3D Organoids from primary melanoma cell lines and from iPSc-derived neural crest stem cells

We propose to generate three-dimensional tumoroids from the primary melanoma cell lines, as well as 3D organoids from the iPSc-derived neural crest stem cells. We will use the AggreWell system (STEMCELL Technologies) to generate uniform, size-controlled three-dimensional spheroids. After 5 days in the AggreWell plate, the spheroids are moved to a PEG-based artificial ECM hydrogel (Gjorevski et al.; Nature Protocols 2017). The organoids can be cultured for weeks in these PEG-droplets. At different time points during organoid culture, organoids will be used for immunostaining and/or for single-cell sequencing. We will dissociate the PEG gel to obtain single cells by use of the cell-dissociation enzyme TrypLE.