staurosporine, Streptomyces sp. Promega: CellTiter-Glo 3D Cell Viability Assay MilliporeSigma: Cisplatin Thermo Fisher Scientific: Invitrogen™ Hoechst 34580 AnaSpec: Propidium iodide CORNING: Corning Dulbecco’s Phosphate-Buffered Saline

    VIAFILL Reagent Dispenser

    Users can easily alternate between 8 and 16 channel dispensing cassettes for rapid filling of 6 to 1536 well plates. The addition of the stacker allows for filling bulk quantities of both non-lidded and lidded plates.

    The addition of a plate stacker increases throughput and allows for unattended operation. Well plates are conveniently stored in the plate stacker using removable chimneys designed for either 25 or 50 plates.

    Part No. 5600

    Visit product page
    VIAFILL touch screen rapid reagent dispenser.

    Dispensing Cassette, 16 channel

    Small bore (.012"), 0.5 - 999μL, pre-sterilized, 5 pack

    Part No. 5742

    VIAFLO 96/384, Handheld Electronic Pipette

    With this 24, 96 and 384 channel pipette, INTEGRA meets the need for an affordable, compact and easy to use pipette that provides increased productivity. The unique operating concept makes VIAFLO 96/384 as easy to use as any traditional handheld pipette.

    Part No. 6001 / 6031

    See how it works
    The VIAFLO 96/384 is handheld, electronic pipette

    384 Channel Pipetting Head, 12.5 µl

    Users can simply change the pipetting head of their VIAFLO 96 and VIAFLO 384 to guarantee optimal matching of the available volume range to the application performed. Changing a pipetting head of VIAFLO 96 and VIAFLO 384 is easy and takes less than one minute.

    Part No. 6131

    Find out more about VIAFLO 96/384 Pipetting Heads Specifications
    VIAFLO 96/384 96 channel pipetting head

    Adjustable spring loaded plate holders

    For 24, 96 and 384 channel pipetting. Available for positions A (6225) and B (6226) these plate holders feature a slide function to pipet into 1536 well plates with a 384 channel pipetting head. A spring load mechanism secures the microplate in place. Both holders are adjustable in y direction to allow an optimal alignment of the pipetting head with the target 1536 well plate.

    Part No. 6225 / 6226

    All plate holders
    VIAFLO 96/384 adjustable spring loaded plate holder A/B with slide function

    GripTip, 12.5 µl SHORT, Sterile, Filter

    GripTip pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

    Part No. 6475

    Learn more about GripTips pipette tips for Benchtop Pipetting Systems
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    Automation Friendly Reagent Reservoirs, 150 ml

    150 ml Automation Friendly Clear Advantage™ Reservoirs (Polystyrene) which are made of crystal clear 100 % virgin polystyrene and fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.

    The SureFlo™ Array located on the flat bottom 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!

    Find out more about Automation Friendly Reagent Reservoirs
    Reservoir for automation with SureFlo™ anti-sealing array

    CORNING: Corning® 1536-well Spheroid Microplate

    With their novel and proprietary design, these microplates are ideal for generating and analyzing 3D multicellular spheroids in the same microplate. The Ultra-Low Attachment (ULA) surface enables uniform and reproducible 3D multicellular spheroid formation. The black opaque microplate body shields each optically clear, round bottom well from well-to-well cross-talk.

    Source: Website Corning

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    Corning® 1536-well Black/Clear Round Bottom Ultra-low Attachment Spheroid Microplate, with Lid, Sterile

    MilliporeSigma: InSolution Staurosporine, Streptomyces sp.

    InSolution Staurosporine, Streptomyces sp. CAS 62996-74-1, is provided as a 1 mM (100 µg/214 µl) solution of Staurosporine (Cat. No. 569397) in DMSO.

    Source: Website MilliporeSigma

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    InSolution Staurosporine, Streptomyces sp.

    Promega: CellTiter-Glo 3D Cell Viability Assay

    The CellTiter-Glo® 3D Cell Viability Assay is designed for determining cell viability in 3D microtissue spheroids. The assay reagent penetrates large spheroids and has increased lytic capacity—allowing more accurate determination of viability compared to other assay methods.

    Source: Website Promega

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    CellTiter-Glo® 3D Cell Viability Assay

    MilliporeSigma: Cisplatin

    Potent platinum-based antineoplastic agent. Forms cytotoxic adducts with the DNA dinucleotide d(pGpG), inducing intrastrand cross-links.

    Source: Website MilliporeSigma

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    Thermo Fisher Scientific: Invitrogen™ Hoechst 34580

    This bisbenzimide dye, Hoechst 34580, is related to the important minor-groove binding, dsDNA-selective dyes Hoechst 33258 and Hoechst 33342. Hoechst 34580 has somewhat longer-wavelength spectra than the other Hoechst dyes when bound to nucleic acids [Howard Shapiro, personal communication].

    Source: Website Thermo Fisher Scientific

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    AnaSpec: Propidium iodide

    Propidium iodide, 1.0 mg/mL solution in water

    Source: Website AnaSpec

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    Propidium iodide, 1.0 mg/ml solution in water

    CORNING: Corning Dulbecco’s Phosphate-Buffered Saline

    Lower documented Endotoxin content than offerings from other major suppliers Dulbecco's Phosphate Buffered Saline (DPBS) is a buffer solution which can be used to maintain cell culture media in the physiological pH range of 7.0 to 7.6.  It can serve as an irrigating, transporting or diluting fluid while maintaining cell tonicity and viability for a limited period of time.  This formulation contains calcium and magnesium.

    Source: Website Corning

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    Customer’s voice

    This is the best bulk liquid dispenser on the market! I highly recommend the Integra Viafill and stacker for all of your bulk liquid dispensing needs. It's faster, easier, and more robust over other automated liquid handlers. Our HTP lab could not function properly without it. Again, I highly recommend the Integra Viafill for your lab.

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