Extending the choice of ULA plates for primary 3D human liver spheroid formation and experimentation with the Akura™ Spheroid Microplates

Abstract: 3D human liver spheroid formation

3D spheroids can be formed through natural aggregation in specific culturing microplates. The common feature for self-aggregation is the non-adherent surface given by a special coating so that cells do not spread out in 2D but form and reorganize to a spherical 3D tissue structure. By using different cell types seeded in a defined ratio into wells, spheroids can model physiologically relevant organ functions and are referred to as microtissues (MT). Plate design, features, surface property, and production quality largely determine the spheroid quality, reproducibility, and organ-specific function of the spheroid.

"The choice of ultra-low attachment plates impacts primary human hepatocyte spheroid formation, phenotypes, and function" as Xing et al. have presented in a comprehensive study carrying this title by comparing several commercially available 96- and 384-well plates for primary liver spheroid aggregation, culturing and analysis [1]. The study addresses aggregation efficiency and quality, their compatibility for imaging, spheroid viability and robustness over time, as well as the preservation of liver-specific function and response to known toxic compounds.

At InSphero, in contrast to the U-shaped well designs, we invented the Akura™ Plates with conical (V-shaped) well design including SureXchange™ ledges that enable precise medium exchange, compound dosing, and enables compatibility with fully- and semi-automated liquid handling systems. A flat bottom proves superior for imaging. Here, we present historical and comparative data obtained from multiple liver spheroid production runs and their characterization in the Akura™ 96 and 384 Spheroid Microplates.

Using optimized protocols, we demonstrate the compact formation of spheroids with low variation across the complete Akura™ plate with respect to diameter and viability. Further, a high reproducibility over several production runs (up to 30’000 microtissues/per month) is achieved. The uniquely designed conical well enables gentle and precise medium exchange and compound dosing, resulting in a low and consistent coefficient of variation over time for spheroid diameter (< 5%) and viability (< 10%). The response to our reference compound Chlorpromazine (IC50) can be reproduced over tens of experiments and production runs.

The results not only support the conclusion of Xing et al. on the importance of the plate format but also expand the previous study and broaden the choice for spheroid users to select the right plate for their application. Our Akura™ ULA plates with conical shape allow ease of use, imaging, and automation while supporting the physiological function of liver MTs at scale.

[1] Xing, C., Kemas, A., Mickols, E., Klein, K., Artursson, P. , & Lauschke, V.M. (2024). The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid

Download InSphero's poster about the 3D Human Liver Spheroid Formation

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