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iCell® Hepatocytes 2.0: Now in 3D!


Figure 1: iCell Hepatocytes 2.0 Are Capable of Forming 3D Spheroids
(A) Cells are initially thawed and plated on collagen I so that they can fully recover from cryopreservation and establish an interconnected, confluent 2D culture. (B) Cells are then gently detached and re-plated into a spheroid forming culture plate. 3D spheroids are generated within 1 – 2 days.

The world of iPSC-derived hepatocytes is no longer flat (Figure 1). CDI has identified protocols enabling the formation of stable 3D spheroids in culture.

A growing body of literature suggests that moving beyond traditional static-plated culture yields a more liver-like environment for hepatocyte assays and generates more predictive biology in vitro. The combination of iPSC technology with advanced culture techniques offers advantages over existing models via:

  • A uniform cell source in the form of iPSC-derived hepatocytes removing the inter-lot and inter-donor variability observed with primary hepatocytes
  • Enhanced functional maturity of the cells imparted by the increased complexity of the culture conditions leading to enhanced predictive power

Conditions developed allow for a tunable spheroid size with maintenance of viability (Figure 2) and put the control over engineered tissue in the hands of the user. This novel workflow allows for the generation of iCell® Hepatocytes 2.0 microtissues in low-attachment plates.

CDI is in the process of functionally characterizing these iCell spheroids with the expectation that they will exhibit prolonged culturability without using a Matrigel® overlay–beyond the already impressive 21 – 28 day lifespan observed in 2D culture on collagen-coated plates. In addition to enhanced culturability, preliminary data suggest that 3D cultures demonstrate increased functional maturity (Figure 3).

See the Protocol: Modeling 3D Liver Tissue: 3D Hepatocyte Spheroids in Low Attachment Plates


Figure 2: The Size of iCell Hepatocytes 2.0 Spheroids Can Be Easily Controlled
(A) The spheroid size can be tuned by adjusting the number of cells seeded into low-attachment wells before spheroid formation. (B, C) Viability correlates with cells per aggregate, suggesting that the microtissues maintain cell health throughout (CellTiter-Glo™ Assay, Promega).


Figure 3: iCell Hepatocytes 2.0 Spheroids Exhibit Highly Reproducible CYP3A4 and CYP1A2 Induction
(A) CYP3A4 induction in response to a rifampicin titration and (B) CYP1A2 induction in response to an omeprazole titration were measured by luminescent functional readout of activity (P450-Glo™ CYP3A4 Assay and P450-Glo CYP1A2 Assay, Promega, respectively).




Contact CDI’S Technical Support to learn more about 2D and 3D culture of iCell Hepatocytes 2.0 and other ways CDI can further your research through iPS cell technology.