An in vitro 3D Model of Functional Human Liver Organoids

Abstract

Introduction: The development of adequate model systems to investigate human liver function and metabolism in health and disease has proved challenging. Three-dimensional (3D) liver tissues reconstituted from liver cells, termed liver organoids, have enormous potential for investigating aspects of liver disorders and drug toxicity. The aim of our study is by integration of multiple novel surgical techniques, engineering of growth factors and morphogens bound to extracellular matrix, direct cell reprogramming to produce functional 3D liver organoids. Methods: Tissue from 20 liver resections were used for hepatocyte isolations. Tissue underwent a two-step EDTA/collagenase digestion. Cell viability was determined by ATP luminescence and 7AAD. qPCR, FACS, Western blot, imunofluorescence analysis and biochemical assays were undertaken to ascertain cellular phenotype and function in 3D cultures. Additionally, liver development-associated signaling pathways were tested. Results: We demonstrate that there is direct correlation between liver status, the number and viability of isolated hepatocytes and their functional properties in 2D and 3D environment. FACS analysis indicated that prolonged cultivation in 2D leads to changes in cell populations. We also generated functional liver 3D organoids that maintained viability up to three weeks, produced albumin and expressed liver specific genes HNF4, CK19, AAT, CYP3A4. Conclusions: We have successfully established a method for the culture of human liver organoids in novel engineered 3D environments that can be used for biomarker discovery, drug toxicity studies and liver transplantation.