Research Article Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells Matthias Bartneck, 1 Klaudia Theresa Warzecha, 1 Carmen Gabriele Tag, 1,2 Sibille Sauer-Lehnen, 1,2 Felix Heymann, 1 Christian Trautwein, 1 Ralf Weiskirchen, 2 and Frank Tacke 1 1 Department of Medicine III, RWTH University Hospital Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany 2 Institute of Molecular Pathobiochemistry, Experimental Gene Terapy and Clinical Chemistry, RWTH University Hospital Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany Correspondence should be addressed to Frank Tacke; frank.tacke@gmx.net Received 17 April 2015; Revised 23 June 2015; Accepted 24 June 2015 Academic Editor: Nady Braidy Copyright © 2015 Matthias Bartneck et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hepatic stellate cells (HSC) are the main efector cells for liver fbrosis. We aimed at optimizing HSC isolation by an additional step of fuorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fbrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a signifcantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60–95%), primarily by excluding doublets of HSC and Kupfer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fbrosis. Viability, migratory properties, and HSC transdiferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, diferentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS signifcantly increases cell purity by removing contaminations from other cell populations especially KC, without afecting HSC viability, migration, or diferentiation. 1. Introduction Hepatic stellate cells (HSC) are the main efector cells in liver fbrosis [1]. In homeostatic conditions, they reside in the perisinusoidal space of Diss´ e, store vitamin A, and are involved in maintaining tissue integrity [2]. In case of liver injury, HSC can be activated by diferent stimuli such as macrophages [3] or danger-associated signals [4]. Activated HSC were found to release proinfammatory mediators and transdiferentiate into myofbroblasts, which are highly pro- liferative and produce large amounts of extracellular matrix proteins such as collagen types I and III. Tis process leads to the excess production of hepatic connective tissue, ultimately leading to hepatic fbrosis, and reduced in liver functionality [5]. Activated HSC are considered one of the major target cells for antifbrotic therapies, because they are the main contrib- utors of hepatic extracellular matrix [6]. In order to study HSC biology and to evaluate therapeutic strategies afecting HSC activation or functionality, primary HSC isolation from human, mouse, or rat liver is an evitable tool in experi- mental fbrosis research. Early attempts to isolate HSC from mouse or rat livers were based on centrifugal fractionation and/or centrifugal elutriation [7, 8]. Subsequent methods incorporated the simultaneous isolation of diferent hepatic cell populations based on density gradient centrifugation with Stractan [9]. With the rise of fow cytometry and fow cytometric cell sorting, early attempts for fow cytometric cell sorting were based on the strong sideward scattering of HSC due to the specifc intracellular (retinol) droplets [10]. Later Hindawi Publishing Corporation Analytical Cellular Pathology Volume 2015, Article ID 417023, 13 pages http://dx.doi.org/10.1155/2015/417023