78 Chapter 3 (Sigma Aldrich), 10% RSpondin Conditioned Medium (provided by Calvin J. Kuo), 30% Wnt3a CM (provided by Hans Clevers), 100 ng/ml Noggin (Petrotech) 50 ng/ ml HGF (Peprotech), 100 ng/ml FGF-10 (Peprotech), 50 ng/ml EGF (Peprotech), 10nM Leu-gastrin. Three days after the isolation Noggin and Wnt3a CM were removed from the medium. The medium was changed every 2-3 days. Liver progenitor organoids were passaged every 6-7 days. Insult-exposed organoids were grown in the same medium but in the presence of no amino acids. Organoid imaging Organoid cultures were followed over time with an AxioObserver Z1 compound microscope (Carl Zeiss), 2.5x and 5x objectives and an AxioCam MRm3 CCD camera (Carl Zeiss) in brightfield. Raw images were processed using the ZEN 3.1 blue edition software. The whole well was imaged by selecting 9 tiles that were later on stitched together. On average 20 different Z-stacks were imaged in order to ensure all organoids in the Matrigel dome were detected. The number and size of organoids were assessed in the whole well. For the first case study, 95 organoids (previously measured by OrganelX) were manually counted. For the second case study, and tracking in time of the organoids, a set of 25 organoids were randomly selected and measured. Images, of the same cultures, were acquired every 24 h and the same 25 organoids were measured at every time point. For manual measurement of the organoids, ZEN3.1 (blue edition) from Carl Zeiss Microscopy GmbH was used. The outline of the organoids was traced using the draw spline tool and the area was calculated by the software. The OrganelX e-Science service For the purpose of studying organoids in culture the free e-Science webpage* offers different functionalities. The service offered by the website begins after uploading a CZI file by the user. A CZI file, which is suitable for microscope image data and captured by the Carl Zeiss microscope, contains tile images for each stack as well as image stacks. It is to be mentioned that the system will be further developed to include different types of images files. In addition to the CZI file, the user needs to provide a short experiment ID and sample ID. This is beneficial for future comparisons between experiments and samples in experiment. The user will be redirected to a page that tracks the request’s progress and presents the final results in an interactive way. Organoids in each image stack are automatically detected and segmented using Mask-RCNN17. Mask-RCNN is a state-of-the-art instance segmentation
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