Date: Saturday, May 30, 2020
Session Time: 3:15pm-4:00pm
Presentation Time: 3:30pm-4:00pm
*Purpose: Animal models used for evaluating the mechanisms of liver disease do not fully mimic the complex architecture and metabolic functions of the human liver. Currently available 2D in vitro human primary hepatocyte (HC) culture systems are inadequate due to the rapid loss of HC function. Novel 3D-structured in vitro human liver models that can recapitulate the in vivo liver microenvironment and stably maintain the long-term pathophysiological function of the liver are critically needed.
*Methods: To achieve major improvements of the human HC culturing system, we incorporated primary human HCs, HPV-E6/7 expression lentivirus-immortalized hepatic stellate cells (HSCs), and human telomerase expression lentivirus-immortalized liver sinusoidal endothelial cells (LSECs) to generate 3D-liver spheroids. We studied molecular mediators that are capable of preserving the in vivo phenotype of liver spheroids with 3 different chemicals (C), such as TGFbeta inhibitor (SB431542), adenylyl cyclase agonist (Forskolin), and gamma-secretase inhibitor (DAPT). Furthermore, we developed a 3-Stage liver organoid differentiation method.
*Results: 3D-liver spheroids were kept in media with 2C or 3C for 3 weeks. Best-sustained HC function was achieved with 3C medium, as demonstrated by increased expression of ALBUMIN (ALB), CYP2B6 (phase I enzyme), and UGT1A3 (phase II enzyme) on day 21 (Figure-1B). Addition of DAPT in the medium together with SB431542 and Forskolin (3C) repressed COL1A1 and COL12A1 in liver spheroids.The 3-Stage-culture method spheroids exhibited the features of human hepatic architecture and survived up to 40 days with robust expression of P450-enzyme and ALB. On day 35-40, CYP3A4-positive cells and ALB-positive cells were distributed at the distinct regions of the spheroid, which indicated the establishment of HC functional zonation (Figure1M).
*Conclusions: This established 3-Stage human 3D-liver organoids culturing system will greatly facilitate the investigation of liver disease mechanisms, providing a model with prolonged liver function for discovery and development of new treatments.
To cite this abstract in AMA style:Zhang W, Isidan A, Chen AM, Smith LJ, Francis HL, Alpini G, Gramelspacher E, Li P, Ekser B. Prolonged Functional Human 3D Liver Organoids as an In Vitro Liver Disease Model [abstract]. Am J Transplant. 2020; 20 (suppl 3). https://atcmeetingabstracts.com/abstract/prolonged-functional-human-3d-liver-organoids-as-an-in-vitro-liver-disease-model/. Accessed April 15, 2021.
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