GravityPLUS™ Hanging Drop System

The industry-leading hanging drop cell culture plate for reliable, scaffold-free 3D cell-based assays

  • SureDrop™ technology assures precise microtissue size and unparalleled drop stability – always!
  • Complete system includes the GravityTRAP™ plate for long-term culture, assays and imaging
  • Proven reliability and compatibility with standard liquid-handling systems

 

  • 3D cell culture made easy

    Scaffold-free re-aggregation of single cells into functional 3D microtissues has been a tricky task in the past. With the introduction of the GravityPLUS™ platform in 2009, InSphero made reliable, automation-compatible and affordable 3D cell culture in hanging drops a reality. Since its introduction, the patented design of the GravityPLUS™ Hanging Drop System has proven its ease of use and versatility in laboratories ranging from academic research to high-throughput screening (view references). It is the only platform offering the consistency and automation compatibility necessary to produce our assay-ready 3D InSight™ Microtissues, in use today by all top 15 global pharmaceutical, chemical and cosmetics companies.

    webinar

    3D101 - Learn about strategies for generating more biologically relevant 3D cell culture models, and get an introduction to InSphero hanging drop and ultra-low attachment plates for producing scaffold-free spheroids.  This 2-part webinar features Dr. Jens M. Kelm and Dr. Yue Geng of InSphero (total view time 50 min).

    • Two plates, one platform

      Hanging Drop Culture Plates- GravityPLUS Hanging Drop System

      The patented GravityPLUS™ Hanging Drop System makes hanging-drop cell culture easy.  It is ideal for use with primary cells, generating more complex co-culture spheroid models, and for use with cell lines that fail to form spheroids in ultra-low-attachment (ULA) plates.  A typical culture workflow is outlined in the diagram below.
      Hanging Drop Culture Workflow with the GravityPLUS Hanging Drop System


      Production starts in the GravityPLUS™ Plate by seeding 40 μL of cell suspension through the SureDrop™ Inlet using your preferred liquid handling device – from a manual pipette to a 96-tip robot. Within 2-4 days for most cell types, a single microtissue will mature within each drop. For long-term culture or differentiation, you can even remove and replenish up to 50% of the medium within the drop through the top inlet. 

      Watch time-lapse video of microtissue formation in the GravityPLUSTM Hanging Drop System.

      Hanging Drop Culture Video Timelapse of Microtissue Formation


      Once the microtissues are formed, they can easily be harvested into the companion GravityTRAP™ Culture and Assay Plate with a simple media addition step. The proprietary non-adhesive coating of the GravityTRAP™ plate allows for culturing over weeks without attachment and decomposition of the microtissues. In addition, its unique design allows reliable medium exchange without microtissue aspiration and excellent imaging, e.g. high-content analysis, through a 1 mm diameter window at the well bottom.

      Hanging Drop Culture- SureXchange Ledge & Culture Chamber    Hanging Drop Culture- Brightfield Spheroid Imaging

    • More than just a plate. A complete 3D culture system.

      Our mission is to make 3D cell culture simple, and we won’t put a product into our customers’ hands that in the end makes life more complicated. Below are a few things to consider prior to simply choosing a cheaper plate. With the GravityPLUS™ Hanging Drop System you get:

      • A complete platform – For starters, each GravityPLUS™ Hanging Drop System 10-pack includes 10 GravityPLUS™ Hanging Drop Plates with base plates and lids, 10 GravityTRAP™ Assay Plates, 10 humidifier pads, disposable tweezers, and instruction manual. 

      • The SureDrop™ Inlet – The name says it all. What good is a hanging drop plate that makes it difficult to form drops? You may notice we don’t go to great lengths to explain an exact pipetting angle, or how to fix missing drops or those stuck mid-channel. That’s because the patented hourglass shape of the SureDrop™ Inlet makes forming a hanging drop simple and reliable...every time. Spring-mounted inlets elastically adjust to downward pressure, ensuring solid contact between the inlet and pipette tip for more efficient pipetting, be it manual or automated. Minimizing drop volume variability results in more precise control over microtissue size, meaning fewer replicates for you to achieve statistical significance. The SBS-standard format increases throughput, saves you handling time, and reduces frustration. 

      • Unparalleled drop stability – You deserve more hanging and less dropping from your hanging drop plate.  The SureDrop™ Inlet design provides maximum surface tension between liquid and plastic, resulting in the most stable drop possible. It’s so stable, you can remove and replace up to 50% of the medium in the drop to replenish medium, add growth factors, or dose with compounds if necessary. Work with the peace of mind that your drops will stay where intended.

      • Improved humidity control – Evaporation of medium from the hanging drop during spheroid maturation or long-term incubation in the drop can alter the ionic strength of the medium and impact the consistency of spheroid formation and size. The greatest air/liquid interface and source of evaporation exists beneath the inlet, on the surface of the hanging drop. Rather than attempting to provide humidity from an upper channel liquid reservoir, the GravityPLUS™ System includes a humidifier pad for each plate that is saturated with buffer and placed in the system’s base plate. This supplies humidity from below where it’s needed most, minimizing evaporation for optimal culture conditions.

      • Simple, efficient spheroid transfer – When it comes to what to do with your spheroid after it has formed, other vendors simply leave you hanging. The GravityPLUS™ Plate includes three pins that ensure perfect alignment of each hanging drop above a corresponding well of the companion GravityTRAP™ plate at just the right height. Simply add medium (70 μl) through the SureDrop™ Inlet, and when the growing drop exceeds the surface tension, spheroids drop safely to the GravityTRAP™ well below, always!

      • The GravityTRAP™ Plate – The reality is, very few experiments are conducted on spheroids in the hanging drop. With other plates, you need to transfer your spheroid to a separate low-attachment plate not designed for use with spheroids, making everything you do past that point more difficult than it needs to be. The GravityTRAP™ receiver plate completes the culture system concept, enabling long-term culture, assay, and analysis of spheroids. Each 96-well GravityTRAP™ plate is ultra-low attachment treated, preventing spheroids from adhering to the well bottom.  The flat-bottom wells include a tapered SureXchange™ design allowing medium exchange without the risk of disturbing or losing spheroids, and the 1 mm diameter circular, clear plastic window in the bottom of each well makes spheroids easier to find, image, and measure.

        Hanging Drop Culture- SureXchange Ledge & Culture Chamber     Hanging Drop Culture- Brightfield Spheroid Imaging

      • InSphero 3D InSight™ - When you buy from InSphero you get more than a complete 3D culture system, you receive support from the world’s leading 3D cell culture company. InSphero tissue engineering experts have formulated 3D-optimized growth media, and partnered with Promega, PerkinElmer, Seahorse Bioscience and other leading technology providers to develop 3D-optimized protocols for key applications, ensuring you can make the most of your microtissues.

       

    • It all adds up

      Why is the GravityPLUS® Hanging Drop System more expensive than other hanging drop or ULA plates?  On the surface, the cost is more for the InSphero system.  But if you sum up the time and materials lost correcting for other imperfect platforms (not to mention your frustration), plus the cost of having to purchase additonal assay plates, you begin to see a true cost comparison.  The simple, fit-for-purpose design of the GravityPLUS® Hanging Drop System lets you spend less time making spheroids, and more time analyzing a more biologically relevant in vitro 3D model.

      We recommend trying GravityPLUS® if you:

      • Are new to 3D cell culture and want to get started using the hanging drop method
      • Have experienced inconsistency with another hanging drop platform
      • Have tried your cell line(s) in ultra-low attachment plates and they failed to form spheroids (ULA plates)
      • Are using primary cells or want to generate more complex co-culture models
       
      Learn more about 3D cell culture basics.

    • Scaffold-free 3D cell culture of any cell type, including primary and stem cells, cell lines and genetically modified cells
    • Compound testing and de-risking
    • Cell-biology studies and tissue engineering
    • Stem-cell differentiation
    • High-content analysis and microscopy
    • Migration from scaffold-based processes to scaffold-free production for scale-up
  • Frequently Asked Questions regarding the GravityPLUS™ Hanging Drop System

    A detailed protocol for production and transfer of microtissues in the GravityPLUS™ Hanging Drop System is provided in the product manual. Below are answers to some frequently asked questions to help get you started. See our introductory video for a brief demonstration.

    Q: Could you recommend a cell concentration for my cell suspension for the generating of microtissues?

    A: For long-term growth profiling, we recommend to start with low cell numbers (250 – 500 cells per drop of 40 µl). If use of non-proliferating cells or rapid production of larger microtissues is required, start with higher numbers (from 2500+ cells per 40 µl). Generally we recommend to try different concentrations for defining your optimal range when using new cell types.

    Q: What is the SureDrop™ inlet?

    A: InSphero’s GravityPLUS™ Hanging Drop Plate features the patent-pending SureDrop™ microtechnology, which allows for precise dispensing into and aspirating from hanging drops. As the drop volume corresponds to the seeding cell number, microtissues produced in GravityPLUS™ Hanging Drop Plates display outstanding size consistency, with variation in diameter of 5% and less across an entire 96-well plate. To assure the SureDrop™ seal, it is important that the pipette tips make sufficient contact with the well surface to assure complete liquid transfer and uniform drop formation. The spring loaded wells elasticity adjust to ensure contact is maintained when downward pressure is applied during pipetting.

    Hanging Drop Culture- SureDrop Inlet

    Q: What is the optimal volume per drop in the GravityPLUS™ Hanging Drop Plate?

    A: To achieve an optimal and stable hanging drop, gently deliver 40 µl (pipetting speed < 50 µl/sec) of cell suspension into each well of the GravityPLUS™ Hanging Drop Plate. Important: For the homogeneity of forming microtissues it is essential to assure a homogeneous distribution of the cells by gently pipetting up and down prior to seeding into the GravityPLUS™ Hanging Drop Plate.

    Q: Why do you recommend pre-wetting GravityTRAP™ wells prior to microtissue transfer?

    A: Pre-wetting the wells of the GravityTRAP™ Plate is required prior to transfer to prevent inclusion of air-bubbles. For that, apply 40 µl of 3D InSight™ Tumor Re-aggregation medium (CS-07-111-02) to each well by placing the tips far into the wells. Remove the pre-wetting solution by placing the tip at the ledge of the upper cavity of the well. Aspirate medium until is completely removed from each well. A negligible amount (< 5-7 µl) may remain in the bottom of the chamber.

    Hanging Drop Culture- Medium Removal and Addition

    Q: How do you transfer microtissues into the receiver (GravityTRAPTM) plate?

    A: Place the raster plate of the GravityPLUS™ Plate onto the GravityTRAP™ Plate by positioning the three pins into the corresponding holes on the top surface of the GravityTRAP™ Plate. The drops under the GravityPLUS™ Plate will then be perfectly aligned with the wells of the GravityTRAP™ Plate underneath. Using a slow pipetting speed (≤ 10 µl /sec), add 70 µl of medium or buffer through the inlet of the GravityPLUS™ Hanging Drop Plate wells. Make sure that the pipette tip forms a tight contact with the well inlets.

    Hanging Drop Culture- Transfer of Microtissues to GravityTRAP Plate

    Q: I am searching for an untreated receiver plate that fits the GravityPLUS™ Hanging Drop Plate. Do you recommend one?

    A: If transfer from the GravityPLUS™ Plate to an untreated receiver plate other than the GravityTRAP™ assay plate is required, we recommend the Nunc™ 96-well plate (e.g., VWR article number 732-2747) which includes receiver holes exactly fitting the alignment pins of the GravityPLUS™ Plate.

    Q: What is the best way to prevent evaporation in the outer wells of my plates?

    A: Evaporation in the outer (perimeter) rows of wells is a phenomenon common to most low-volume culture platforms, and thus requires careful attention to maintaining proper humidity control. Evaporation in the outer rows of hanging drops (GravityPLUS™ Plate) or wells of the GravityTRAP™ Plate although infrequent when following the recommendations below, is a possibility. If not controlled, pronounced evaporation can result in concentration or precipitation of media components (serum, salt) that can impact microtissue formation or health, and can alter the effective concentration of a compound/additive in the medium over the course of a long-term experiment. To provide maximum humidity control when using the GravityPLUS™ Hanging Drop System, we recommend the following:

    • Use an incubator with good humidity control (>95% of rel. humidity), and exercise best practice in maintaining and minimizing loss of humidity.
    • During microtissue formation in the GravityPLUS™ Hanging Drop Plate, always use the supplied humidifier pad in the base plate, thoroughly soaking it with sterile 0.5x diluted PBS. This is key to maintaining proper humidity in the GravityPLUS™ Plate during microtissue maturation in the hanging drop.
    • For incubators with poor humidity control, hypotonic buffer solutions (e.g. 0.2× PBS) may be applied to the humidifier pad.
    • For culture in the GravityTRAP™ Plate, at least 50-70 µl of medium in each well is recommended, and can be increased to a maximum of 80 µl if incubator humidity control is a persistent issue. Medium exchange frequency can also be increased to every other day or daily if conditions dictate. InSphero recommends the use of MicroClime® lids to reduce edge-related evaporation effects during long-term repeat-dosing experimental protocols in the GravityTRAP™ Plate. MicroClime® lids are supplied for use with every plate of InSphero assay-ready 3D InSight™ Microtissues.

     

    Q: Why do my cells not form microtissues?

    A: There are several reasons why a particular cell line, cell type, or co-culture may not re-aggregate and form a microtissue. Pre-treatment/harvesting conditions, cell seeding density (or ratio in co-culture models), media formulation, incubation time, media evaporation, and many other factors can impact the success or failure of microtissue production and culture. Many tumor cell lines are known to readily form microtissues in ultra-low attachment (ULA) treated plates, and we recommend our GravityTRAP™ ULA plates (ISP-09-001) as a starting point if the literature suggests this is the case for your cell line of interest, or if you are unsure. For primary cells, more complex co-cultures, or cell lines that fail to form spheroids in ULA plates, we recommend the GravityPLUS™ Hanging Drop System (ISP-06-001). Some cells may require co-culture with fibroblasts or supplemental growth factors in order to form dense, spheroidal microtissues, or to establish and maintain an organotypic phenotype. Contact our 3D experts if you require additional assistance troubleshooting culture of a particular spheroid model.

    Q: What is the best way to image microtissues in the hanging drop?

    A: Microtissue formation, appearance and growth profiles can be assessed using an inverted brightfield microscope. A long-working-distance objective (LWDO), preferentially of 10x magnification, is required for proper imaging. Depending on the minimal gap (D1) between the objective plane and the microscope stage, the specifications of the objective should include a working distance of minimally 11.5 mm + D1. As long-working distance objectives are commonly shorter in height than regular objectives, a distance ring (R) will be required to elevate the long working distance objective plane to the level of other regular objectives installed on the objective revolver. The included humidifier pad has a rectangular opening in the center to allow easy visualization of hanging drops in the most central wells. If microscopic assessment of all 96 wells is desired, simply transfer the Hanging Drop Plate to an empty, sterile base plate for imaging, then return to the humidified base plate prior to returning to the incubator.

    Hanging Drop Culture Microscopy  Specifications

    Hanging Drop Culture- Base Plate with Humidifier Pad

    MicroClime® is a registered trademark of Labcyte Inc.
 

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