The Akura™ 384 Spheroid Microplate is our ultimate spheroid screening plate unifying robust liquid and spheroid handling with automated high-throughput screening applications. The flat bottom and black walls of the InSphero 384-well plate facilitate fluorescent imaging, while the special well design allows medium exchange, dosing, and endpoint determination in the same plate saving time and resources.
Product Documents
A protocol for the production of spheroids in the Akura™ 384 Plate is provided in the product manual. Below are answers to some frequently asked questions to help get you started.
Pre-wetting the wells of the Akura™ 384 Plate is recommended prior seeding to prevent inclusion of air-bubbles. For pre-wetting, apply 50 µl of PBS to each well by placing the tips near to, but not touching the bottom of the well.
Centrifuge the Akura™ Plate for 2 minutes at 250 RCF and incubate it in a humidified CO2 incubator for at least 1 day. Before cell seeding take the Akura™ Plate from the incubator, centrifuge the Akura™ Plate for 2 minutes at 250 RCF. Aspirate the PBS by placing the tip at the ledge of the upper cavity of the well. Aspirate until the PBS is removed from each well. A small amount of PBS (< 2-3 μl) remains in the bottom of the chamber.
For long-term growth profiling, we recommend starting with low cell numbers (250 – 500 cells per well of 50 µl). If use of non-proliferating cells or rapid production of larger spheroids are required, start with higher numbers (from 2500+ cells per 50 µl). Generally, we recommend trying different concentrations for defining your optimal range when using new cell types.
To achieve optimal conditions, gently deliver 50 µl (pipetting speed < 10 µl/sec) of cell suspension into each well of the Akura™ 384 Plate by placing the pipette tips far into the wells (avoid touching the well bottom).
Important: To obtain spheroids with uniform size and cell composition it is essential to assure a homogeneous distribution of the cells by gently pipetting up and down prior to seeding into the Akura™ 384 Plate.
We recommend to briefly centrifuge the plate after cell seeding to remove any air bubbles and to force the cells to the bottom of the well to promote cell-aggregation and spheroid formation.
For that, place the lid on the plate and spin in a microplate centrifuge for 2 minutes at 250 RCF. Afterwards, incubate the plate in a humidified CO2 incubator at 37 °C for 2-5 days.
Tilt the plate in the incubator to approximately 30° or use Akura™ Tilting Stand (InSphero, CS-AG11) to improve the maturation process.
The Akura™ 96 and 384 Spheroid Microplates are ‘all-in-one’ solutions and can be used for the aggregation of spheroids, long-term culturing experiments due to the stable coating and the unique well design, and for screening and imaging endpoints. As the lower cavity has an optical window of only 1 mm diameter, we recommend not to grow spheroids or organoids, which are larger in diameter than 800 μm.
The unique design of the Akura™ 384 Plate enables the use of semi-and automated liquid handling systems for parallel liquid handling without the risk of spheroid loss. We recommend the INTEGRA VIAFLO 96/384 system as it is a compact, easy-to-use, semi-automated pipette system that can be configured for 384 channels for increased productivity (find more information and a detailed protocol in the Product Manual).
When using automated liquid handling devices, an off-center alignment of the vertical pipette tip of approximately 1 mm is needed for placing the pipette tip at the ledge of the Akura™ 384 Plate, see
Figure 1, left image.
Figure 1: Medium exchange in Akura 384 Spheroid Microplate using automated liquid handling systems.
Note: Alternatively, you can use the Akura™ 384 VIAFLO adapter, which enables a precise positioning of Akura™ Spheroid Microplates on the plate holders of the Integra VIAFLO 96/384 pipetting system. With the adapter, a defined offset between pipette tip and well center is created. This results in a precise positioning of the pipette tip on top of the SureXchange™ ledge in the wells of the plate. For more information, please download the Technical Specifications of the VIAFLO adapter.
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. If not controlled, pronounced evaporation can result in concentration or precipitation of media components (serum, salt) that can impact spheroid 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 Akura™ Plates, we recommend the following:
1. Use an incubator with good humidity control (>95% of rel. humidity), and exercise best practice in maintaining and minimizing loss of humidity (e.g., minimize incubator door opening and closing).
2. For culture in the Akura™ 384 Plate, at least 40-50 μl of medium in each well is recommended. Medium exchange frequency can also be increased to every other day or daily if conditions dictate.
3. We recommend the use of the InSphero Incubox™ (CS-AH11) (Figure 3) to reduce edge effects when performing long-term culture with low-frequency medium exchange.
Figure 2: InSphero Incubox™
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