Cryobiology 54 (2007) 164–172 www.elsevier.com/locate/ycryo 0011-2240/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2006.12.005 Characterization of rat and human KupVer cells after cryopreservation  Peter Walbrun a , Claus Hellerbrand a , Thomas S. Weiss b , Susanne Netter a , Daniel Neumaier c , Erwin Gaebele a , Reiner Wiest a , Juergen Schoelmerich a , Matthias Froh a,¤ a Department of Internal Medicine I, University of Regensburg, 93042 Regensburg, Germany b Center for Liver Cell Research, University of Regensburg, Germany c Institute of Experimental and Applied Physics, University of Regensburg, Germany Received 28 March 2006; accepted 12 December 2006 Available online 8 January 2007 Abstract KupVer cells (KC) are the resident macrophages of the liver and represent about 80% of the total Wxed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physio- logically active substances such as eicosanoids and inXammatory cytokines (IL-1, IL-6, TNF), and produce free radical species. Thus, KC are attractive targets for anti-inXammatory therapies and potential candidates responsible for diVerences in inXammation in liver dis- ease seen between diVerent individuals. However, to perform parallel in vitro experiments with KC from diVerent donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard con- ditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at diVerent time points and cytokine release was analyzed with IL-6 and TNF ELISAs, respectively. Phagocytic capacity was investigated by using a speciWc phagocytosis assay and FACS analy- sis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be com- pared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not diVer in the cultiva- tion proWles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNF or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no diVerences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological char- acteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.  2007 Elsevier Inc. All rights reserved. Keywords: KupVer cells; Cryopreservation; Tumor necrosis factor-; Interleukin-6; Phagocytosis; Bacterial endotoxin KupVer cells (KC) are macrophages that reside in the hepatic sinusoids, predominantly in periportal regions, and are usually attached to endothelial cells. They show recep- tor-mediated, endocytotic activity, which is important for the uptake of foreign particles, mainly microorganisms and bacterial endotoxins. Although KC make up <5% of the liver volume, they represent about 80% of the total Wxed macrophage population in the human body. KC play a major role in physiological processes as well as in the path- ogenesis of several disease states, including endotoxin shock [1] and alcoholic liver disease [23]. They release physiologically active substances such as eicosanoids,  This work was supported in parts by a grant from the Deutsche For- schungsgemeinschaft (FR 1644/4-1). * Corresponding author. Fax: +49 941 944 7011. E-mail address: matthias.froh@klinik.uni-regensburg.de (M. Froh).