Final Report Summary - CLOC (Cultured Liver Organoids for Investigation and Treatment of Inherited Cholestatic Diseases) In this project we aimed to develop novel approaches to treatment of cholestatic liver disorders using cultured liver organoids. In order to progress towards the aims of the project many innovative solutions had to be made. 1. Generation of cell lines. Skin fibroblasts from patients with cholestatic liver disorders have been reprogrammed into Induced Pluripotent Stem Cells (iPSc) and several cell lines have been generated from each patient. Some of the iPSc have been mutation corrected using lentiviral vectors in order to generate control lines. The patient derived and corrected lines are now available for translational projects. 2. Custom-made bioreactor. In order to facilitate long-term cell culture of liver cells we engineered a specific bioreactor that generates controlled environment for unique culture of liver cells. The single or multiple cell types can be cultured in this bioreactor simultaneously which is adapted to generate liver model. 3. iPSc differentiation and culture protocols. Special cell differentiation protocols have been adapted to bioreactor environment in order to generate hepatocyte-only liver model or hepatocyte + endothelial or hepatocyte + cholangiocyte liver models. 4. Generation of scaffolds from biological and artificial materials and matrix repopulation protocols. Specific protocols had to be adapted for the liver model to generate repopulated scaffolds seeded with cells cultured in the bioreactor. 5. Liver cell assays. Many assays were adapted specifically for the bioreactor use in order to assess function of the liver model in real time without disturbing cell culture. 6. Testing of the genetic therapies in the liver model. We were able to adapt the protocols for testing the innovative therapies in the live cultured patient derived cells. The methods and new models developed in this project will allow faster progress in developing therapies for human liver disease and reduce the need for animal studies.
