Studying human hepatic immunology through the isolation and culture of liver slices and purified cellular subpopulations

Abstract

Abstract Although advances in culture techniques have allowed us to better understand hepatocyte inflammatory signaling under both normal and diseased conditions, still little is known about how non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and hepatic macrophages (i.e. Kupffer cells) impact these events in the intact human liver. In order to investigate this network of cellular cross-talk, we have developed two novel ex vivo culture systems. In Method A, 250 μm slices of human liver tissue were cultured in a transwell system. These slices induced inflammatory and tissue repair signals following both physical injury and challenge with pro-inflammatory ligands (polyI:C, LPS, and LTA). Responses following physical injury were consistent with an acute phase response, while pro-inflammatory ligands induced responses consistent with TLR activation and production of type I interferon. In Method B, we perfused wedges of liver tissue and isolated purified hepatocyte and NPC populations. Hepatocytes isolated by this procedure were successfully maintained in culture for up to 14 days on collagen-coated plates. Isolated NPCs were further purified by FACS analysis into individual populations of LSECs, HSCs, and Kupffer cells. Purified Kupffer cells recovered from sorting were also successfully maintained in culture. Together, these systems will ultimately allow us to examine how the presence of intact liver structure and/or various NPC populations impacts liver cellular signaling in response to different immune stimuli.