Abstract 2072: Human liver patient derived organoids: Development and characterization of a new tissue mimetic model

Abstract

Abstract Liver is the largest solid organ and performs hundreds of vital functions including drug metabolism. Liver diseases, including viral infections, cancer and metabolic disorders account for about 2 million deaths annually, worldwide (1). Current models for studying liver biology and drug metabolism include 2D cultured primary cells/cell lines, which do not represent the metabolic and structural complexity of human tissue. In vivo models for liver diseases are time consuming, cost prohibitive and often ineffective. Therefore, better tissue mimetic liver models are needed. Organoids are self-assembled 3D cultured cellular units that may mimic the corresponding human tissue structurally and functionally (2). Patient derived organoids (PDO) are developed from tissue of specific patients and may resemble the source tissue in molecular features, including recapitulation of patient specific responses to therapies. Here, we report development and characterization of novel liver PDO lines from vendor-sourced cryopreserved tissue. We have developed biobanks of undifferentiated as well as differentiated liver PDO models.Human cadaver-sourced liver tissue were obtained with explicit donor consent. Liver PDO were generated following modification of a previously established protocol (3). Briefly, samples were minced, digested to single cells, and filtered. Specific number of cells were mixed with diluted Matrigel (Corning) and added as small drops on cell culture plates and grown in liver PDO specific growth media. Whenever appropriate, PDO were differentiated by culturing in differentiation-specific media in multi-well plates. In all cases, PDO were monitored using brightfield microscopy (Olympus CK40) and live cell imaging (Muvicyte, Perkin Elmer); and passaged every 6-8 days. Undifferentiated PDO were cryopreserved/thawed following in-house SOP to confirm viability. Liver-specific biomarker expression in the PDO lines were confirmed by confocal microscopy (ImageExpress, Molecular Devices). PDO lines were also assayed for ALT activity, albumin production and urea. PDO were cleared for microbial/viral contamination and confirmed as unique lines using single tandem repeat analyses. Transcriptome profiles were analyzed using vendor services. So far, we have successfully generated 3 liver PDO lines, both undifferentiated as well as differentiated. Our liver PDO lines would be highly useful in understanding liver biology and disease models; and will have significant usage in drug testing, including DMPK and ADME/Tox studies. Future efforts would include scaling up, development of liver specific assays and developing PDO from specific liver diseases, including hepatocellular carcinoma, MASLD and NASH.