Human liver slices as an in vitro model to study toxicity-induced hepatic stellate cell activation in a multicellular milieu

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Abstract

Introduction

Hepatic stellate cell (HSC) activation is a key event in wound healing as well as in fibrosis development in the liver. Previously we developed a technique to induce HSC activation in slices from rat liver. Although this model provides a physiologic, multicellular milieu that is not present in current in vitro models it might still be of limited predictive value for the human situation due to species-differences. Therefore, we now aimed to evaluate the applicability of human liver slices for the study of HSC activation.

Method

Liver slices (8 mm diameter, 250 μm thickness) were generated from human liver tissue and incubated for 3 or 16 h with 0–15 μl of carbon tetrachloride (CCl4) after which ATP-content and expression levels of HSC (activation) markers was determined.

Results

Human liver slices remained viable during incubation as shown by constant ATP levels. Incubation with CCl4 caused a dose-dependent decrease in viability and an increase in mRNA expression of the early HSC activation markers HSP47 and αB-crystallin, but not the late markers for HSC activation, αSMA and pro-collagen 1a1. Synaptophysin mRNA expression remained constant during incubation with or without CCl4, indicating a constant number of HSC in the liver slices.

Conclusion

We developed a technique to induce early toxicity-induced HSC activation in human liver slices. This in vitro model provides a multicellular, physiologic milieu to study mechanisms underlying toxicity-induced HSC activation in human liver tissue.

Introduction

Liver fibrosis due to viral or metabolic liver injury is one of the leading causes of death worldwide [1]. It is well known that the injury-induced activation of hepatic stellate cells (HSC) is a key event in the natural process of scar tissue formation as well as excessive fibrogenesis in the liver [1], [2]. However, to date no curative treatment for liver fibrosis is available and patients are dependent on liver transplantations. In order to develop effective anti-fibrotic therapies it is of importance to elucidate the mechanisms underlying initial injury-induced HSC activation and subsequent fibrosis development in human liver. Current in vivo and in vitro models contribute significantly to the understanding of HSC biology. However, experimental animal models for liver fibrosis are difficult to extrapolate to human disease and in vitro cell culture models cannot incorporate cell–cell and cell–extracellular matrix interactions that play an important role in the development of fibrosis. Thus, there persists a need for an in vitro model that can mimic the in vivo situation more closely.

Previously we showed that precision-cut rat liver slices could be used to study early activation of HSC in a physiological milieu [3]. The main advantage of this system over current available in vitro models for the study of HSC activation is that in the liver slice all liver cell types are present in a physiological extracellular matrix. This feature of liver slices provides the opportunity to study HSC activation while preserving cell–cell and cell–extracellular matrix interactions [4]. However, when using rodent liver tissue for this purpose, the model may still be of limited predictive value for the human situation. Therefore, the aim of our present study was to evaluate the applicability of precision-cut liver slices derived from human liver tissue for the study of HSC activation.

The design of the study was two-fold. Firstly, we evaluated the viability of human liver slices during incubation and determined whether responses of the liver slices were dependent on the origin of the liver tissue used. Secondly, we determined if HSC activation could be induced in the liver slices. For this purpose, we incubated the slices with carbon tetrachloride (CCl4), a known fibrogenic compound whose toxicity and fibrogenic activity requires conversion into a free radical by hepatocytes. Its fibrogenic activity, therefore, has been linked to oxidative damage to hepatocytes, yielding lipid peroxides and other mediators that activate HSC [5], [6]. To assess HSC activation in the liver slices heat shock protein 47 (HSP47) and αB-crystallin expression were used as early marker for HSC activation, and mRNA expression of α smooth muscle actin (αSMA) and pro-collagen 1a1 as late marker for HSC activation and fibrogenesis. Synaptophysin mRNA, which is expressed in quiescent as well as activated HSC [7] was used as a marker for the amount of HSC present.

The development of this in vitro model enables the study of human HSC activation in a physiological milieu and may contribute to the understanding of processes underlying early HSC activation in human liver. In addition, the model may contribute substantially to the reduction, refinement, and potential replacement of animal experiments.

Section snippets

Human liver tissue

Human liver tissue was obtained from multi-organ donors (Tx-livers) or from patients after partial hepatectomy because of metastasis of colorectal carcinoma (PH-livers). Consent from the legal authorities and from the families concerned was obtained for the use of Tx-livers for transplantation-related research. The Tx-liver was perfused with cold University of Wisconsin organ storage solution (UW, DuPont Critical Care, Waukegan, IL, USA) in situ before explantation and stored in cold UW until

Characteristics of the human liver tissue used

The characteristics of the human liver tissue used for the experiments with respect to age of the donor or patient, and the ATP-content of the liver slices are indicated in Table 1. No significant differences were observed in these parameters when comparing the different liver origins, although the age of the patients donating liver tissue after partial hepatectomy (PH) tended to be higher than the age of multi-organ donors (Tx). Importantly, neither the marker expression in the liver slices in

Discussion

Previously we have developed a technique to induce activation of HSC in rat liver slices, which provides the opportunity to study early toxicity-induced HSC activation in a physiologic, multicellular milieu [3]. However, due to species-differences this model may still be of limited predictive value for human disease. In the present study, we therefore evaluated the applicability of liver slices derived from human liver tissue for the study of HSC activation.

Acknowledgments

M. van de Bovenkamp was supported by grants of ZON/Mw, and the Johns Hopkins Center for Alternatives to Animal Testing; P. Olinga was supported by Organon NV.

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