We haven’t seen any organoid formation with our hepatocyte cells after 3 days, should we give it more time or start over?

Answer

Normally, 3 days it is very short time, but it also depends on the nature of your original materials (mouse or human hepatocytes) and whether after the isolation the hepatocytes were plated as single cells or clumps of cells. In the case of single human hepatocyte cells it will require more than a week to start observing hepatocyte organoids.

Under conventional 2D monolayer culture conditions, primary human hepatocytes (PHHs) rapidly lose their hepatic phenotypes whereas PHHs cultured in 3D culture can maintain the viability and functions for several weeks.

To support the 3D liver spheroid model in drug discovery and development studies, Corning offers 3D spheroid-qualified primary human hepatocytes using Corning spheroid microplates. Using the protocol described in the Application Note: 3D Primary Human Hepatocytes (PHH) Spheroids Demonstrate Increased Sensitivity to Drug-induced Liver Injury in Comparison to 2D PHH Monolayer Culture, PHHs form small cell clusters, larger cell aggregates, and then spheroids in 6 to 7 days. Once formed, PHH spheroids remain stable over 4 weeks as shown by morphology and size measurement.

Based on this finding, the generation of spheroids can take up to 7 days.

However, those spheroids were generated from primary human hepatocytes. Organoids can be generated from, for example, isolated duct cells. In “Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation”, a protocol for the generation of organoids is described in which the isolated duct cells are cultured in Corning® Matrigel® matrix after which organoids become visible after about 7 days in culture.

For both approaches, the formation of the spheroid/organoid can take up to 7 days. We, therefore, would recommend keeping the cells in culture and reevaluate the presence of organoids at day 7.