Precision-cut Rat Liver Slices Research Articles

P11-15 Toxicological assessment of collagenase and benzonase on rat precision-cut liver slices

P11-15 Toxicological assessment of collagenase and benzonase on rat precision-cut liver slices

Modulation of vascular contraction via soluble guanylate cyclase signaling in a novel ex vivo method using rat precision-cut liver slices.

Fibrotic processes in the liver of non‐alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre‐clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin‐1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin‐1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin‐induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non‐responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.

Glutathione-responsive cyclodextrin-nanosponges as drug delivery systems for doxorubicin: Evaluation of toxicity and transport mechanisms in the liver

The potential mammalian hepatotoxicity of a new class of GSH-responsive cyclodextrin-based nanosponges loaded with the anticancer drug doxorubicin (Dox-GSH-NS) was investigated. Previous studies showed that these nanosponges can release medicaments preferentially in cells having high GSH content, a common feature of chemoresistant cells, and showed enhanced anti-tumoral activity compared to free Dox in vitro and in vivo in cells with high GSH content. Following these promising results, we investigated here the Dox-GSH-NS hepatotoxicity in human HepG2 cells (in vitro) and in the organotypic cultures of rat precision-cut liver slices (PCLS, ex vivo), while their accumulation in rat liver was assessed in vivo. Moreover, the transport in Dox uptake, as well as its efflux, was studied in vitro. Overall, benefiting of the integration of different investigational models, a good safety profile of Dox-GSH-NSs was evidenced, and their hepatotoxicity resulted to be comparable with respect to free Dox both in vitro and ex vivo. Furthermore, in vivo studies showed that the hepatic accumulation of the Dox loaded in the NS is comparable with respect to the free drug. In addition, Dox-GSH-NSs are taken up by active mechanisms, and can escape the efflux drug pump, thus, contributing to overcoming drug resistance.

In vitro and ex vivo anti-fibrotic effects of LY2109761, a small molecule inhibitor against TGF-β

Fibrosis is a pathophysiological state characterized by the excessive formation/deposition of fibrous extracellular matrix. Transforming growth factor-beta (TGF-β) is a central profibrotic mediator, and targeting TGF-β is a promising strategy in the development of drugs for the treatment of fibrosis. Therefore, the effect of LY2109761, a small molecule inhibitor against TGF-β with targets beyond TGF-β signaling, on fibrogenesis was elucidated in vitro (HepG2 cells and LX-2 cells) and ex vivo (human and rat precision-cut liver slices). Our results displayed an anti-fibrotic effect of LY2109761, as it markedly down-regulated gene and protein expression of collagen type 1, as well as gene expression of the inhibitor of metalloproteinases 1. This effect on fibrosis markers was partially mediated by targeting TGF-β signaling, seeing that LY2109761 inhibited TGF-β1 gene expression and SMAD2 protein phosphorylation. Interestingly, particularly at a high concentration, LY2109761 decreased SMAD1 protein phosphorylation and gene expression of the inhibitor of DNA binding 1, which appeared to be TGF-β-independent effects. In conclusion, LY2109761 exhibited preclinical anti-fibrotic effects via both TGF-β-dependent and -independent pathways. These results illustrate that small molecule inhibitors directed against TGF-β could possibly influence numerous signaling pathways and thereby mitigate fibrogenesis.

Antagonistic and synergistic interactions during the binding of binary mixtures of polycyclic aromatic hydrocarbons to the aryl hydrocarbon receptor

In order to assess the potential of polycyclic aromatic hydrocarbons (PAHs) to interact with each other, benzo(a)pyrene (B(a)P) was incubated either alone or in combination with other isomeric 5-ring PAHs in precision-cut rat liver slices. At the end of the incubation, the slices were removed and the O-deethylation of ethoxyresorufin (EROD) was determined in microsomal preparations. The BP-mediated rise in EROD activity was suppressed in the presence of dibenzo(a,j)anthracene, dibenzo(a,c)anthracene and picene, whereas it was increased in the presence of pentacene. In the case of benzo(b)chrysene, benzo(c)chrysene and benzo(g)chrysene the effect was concentration-dependent with both antagonism and synergism being observed. The binding of B(a)P to the aryl hydrocarbon (Ah) receptor was similarly modulated by other PAHs. No correlation was evident between binding avidity of the PAH to the Ah receptor and either its potential for interaction or nature of interaction, e.g. synergism or antagonism. These interactions were also independent of the molecular shape (ring arrangement) of the 5-ring isomeric PAHs. Bearing in mind the role of the Ah receptor in chemical carcinogenesis, it may be concluded that interactions at the Ah receptor site may contribute to the well-established modulation of the carcinogenicity of one PAH in the presence of another.

Catalase increases ethanol oxidation through the purine catabolism in rat liver

Hepatic ethanol oxidation increases according to its concentration and is raised to near-saturation levels of alcohol dehydrogenase (ADH); therefore, re-oxidation of NADH becomes rate limiting in ethanol metabolism by the liver. Adenosine is able to increase liver ethanol oxidation in both in vivo and in vitro conditions; the enhancement being related with the capacity of the nucleoside to accelerate the transport of cytoplasmic reducing equivalents to mitochondria, by modifying the subcellular distribution of the malate-aspartate shuttle components. In the present study, we explored the putative effects of adenosine and other purines on liver ethanol oxidation mediated by non-ADH pathways. Using the model of high precision-cut rat liver slices, a pronounced increase of ethanol oxidation was found in liver slices incubated with various intermediates of the purine degradation pathway, from adenosine to uric acid (175–230%, over controls). Of these, urate had the strongest (230%), whereas xanthine had the less pronounced effect (178% over controls). The enhancement was not abolished by 4-methylpyrazole, indicating that the effect was independent of alcohol dehydrogenase. Conversely, aminotriazole, a catalase inhibitor, completely abolished the effect, pointing out that this enhanced ethanol oxidation is mediated by catalase activity. It is concluded that the H2O2 needed for catalase activity is derived from the oxidation of (hypo)xanthine by xanthine oxidase and the oxidation of urate by uricase. The present and previous data led us to propose that, depending on the metabolic conditions, adenosine might be able to stimulate the metabolism of ethanol through different pathways.

Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound

Precision cut liver slices (PCLiS) represent a promising tool in reflecting hepatotoxic responses. However, the culture of PCLiS varies considerably between laboratories, which can affect the performance of the liver slices and thus the experimental outcome. In this study, we describe an easily accessible culture method, which ensures optimal slice viability and functionality, in order to set the basis for reproducible and comparable PCLiS studies. The quality of the incubated rat PCLiS was assessed during a 24h culture period using ten readouts, which covered viability (lactate dehydrogenase-, aspartate transaminase- and glutamate dehydrogenase-leakage, ATP content) and functionality parameters (urea, albumin production) as well as histomorphology and other descriptive characteristics (protein content, wet weight, slice thickness). The present culture method resulted in high quality liver slices for 24h. Finally, PCLiS were exposed to increasing concentrations of acetaminophen to assess the suitability of the model for the detection of hepatotoxic responses. Six out of ten readouts revealed a toxic effect and showed an excellent mutual correlation. ATP, albumin and histomorphology measurements were identified as the most sensitive readouts. In conclusion, our results indicate that rat PCLiS are a valuable liver model for hepatotoxicity studies, particularly if they are cultured under optimal standardized conditions.

Rat precision-cut liver slices predict drug-induced cholestatic injury

Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 μM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC.

Up-regulation of CYP1A1 and phase II enzymes by 5-ring isomeric polycyclic aromatic hydrocarbons in precision-cut rat hepatic slices: Importance of molecular shape

The objectives of the present study were two-fold: (a) to evaluate the role of molecular shape on the interaction of polycyclic aromatic hydrocarbons (PAHs) with the Ah receptor and CYP1A1 upregulation, and (b) to evaluate the potential of PAHs to induce epoxide hydrolase and glutathione S-transferase, two major enzymes involved in their metabolism. In order to achieve these objectives, precision-cut rat liver slices were incubated with a range of concentrations of seven 5-ring isomeric PAHs, namely benzo[c]chrysene, benzo[b]chrysene, benzo[g]chrysene, dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, picene and pentacene, for 24h. All compounds, with the exception of pentacene, elevated the O-deethylation of ethoxyresorufin, an activity associated with CYP1A1; induction of this enzyme by the various PAHs correlated with their avidity for the Ah receptor. None of the PAHs studied increased epoxide hydrolase activity, monitored using benzo[a]pyrene 4,5-oxide. Of the seven PAHs, only benzo[g]chrysene elevated glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole as substrates. No relationship could be established between length or length/width and interaction with the Ah receptor and CYP1A1 up-regulation indicating that other structural or electronic factors are likely to be more important. Finally, 5-ring PAHs are poor inducers of the epoxide hydrolase and glutathione S-transferase enzyme systems.

Synthesis, antimycobacterial and cytotoxic activity of α,β-unsaturated amides and 2,4-disubstituted oxazoline derivatives

The synthesis of six α,β,-unsaturated amides and six 2,4-disubstituted oxazolines derivatives and their evaluation against two Mycobacterium tuberculosis strains (sensitive H37Rv and a resistant clinical isolate) is reported. 2,4-Disubstituted oxazolines (S)-3b,d,e were the most active in the sensitive strain with a MIC of 14.2, 13.6 and 10.8μM, respectively, and the compounds (S)-3d,f were the most active against resistant strain with a MIC of 6.8 and 7.4μM. The ex-vivo evaluation of hepatotoxicity on precision-cut rat liver slices was also tested for the α,β-unsaturated amides (S)-2b and (S)-2d,f and for the oxazolines (S)-3b and (S)-3d,f at different concentrations (5, 15 and 30μg/mL). The results indicate that these compounds possess promising antimycobacterial activity and at the same time are not hepatotoxic. These findings open the possibility for development of new drugs against tuberculosis.

Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis

Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and studied the efficacy of multiple putative antifibrotic compounds.Our results demonstrated that human PCLS remained viable for 48h and the early onset of fibrosis was observed during culture, as demonstrated by an increased gene expression of Heat Shock Protein 47 (HSP47) and Pro-Collagen 1A1 (PCOL1A1) as well as increased collagen 1 protein levels. SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK) showed a marked decrease in HSP47 and PCOL1A1 gene expression, whereas specific inhibitors of Smad 3 and Rac-1 showed no or only minor effects. Regarding the studied antifibrotics, gene levels of HSP47 and PCOL1A1 could be down-regulated with sunitinib and valproic acid, while PCOL1A1 expression was reduced following treatment with rosmarinic acid, tetrandrine and pirfenidone. These results are in contrast with prior data obtained in rat PCLS, indicating that antifibrotic drug efficacy is clearly species-specific.Thus, human PCLS is a promising model for liver fibrosis. Moreover, MAPK signaling plays an important role in the onset of fibrosis in this model and transforming growth factor beta pathway inhibitors appear to be more effective than platelet-derived growth factor pathway inhibitors in halting fibrogenesis in PCLS.

Synergistic and antagonistic interactions of binary mixtures of polycyclic aromatic hydrocarbons in the upregulation of CYP1 activity and mRNA levels in precision-cut rat liver slices

The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision-cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O-deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O-deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds. In contrast, benzo[b]fluoranthene and, at high doses only, dibenzo[a,l]pyrene gave rise to antagonism, whereas 1-methylphenanthrene had no effect at all concentrations studied. When CYP1A1 mRNA levels were monitored, benzo[b]fluoranthene gave rise to an antagonistic response when incubated with benzo[a]pyrene, whereas all other compounds displayed synergism, with 1-methylphenathrene being the least effective. A similar picture emerged when CYP1B1 mRNA levels were determined, though the effects were less pronounced. In conclusion, it has been demonstrated that the benzo[a]pyrene-mediated upregulation of CYP1, at the mRNA and activity levels, is synergistically and antagonistically modulated by other PAHs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 764-775, 2017.

Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium

Precision-cut liver slices (PCLS) are an ex vivo model for metabolism and toxicity studies. However, data on the maintenance of the morphological integrity of the various cell types in the slices during prolonged incubation are lacking. Therefore, our aims were to characterize morphological and functional changes in rat PCLS during five days of incubation in a rich medium, RegeneMed®, and a standard medium, Williams' Medium E. Although cells of all types in the slices remain viable, profound changes in morphology were observed, which were more prominent in RegeneMed®. Slices underwent notable fibrosis, bile duct proliferation and fat deposition. Slice thickness increased, resulting in necrotic areas, while slice diameter decreased, possibly indicating cell migration. An increased proliferation of parenchymal and non-parenchymal cells (NPCs) was observed. Glycogen, albumin and Cyp3a1 were maintained albeit to a different level in two media. In conclusion, both hepatocytes and NPCs remain viable and functional, enabling five-day toxicity studies. Tissue remodeling and formation of a new capsule-like cell lining around the slices are evident after 3–4days. The differences in effects between media emphasize the importance of media selection and of the recognition of morphological changes in PCLS, when interpreting results from toxicological or pharmacological studies.

Lack of anti-inflammatory effect of coenzyme Q10 supplementation in the liver of rodents after lipopolysaccharide challenge

Summary Background & aims Sepsis is characterized by a systemic dysregulated inflammatory response and oxidative stress. A large body of evidence supports a key role of mitochondrial dysfunction during the various phases of sepsis (early and late-phase of sepsis-associated multiorgan failure). Coenzyme Q10 (CoQ10) is a key cofactor in the mitochondrial and respiratory chain, and is depleted in septic shock patients. However its effect on acute sepsis remains unexplored. The reduced form of CoQ10 (Qx) has been shown to lessen pro-inflammatory response in macrophages or fibroblasts in culture. The aim of the study is to investigate the effect of Qx on hepatic inflammation in models of acute LPS-induced inflammation. Methods We have conducted 2 experiments: one in vitro using rat precision-cut liver slices (PCLS) incubated with LPS and Qx, and one in vivo experiment using mice fed with Qx for 2 weeks and killed 1 h after LPS administration. Results LPS challenge induced hepatic inflammatory stress both in vitro and in vivo (increased TNFα, nitrite and PGE2 release and/or expression). Incubation of PCLS with Qx fails to modulate hepatic inflammatory stress. Furthermore, Qx supplementation is not able to counteract hepatic LPS-induced acute inflammation in vivo. Conclusion Coenzyme Q10 is not able to counteract LPS-induced hepatic acute inflammation.

P0440 : Antifibrotic efficacy of TGF-beta receptor antagonist in precision-cut human and rat liver slices

P0440 : Antifibrotic efficacy of TGF-beta receptor antagonist in precision-cut human and rat liver slices

Improved synthesis of N-benzylaminoferrocene-based prodrugs and evaluation of their toxicity and antileukemic activity.

We report on an improved method of synthesis of N-benzylaminoferrocene-based prodrugs and demonstrate its applicability by preparing nine new aminoferrocenes. Their effect on the viability of selected cancer cells having different p53 status was studied. The obtained data are in agreement with the hypothesis that the toxicity of aminoferrocenes is not dependent upon p53 status. Subsequently the toxicity of a selected prodrug (4) was investigated ex vivo using rat precision cut liver slices and in vivo on hybrid male mice BDF1. In both experiments no toxicity was observed: ex vivo, up to 10 μM; in vivo, up to 6 mg/kg. Finally, prodrug 4 was shown to extend the survival of BDF1 mice carrying L1210 leukemia from 13.7 ± 0.6 days to 17.5 ± 0.7 days when injected daily 6 times at a dose of 26 μg/kg starting from the second day after injection of L1210 cells.

Compound-Specific Toxicities Detected in CFU-GM, Rat Kidney NRK Cells, Rat Bladder RBLAK Cells, and Rat Liver Slices following Batracylin or N-Acetyl Batracylin Exposure

The investigational anticancer agent batracylin (BAT; 8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) causesγ-H2AX foci development in exposed tumor cells and has demonstrated activity against solid tumors and adriamycin-resistant leukemia. Reports indicate BAT has wide interspecies variation of adverse effects, including myelosuppression, kidney, bladder, and liver damage, including biliary hyperplasia. The effects of BAT and its metabolite N-acetyl batracylin (NAB) were evaluated in the CFU-GM bone marrow toxicity assay, rat kidney (NRK) cells, bladder epithelial (RBLAK) cells, and rat precision cut liver slices (PCLS). Exposure effects were evaluated biochemically and histologically. Human, dog, and rat exhibited similar CFU-GM IC90 values for BAT (21–29 μM). The ATP assay andγ-H2AX staining showed time- and concentration-dependent toxicity in RBLAK (more severe than NRK at <72 hr) NRK and cells (IC50<20 μM after 96 hr BAT exposure). BAT (5 μM and 25 μM) caused biochemical and histology changes to PCLS by day 3 and 25 μM produced centrilobular hepatotoxicity. NAB (≤5 μM) produced no toxicity in CFU-GM, NRK, or RBLAK cells. However, both BAT and NAB caused biliary epithelial cell proliferation in PCLS. Our studies demonstrated species similarities in sensitivity to BAT-induced myelosuppression, and implicate the metabolite NAB in biliary hyperplasia.

Precision-cut liver slices as a model for the early onset of liver fibrosis to test antifibrotic drugs

Induction of fibrosis during prolonged culture of precision-cut liver slices (PCLS) was reported. In this study, the use of rat PCLS was investigated to further characterize the mechanism of early onset of fibrosis in this model and the effects of antifibrotic compounds. Rat PCLS were incubated for 48h, viability was assessed by ATP and gene expression of PDGF-B and TGF-β1 and the fibrosis markers Hsp47, αSma and Pcol1A1 and collagen1 protein expressions were determined. The effects of the antifibrotic drugs imatinib, sorafenib and sunitinib, PDGF-pathway inhibitors, and perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone, TGFβ-pathway inhibitors, were determined. After 48h of incubation, viability of the PCLS was maintained and gene expression of PDGF-B was increased while TGF-β1 was not changed. Hsp47, αSma and Pcol1A1 gene expressions were significantly elevated in PCLS after 48h, which was further increased by PDGF-BB and TGF-β1. The increased gene expression of fibrosis markers was inhibited by all three PDGF-inhibitors, while TGFβ-inhibitors showed marginal effects. The protein expression of collagen 1 was inhibited by imatinib, perindopril, tetrandrine and pirfenidone. In conclusion, the increased gene expression of PDGF-B and the down-regulation of fibrosis markers by PDGF-pathway inhibitors, together with the absence of elevated TGF-β1 gene expression and the limited effect of the TGFβ-pathway inhibitors, indicated the predominance of the PDGF pathway in the early onset of fibrosis in PCLS. PCLS appear a useful model for research of the early onset of fibrosis and for testing of antifibrotic drugs acting on the PDGF pathway.

Proteomic profiling in incubation medium of mouse, rat and human precision‐cut liver slices for biomarker detection regarding acute drug‐induced liver injury

Drug-induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision-cut liver slices (PCLS) exposed to liver injury-inducing drugs for biomarker identification, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PCLS were incubated with acetaminophen (APAP), 3-acetamidophenol, diclofenac and lipopolysaccharide for 24-48 h. PCLS medium from all species treated with APAP demonstrated similar changes in protein profiles, as previously found in mouse urine after APAP-induced liver injury, including the same key proteins: superoxide dismutase 1, carbonic anhydrase 3 and calmodulin. Further analysis showed that the concentration of hepcidin, a hepatic iron-regulating hormone peptide, was reduced in PCLS medium after APAP treatment, resembling the decreased mouse plasma concentrations of hepcidin observed after APAP treatment. Interestingly, comparable results were obtained after 3-acetamidophenol incubation in rat and human, but not mouse PCLS. Incubation with diclofenac, but not with lipopolysaccharide, resulted in the same toxicity parameters as observed for APAP, albeit to a lesser extent. In conclusion, proteomics can be applied to identify potential translational biomarkers using the PCLS system.

36. Systematic analysis of gene expression changes due to chilling injury in precision-cut liver slices

Chilling injury is one of the limiting steps for successful vitrification of cells, organs and tissue. Chilling injury is known to be caused by the decrease of temperature per se. Although some research has been done to study chilling injury in cryopreserved oocytes and sperm cells amongst others, the exact mechanism especially in the intact organ is yet unknown. Precision-cut liver slices (PCLS), contain all liver cell types and microstructures and closely resemble the organ from which they are derived. Therefore, it is an ideal model to study the mechanism of chilling injury in the intact organ. Using PCLS as a tool we aim at identifying the critical genes and signaling pathways responding to chilling injury by microarray analysis to enhance insight into its mechanism and to facilitate development of vitrification methods for integrated tissue and organs. Chilling injury was induced in rat PCLS loaded with cryoprotectant solutions (Sol Y or Sol Z, consisting of M22 without N-methyl-formamide and polyvinylpyrrolidone, and without or with sucrose respectively) and chilled to −15 °C for 10 min. At this temperature, no ice formation occurred as was shown by differential scanning calorimetry. The cryoprotectant solutions induced little or no damage as indicated by ATP levels. Principle Component Analysis indicated that gene expression changes induced in chilled PCLS compared to untreated controls were clearly distinguishable from those induced by the cryoprotectants alone. Pathway analysis in metacore revealed that immune innate and defense responses as well as cell cycle G1/S phase transition, MAP Kinase and lipid/fatty acid biosynthesis and metabolism were affected by chilling. Among the up-regulated pathways, cell surface receptor linked signaling pathways accounted for 68% of all processes, indicating that the cell membrane could be the primary site and sensor for chilling injury. Furthermore, we also found some indication that chilling might cause DNA damage, since DNA-damage inducible genes were up-regulated. In addition, chilling decreased gene expression of a set of enzymes required for cholesterol biosynthesis. Besides, pathway analysis indicated decreased fatty acid oxidation and unsaturated fatty acid biosynthesis. These observations could explain the decreased ratio of unsaturated fatty acid to saturated fatty acid and the decreased cholesterol content in cells after cryopreservation that was reported by others. Most importantly, we were able to observe the responses from the different cell types such as the increased differentiation of stellate cells, and immune responses (e.g. MHC class II activation) in Kupffer cells suggesting the involvement of all liver cell types in the injury. In conclusion, a broad spectrum of changes in the tissue at the gene level was identified that were specifically due to chilling, as well as previously unknown changes in the different cell types. This is the first report of a systematic investigation on the mechanism of chilling injury investigated in integrated tissue by microarray analysis under conditions in which other sources of injury are minimal.