Overview of Akura™ 3D SureKit
The Akura™ 3D SureKit provides a validated all-in-one solution to generate and culture organotypic 3D spheroids from primary human hepatocytes.
The 3D SureKit contains frozen primary human hepatocytes and specialized media to aggregate the cells and culture-formed spheroids. 3D optimized Akura™ 96 plates are included to allow for homogenous aggregation and loss-free media exchanges. Reference characterization data and dose-response curves for model DILI compounds are provided as benchmark data for your in-house experiments.
- Hepatocytes guaranteed to form 3D spheroids
- The best-in-class 3D cell culture compatible Akura™ 96 Spheroid Microplates, which guarantee homogenous aggregation of cells, minimal tissue loss during media exchanges, and an optimized geometry designed to minimize media evaporation
- InSphero proprietary aggregation and culture media are essential for generating and maintaining spheroids
- Access to QC data and protocols
Note: This Akura™ 3D SureKit contains validated hepatocytes from a single donor. If you would like to work with more complex models or microtissues from pooled donors, please check our 3D InSight™ products or talk to our customer service representative.
Components of the Akura™ 3D SureKit
- 3 x Akura™ 96 Spheroid Microplates
- 1 x Akura™ Tilting Stand
- Primary human hepatocytes, cryopreserved (> 1 million per vial)
- 40 ml Aggregation base medium stored at 4°C
- 12.5 ml Aggregation supplement stored at -20°C
- 200 ml Maintenance base medium stored at 4°C
- 10 ml Maintenance supplement stored at -20°C
Features of the Akura™ 3D SureKit
Ease of use and high reproducibility by using pre-qualified cells and thoroughly tested protocols
Flexibility to perform your assays at your convenience, you can store the kit until just before performing your assays.
Options to modify hepatocytes before or during the aggregation process (e.g. by viral transduction methods or gene-editing systems)
Possibility to tailor the spheroid size by varying seeding cell numbers
Option to customize spheroid composition by introducing additional cell types
An advantage to benchmark generated liver spheroids to characterization data (morphology, cell health, functionality, and sensitivity towards a panel of standard DILI compounds)
Our manuals and protocols contain valuable information and hands-on advice to get you started immediately.
A detailed protocol for the production of hepatocyte spheroids is provided in the product manual
FAQs of the Akura™ 3D SureKit
Q: What is the optimal volume per well in the Akura™ 96 Plate within the Akura 3D™ SureKit?
A: To achieve optimal volume per well, 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.
Q: Why do you recommend centrifuging the Akura™ 96 Plate in the Akura 3D™ SureKit after cell seeding?
A: We recommend briefly centrifuging the plate after cell seeding to remove any air bubbles and to force the cells to the bottom of the well in order to promote cell aggregation and spheroid formation.
For that, place the lid on the plate and spin it in a microtiter-plate centrifuge for 2 minutes at 250 RCF. Afterward, incubate the plate in a humidified CO2 incubator at 37 °C for 2-5 days.
Q: How do I exchange the medium in the Akura™ 96 Plate in the Akura 3D™ SureKit without disturbing or losing the spheroids?
A: To prevent spheroid/organoid 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, see figure below, and remove the medium at a low pipetting speed (>30 µl/sec). A minimal volume of ~ 5-7 µl will remain in the well.
Then, add 70 µl of the fresh medium by placing the pipette tip at the ledge, using 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.
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.
If not controlled, pronounced evaporation can result in the 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:
- Use an incubator with good humidity control (>95% of rel. humidity), and exercise best practices in maintaining and minimizing loss of humidity (e.g. minimize incubator door opening and closing).
- For culture in the Akura™ 96 Plate, at least 50-70 µl of the 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.
- We recommend the use of the InSphero Incubox™ to reduce edge effects when performing long-term culture with the low-frequency medium exchange. The Incubox™ is available here