The Akura™ 96 Spheroid Microplate is the super convenient culturing plate for your daily spheroid/organoid culturing in every lab environment, from manual to automated handling. The plate is designed for lossless spheroid/organoid formation and handling as well as reliable cell culturing and analysis.
Product Documents
A detailed protocol for the production of spheroids in the Akura™96 Spheroid Microplate is provided in the product manual. Below are answers to some frequently asked questions to help get you started.
The Akura™ Spheroid Microplate features a unique conical well design with a SureXchange™ ledge that enables near-complete medium exchange while reliably retaining spheroids in the wells.
In conventional round-bottom ULA plates (a) spheroids disintegration can occur due to shear forces on tissue surface, and (b) spheroids can accidentally be aspirated during medium exchange when removing more than 50% of the medium in the wells.
Figure 1: A. Spheroid disintegration with flow due to shear forces on tissue surface. B. Accidental aspiration during medium exchange.
Not all cells aggregate to spheroids. First, it highly depends on the architecture, function, and morphology of the tissue/organ of which they are isolated from and how the isolation affected the cells. Further, we see variations in successful aggregation between donors for the same cell type. Plateable cells, in general, have a good chance to form spheroids. To achieve optimal spheroid formation, it may be necessary to modify the aggregation conditions such as modifying the cell concentration or media composition, or by the addition of supporting cell types (e.g., matrix secreting cells) or supplements (e.g., ECM). For new cell types we recommend trying a variety of aggregation conditions.
For long-term growth profiling, we recommend starting with low cell numbers (250 – 500 cells per well of 70 µl). If use of non-proliferating cells or rapid production of larger spheroids are required, start with higher numbers (from 2500+ cells per 70 µl). Generally, we recommend trying different concentrations for defining your optimal range when using new cell types.
To achieve optimal conditions, gently deliver 70 µl (pipetting speed < 10 µl/sec) of cell suspension into each well of the Akura™ 96 Plate by placing the pipette tips near, but not touching, the bottom of the wells.
Important: For 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™ 96 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 microtiter-plate 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.
To prevent spheroid loss during the exchange of media, the SureXchange™ ledge at the inside wall of each well serves as an anchoring point for the pipette tip. Just place the tip at the ledge of the well and remove the medium at low pipetting speed (>30 µl/sec). A minimal volume of ~5-7 µl will remain in the well.
Then, add 70 µl of fresh medium by placing the pipette tip at the ledge, use dispensing rate <50 µl/sec.
Important: when using automated liquid handling devices, an off-center alignment of the vertical pipette tip will achieve the same effect.
Figure 2: SureXchange™ ledge for manual handling.
Evaporation in the outer (perimeter) rows of wells is a common phenomenon 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:
To provide maximum humidity control when using the Akura™ Plates, we recommend the following:
Figure 3: InSphero Incubox™ to prevent evaporation in the outer wells of the Akura™ Plates.
In order to achieve optimal results, a few relatively simple changes need to be made by a knowledgeable instrument operator. By adhering to the suggestions below, the Akura™ 96 Plate can be used successfully with nearly all high content imaging platforms:
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