Precision-cut liver slices: a versatile tool to advance liver research
Human precision-cut liver slices represent a robust and versatile ex vivo model which retains the complex and multi-cellular histoarchitecture of the hepatic environment. As such, they represent an ideal model to investigate the mechanisms of liver injury and for the identification of novel therapeutic targets.
KeywordsLiver slices Organotypic system Liver disease model
There is an urgent need for reliable systems to understand the pathogenesis of liver diseases, such as viral hepatitis, NASH, cancer, fibrosis and alcohol-induced liver disease. The in vitro cellular models and in vivo animal models currently utilised in liver research do not fully mimic the complex pathogenesis of liver injury and disease progression, particularly the development of fibrosis that occurs in humans. Lack of this understanding hinders the development of biomarkers both prognostic and diagnostic. Moreover, the pre-clinical testing of potential therapeutic targets in animal models can result in the identification of molecules that do not always show efficacy or worse still provoke toxic events in humans . Recapitulating liver pathophysiology is challenging because of the intricacy of cellular composition and cell heterogeneity, the active role of the extracellular matrix and the complex tissue arrangement. Furthermore, immunity in man varies significantly from mouse models in terms of composition, strength and breadth, which is a problem that has to be addressed as inflammation and dysfunctional host immunity are major drivers of liver injury and disease.
The precision-cut slices of kidney, intestinal and lung tissues  have been very successfully used to study multiple multicellular pathological processes such as in fibrosis and as shown below, liver slices are now emerging as a versatile tool to understand disease pathways in several liver conditions.
Alcoholic liver disease (ALD)
Non-alcoholic fatty liver disease (NAFLD)
Alternatively, use of liver explants from patients with NASH cirrhosis undergoing transplantation would help to assess if there was an opportunity for therapeutic interventions at this late stage of disease or whether the reversibility is restricted to earlier stages.
Fibrosis and cirrhosis
There is an increasing evidence that different chronic liver diseases are characterised by different pro-fibrogenic mechanisms and while cirrhosis is the common result of progressive fibrogenesis, there are distinct patterns of fibrosis development associated with different etiologies. The induction of fibrosis from toxic agents can be demonstrated in the hPCLS, as the key events and the cellular components of fibrogenesis are maintained and remain active. Within each slice, the hepatocytes are metabolically fully competent and the other cell types retain their functionality including the inflammatory response induced by Kupffer cells and the development of fibrosis mediated by the activation of hepatic stellate cells and myofibroblasts. We have previously used mouse PCLS model to induce fibrogenesis with leptin and assess the ability of Osteopontin neutralisation to abrogate this response  confirming its utility as a pre-clinical model for anti-fibrotic therapies. Recent evidence suggests, however, that fibrogenic processes may be different in rodents. A recent study assessing the anti-fibrotic effects of LY2109761, a TGF-Beta inhibitor using human and rat PCLS showed that whilst LY2109761 was anti-fibrotic in both rat and human PCLS, there were considerable differences between the regulation of alpha-SMA and HSP47 in the responses to the treatment. Differences in fibrotic response between rat and human PCLS were also demonstrated earlier by the same group, demonstrating the strength of the PCLS model in fibrotic research while underlining the inter-species differences in mechanisms of fibrosis [8, 9].
Due to the versatility of the PCLS model, slices can also be prepared from diseased explants from patients with cirrhosis undergoing transplantation to allow the study of more advanced stages. An example is the study performed by Guyot et al. using ‘cirrhotic’ slices shows the suitability of this model to follow the processes of tissue remodelling by myofibroblasts and other hepatic cell types, allowing the comparison of cell behaviours between different pathological situations .
The hPCLS system has also been shown to have considerable utility in research studies in viral hepatitis. Liver slices are the ideal ex vivo model to investigate the mechanisms of viral infection and replication, viral life cycle and dynamics of virus propagation in native tissue and to evaluate the efficacy of new antiviral drugs. The infection of slices with hepatitis C virus (HCV) has been achieved using primary viral isolates from HCV-infected patient serum with high viral load not treated with antiviral drugs or with cell culture-grown viral strains of different genotypes (2a, 1a, 1b). In the study by Lagaye et al. the slice culture has been followed for up to 10 days and intracellular replication of HCV RNA, expression of viral proteins, extracellular production of infective particles demonstrated. Moreover, the slice supernatants of the cultured slices after infection were also able to further infect other liver slices . In our laboratory we are currently developing the protocol for the infection of hPCLS with hepatitis B virus isolated from HepG2.2.15 cell culture and encouraging results have been achieved including cccDNA production (unpublished data). The infection of liver slices can also be performed using other types of viruses and we have tested both adenoviruses and adeno-associated viruses with consistent results. Genetic manipulation of the slices opens further the applicability of this powerful tool.
Cholestatic disease and drug-induced liver injury
Human PCLS have been shown to produce bile acids and to be sensitive to drug-induced cholestasis . Their capability of taking up bile acids or to express genes involved in bile acid transport and metabolism in a physiological and stable level validates the model for research in other important areas of hepatology, including drug-induced liver injury (DILI). Understanding the mechanisms of DILI is a major challenge to contemporary hepatology. DILI accounts for more than 50% of the cases of acute hepatic failure and is associated with a case-fatality rate of 10–50% depending on the drug involved. Hepatic drug reactions are difficult to diagnose as they can mimic hepatobiliary disease which makes distinguishing DILI and spontaneous hepatic disease difficult. The currently available diagnostic tests for DILI report on dying cells and signal the presence of injury after considerable damage has taken place. The hPCLS could be used to provide detailed insight into molecular mechanisms underlying the toxic endpoints of DILI to identify early signatures of liver-specific toxicity.
Hepatocellular carcinoma (HCC)
Besides antineoplastic drugs, oncolytic viruses have been successfully tested on hPCLS derived from HCC tumors. Zimmerman et al.  used the slice model as a predictive test for patient-specific tumor response and to evaluate the safety of an oncolytic vaccine viruses. Given the importance of the immune system in HCC , the development of potential immunotherapeutic options such as checkpoint inhibitors for HCC will be accelerated by availability of relevant immunocompetent models of HCC to test the rationale of combination therapies, early identification of biomarkers and designing personalised approaches. The use of hPCLS co-cultured with matched peripheral leukocytes may hold the key for assessing cancer-immune cell interactions and this is an area of current research interest for the hPCLS system.
Local access to freshly resected (human) tissue is one of the main limitations of this technique; the procedure of acquiring fresh tissue samples requires co-ordination and good communication between the stakeholders, including surgeons, histopathologists, nurses and researchers.
Further to this, the ‘healthy’ tissue taken from resected samples is not truly healthy, as it is inevitably from the distal portion of a diseased liver. This can bring variability between experiments and subjects. The same applies to the use of diseased (cirrhotic/HCC) slices, as oftentimes these patients have previously undergone multiple different (chemo)therapeutic/ablative strategies prior to surgery. This can not only add inconsistency between samples but also compromise the physical (structural integrity/fat content) and physiological properties of the tissue. Other personalised approaches do not suffer from these drawbacks such as the use of induced pluripotent stem cells (iPSC) which are flexible and feasible for personalised medicine, but it must be stressed that iPSC lack the original cellular and genetic landscape of the diseased tissue.
Chronic liver disease and cirrhosis are a major cause of illness and death worldwide and this is projected to substantially increase in the future. Considering NAFLD alone, forecasts predict an exponential rise in disease burden from 83.1 million in 2015 to 100.9 million by 2030. Primary liver cancer rates are also increasing and HCC is currently the second leading cause of global cancer mortality. As such, there are multiple needs for models of liver disorders to study prognostic and diagnostic markers and for drug discovery.
A (hu)man is not a big mouse and inter-species differences in disease processes and immunity amplify small differences in complex pathological pathways and drug responsiveness. Preservation of the original 3D architecture and the lobular structure of the liver in the human precision-cut liver slices provide the essential requirement for a good pre-clinical model and use of this technique will increase our understanding of the pathophysiology of different liver diseases.
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Human and animal rights statement
This article does not contain any direct studies with human or animal subjects.
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