The role of liver sinusoidal endothelial cells in liver remodeling after injury

Liver transplantation is the optimal treatment for patients with end-stage liver disease,metabolic liver diseases,and hepatic malignancies that are not amenable to resection.Hepatic ischemia-reperfusion injury(IRI)is the main problem in liver transplantation and liver resection,leading to parenchymal cell injury and organ dysfunction.The damage of liver sinusoidal endothelial cells(LSECs)is a critical event in IRI.LSECs work as an important regulating factor of liver regeneration after partial hepatectomy.This review primarily describes the mechanisms of LSECs injury in IRI and explores the roles of LSECs in liver regeneration,and briefly introduces the protective strategies targeting LSECs damaged in IRI.

Effects of ethanol on liver sinusoidal endothelial cells-fenestrae of rats

Important advances have been made in research into the mechanism of alcoholic liver disease (ALD) over the past few years,but the role of liver sinusoidal endothelial cell (LSEC) in ALD has not been elucidated adequately. This study was undertaken to investigate the effect of ethanol on fenestrae of LSECs in rats. METHODS: A rat model of alcoholic liver disease was established by means of direct intragastric instillation of ethanol. Fifty-five rats of experimental (35 rats) and control (20) groups were sacrificed at the end of 4,8,12 weeks respectively, and also at the end of 12-week abstinence. After heart perfusion, the liver tissue was fixed and stained with hematoxylin and eosin for observation of serial changes of LSEC-fenestrae under a transmission electron microscope. RESULTS: Normal LESC was flat with a nucleus and organelles arranged regularly. The distal cytoplasm displayed as a lamina with many fenestrae, lacking the basement membrane(BM) underneath the endothelium. At the end of 4-week alcohol feeding, the number of fenestrae decreased at the distal cytoplasm in some LSECs, without the formation of the BM underneath the endothelium. At the end of 8 weeks, the number of fenestrae decreased significantly or even disappeared. The BM began to develop incompletely underneath the endothelium, while the active fibroblast appeared. At the end of 12 weeks, the number of fenestrae decreased more significantly and the complete BM could even be seen. But the changes were mostly limited in the single or adjoining sinus, and fibrosis was scarcely formed. At the end of 12-week abstinence, defenestration and formation of the endothelial BM lightened significantly. CONCLUSIONS:Defenestration and formation of the BM in LSECs develop gradually with the chronic stimulation of ethanol. Hepatic sinusoidal capillarization and fibrosis will be seen if their state is more serious. These early changes, i. e., limited and regional defenestration and capillarization may be the basis of alcoholic peri-fibrosis. This kind of he- patic fibrosis is reversible after removal of etiological factors.

Discussion on Serum Technique and Cryopreservation Technique in the Culture of Liver Sinusoidal Endothelial Cells

Hepatic sinusoidal endothelial cells are a kind of highly differentiated cells in hepatic sinusoids, which play an important role in the occurrence and development of liver fibrosis. Hepatic sinusoidal endothelial cells are often used in the study of liver fibrosis. In the process of culturing liver sinusoidal endothelial cells, the treatment of serum for culture and cryopreservation of cells are relatively complicated and error-prone. If the technical details of serum treatment and cell cryopreservation are not handled properly, it will have a more obvious effect on the effect of hepatic sinusoidal endothelial cell culture. We have gained experience in the theoretical study of liver fibrosis and the operation of cell culture experiments, and this paper summarized the details of liver sinusoidal endothelial cell culture such as the problems of serum preservation, thawing, precipitates and heat inactivation, as well as methods and precautions for cell cryopreservation.

Structural and functional aspects of the liver and liver sinusoidal cells in relation to colon carcinoma metastasis

Nowadays, liver metastasis remains difficult to cure. When tumor cells escape and arrive in the liver sinusoids, they encounter the local defense mechanism specific to the liver. The sinusoidal cells have been widely described in physiologic conditions and in relation to metastasis during the past 30 years. This paper provides an 'overview' of how these cells function in health and in diseases such as liver metastasis.

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

AIM: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis(PBC).METHODS: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B(HBV) virus,3 patients with hepatitis C virus(HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis(AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targetedby autoantibodies present in the patients sera was performed by indirect immunofluorescence(IIF) analysis using paraffin-embedded liver sections as a substrate.Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy.RESULTS: In total, 18/21(85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21(47.6%) in lymphocytes, 8/21(38%) in cholangiocytes and 7/21(33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G(IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining(20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody,positive staining was observed in 75% of lymphocytes,62.5% of cholangiocytes, 37.5% of hepatocytes and50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections,weak positive staining of cholangiocytes was observed in 3/21(14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients.CONCLUSION: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

Pathological process of liver sinusoidal endothelial cells in liver diseases

Cirrhosis develops from liver fibrosis and is the severe pathological stage of all chronic liver injury. Cirrhosis caused by hepatitis B virus and hepatitis C virus infection is especially common. Liver fibrosis and cirrhosis involve excess production of extracellular matrix,which is closely related to liver sinusoidal endothelial cells(LSECs). Damaged LSECs can synthesize transforming growth factor-beta and platelet-derived growth factor,which activate hepatic stellate cells and facilitate the synthesis of extracellular matrix. Herein,we highlight the angiogenic cytokines of LSECs related to liver fibrosis and cirrhosis at different stages and focus on the formation and development of liver fibrosis and cirrhosis. Inhibition of LSEC angiogenesis and antiangiogenic therapy are described in detail. Targeting LSECs has high therapeutic potential for liver diseases. Further understanding of the mechanism of action will provide stronger evidence for the development of anti-LSEC drugs and new directions for diagnosis and treatment of liver diseases.

Effects of Astragalus Polysaccharide on Mechanical Characterization of Liver Sinusoidal Endothelial Cells by Atomic Force Microscopy at Nanoscale

Objective： To study the effects of Astragalus polysaccharide （APS）, the primary effective component of the Chinese herb medicine Astragalus membranaceus （frequently used for its anti-hepatic fibrosis effects）, on nanoscale mechanical properties of liver sinusoidal endothelial cells （SECs）. Methods： Using endothelial cell medium as the control, 5 experimental groups were established utilizing different concentrations of APS, i.e. 12.5, 25, 50, 100, and 200μg/mL. By using atomic force microscopy along with a microcantilever modified with a silicon dioxide microsphere as powerful tools, the value of Young＇s modulus in each group was calculated. SAS 9.1 software was applied to analyze the values of Young＇s modulus at the pressed depth of 300 nm. Environmental scanning electron microscopy was performed to observe the surface microtopography of the SECs. Results： The value of Young＇s modulus in each APS experimental group was significantly greater than that of the control group： as APS concentration increased, the value of Young＇s modulus presented as an increasing trend. The difference between the low-concentration （12.5 and 25 μg/mL） and high-concentration （200μg/mL） groups was statistically significant （P〈0.05）, but no significant differences were observed between moderate-concentration （50 and 100μg/mL） groups versus low- or high-concentration groups （P〉0.05）. Surface topography demonstrated that APS was capable of increasing the total area of fenestrae. Conclusions： The values of Young＇s modulus increased along with increasing concentrations of APS, suggesting that the stiffness of SECs increases gradually as a function of APS concentration. The observed changes in SEC mechanical properties may provide a new avenue for mechanistic rasearch of anti-hepatic fibrosis treatments in Chinese medicine.

Mechanotransduction of liver sinusoidal endothelial cells under varied mechanical stimuli

Liver sinusoidal endothelial cells(LSECs)are the gatekeeper of liver to maintain hepatic homeostasis.They are formed into the highly specialized endothelium between vascular lumen and the space of Disse and are mechanosensitive to respond varied microenvironments.Shear stress and mechanical stretch induced by blood perfusion and substrate stiffness enhancement derived from deposition of extracellular matrix(ECM)are major mechanical stimuli that surround LSECs.This review introduces how LSECs respond to the external forces in both physiological and pathological cases and what is the interplay of LSECs with other hepatic cells.Molecular mechanisms that potentiate LSECs mechanotransduction are also discussed.

Reversible sinusoidal obstruction syndrome associated with tacrolimus following liver transplantation

Sinusoidal obstruction syndrome(SOS), previously known as hepatic veno-occlusive disease, is a rare disorder in solid organ transplant patients, and is an uncommon complication after liver transplantation. Severe SOS with hepatic failure causes considerable mortality. Tacrolimus has been reported to be an offending agent, which potentially plays a role in the pathophysiological process of SOS. SOS due to tacrolimus has been reported in lung and pancreatic transplantations, but has never been described in a liver transplant recipient. Herein, we present a case of SOS after liver transplantation, which was possibly related to tacrolimus. A 27-year-old man developed typical symptoms of SOS with painful hepatomegaly, ascites and jaundice after liver transplantation, which regressed following withdrawal of tacrolimus. By excluding other possible predisposing factors, we concluded that tacrolimus was the most likely cause of SOS.

Sinusoidal endotheliitis as a histological parameter for diagnosing acute liver allograft rejection

AIM To investigated the feasibility of using sinusoidal endotheliitis(SE) as a histological marker for liver allograft rejection.METHODS We compared the histological features of 88 liver allograft biopsies with acute cellular rejection(ACR) and 59 cases with no evidence of ACR. SE was scored as:(1) focal linear lifting up of the endothelial cells by lymphocytes with no obvious damage to adjacent hepatocytes;(2) focal disruption of the endothelial lining by a cluster of subendothelial lymphocytes(a group of > 3 lymphocytes); and(3) severe confluent endotheliitis with hemorrhage and adjacent hepatocyte loss.RESULTS The sensitivity and specificity of SE was 81% and 85%, respectively. Using SE as the only parameter, the positive predictive value for ACR(PPV) was 0.89, whereas the negative predictive value for ACR(NPV) was 0.75. The correlation between RAI and SE was moderate(R = 0.44, P < 0.001)(Figure 3A), whereas it became strong(R = 0.65, P < 0.001) when correlating SE with the venous endotheliitis activity index only.CONCLUSION Our data suggest that SE scoring could be a reliable and reproducible supplemental parameter to the existing Banff schema for diagnosing acute liver allograft rejection.

Liver stiffness and perfusion changes for hepatic sinusoidal obstruction syndrome in rabbit model

BACKGROUND Hepatic sinusoidal obstruction syndrome(SOS)is caused by damage to hepatic sinusoidal endothelial cells that results in fibrous obliteration of intrahepatic venules and necrosis of hepatocytes.Currently the diagnosis is primarily based on nonspecific clinical features and invasive liver biopsy.Therefore,noninvasive imaging methods are required for the early diagnosis and severity assessment of hepatic SOS.AIM To determine the effectiveness of supersonic shear wave imaging(SSI)and dual energy computed tomography(DECT)for diagnosing hepatic SOS using a rabbit model.METHODS Among nine New Zealand white rabbits(3-4 kg,male),three in control group ingested normal saline for 20 d and six in the SOS group ingested 6-thioguanine(5 mg/kg/d)for 20 d.Liver stiffness was measured using SSI on days 0,3,10,and 20.On the same days,liver perfusion was evaluated from virtual monochromatic images of 55 keV and iodine map using DECT.Morphologic changes in the liver were assessed using CT.Final pathology scores were compared between the two groups.Liver stiffness and perfusion parameters were compared according to the groups,days,and pathology scores.RESULTS Final pathology scores were significantly higher in the SOS than the control group(median 22 vs 2,P=0.024).No gross morphologic changes were seen in livers.Liver stiffness,Hounsfield Unit values,and iodine concentrations were higher in the SOS compared to the control group on days 10 and 20(all,P≤0.007).Compared to day 0,liver stiffness and perfusion parameters were higher on day 20 in the SOS group(all,P≤0.001).Correlation coefficients for liver stiffness(r=0.635),Hounsfield Unit values(r=0.587),and iodine concentration(r=0.611)with final pathology scores were positive without significance(all,P>0.05).CONCLUSION Liver stiffness and perfusion parameters were significantly increased in the livers of a rabbit SOS model.SSI and DECT might aid in early diagnosis of hepatic SOS.

罗格列酮通过诱导HO-1减轻大鼠肝缺血再灌注损伤的作用机制

目的观察过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor,PPAR-γ)激动剂罗格列酮是否通过调控血红素加氧酶1(heme oxygenase 1,HO-1)活性来减轻大鼠肝缺血再灌注损伤(ischemia reperfusion injury,IRI)。方法建立大鼠70%肝脏热缺血再灌注(ischemia/reperfusion,I/R)模型和缺氧缺糖/复氧复糖(oxygen-glucose deprivation/reperfusion,OGD/R)诱导的大鼠肝窦内皮细胞(liver sinusoidal endothelial cells,LSECs)损伤模型,随机分为假手术组、模型组、罗格列酮预处理组和锌原卟啉(zinc protoporphyrin,ZnPP)组(n=6)。全自动生化分析仪检测大鼠血清ALT、AST水平;HE染色评估肝组织病理学损伤;Western blot检测PPAR-γ和HO-1蛋白表达水平;CCK8法测定LSECs的细胞活力,流式细胞仪测定LSECs中活性氧(reactive oxygen species,ROS)含量。结果与I/R组相比,罗格列酮预处理能显著降低肝IRI大鼠ALT、AST水平(P<0.01),减少肝细胞凋亡并减轻肝组织IRI(P<0.01)。Western blot结果显示,罗格列酮能上调PPAR-γ和HO-1蛋白的表达(P<0.01)。此外,罗格列酮预处理(10,30μmol/L)能改善OGD/R诱导的LSECs存活率,显著降低细胞ROS含量,并呈剂量反应相关性(P<0.01)。使用ZnPP阻断HO-1活性后,罗格列酮对大鼠肝IRI的保护作用均消失。结论罗格列酮通过上调HO-1活性介导抗氧化和抗炎作用,减轻大鼠肝IRI。

Autophagy in liver diseases

Autophagy is the liver cell energy recycling system regulating a variety of homeostatic mechanisms.Damaged organelles,lipids and proteins are degraded in the lysosomes and their elements are re-used by the cell.Investigations on autophagy have led to the award of two Nobel Prizes and a health of important reports.In this review we describe the fundamental functions of autophagy in the liver including new data on the regulation of autophagy.Moreover we emphasize the fact that autophagy acts like a two edge sword in many occasions with the most prominent paradigm being its involvement in the initiation and progress of hepatocellular carcinoma.We also focused to the implication of autophagy and its specialized forms of lipophagy and mitophagy in the pathogenesis of various liver diseases.We analyzed autophagy not only in well studied diseases,like alcoholic and nonalcoholic fatty liver and liver fibrosis but also in viral hepatitis,biliary diseases,autoimmune hepatitis and rare diseases including inherited metabolic diseases and also acetaminophene hepatotoxicity.We also stressed the different consequences that activation or impairment of autophagy may have in hepatocytes as opposed to Kupffer cells,sinusoidal endothelial cells or hepatic stellate cells.Finally,we analyzed the limited clinical data compared to the extensive experimental evidence and the possible future therapeutic interventions based on autophagy manipulation.

Neuropilin-1: A feasible link between liver pathologies and COVID-19

The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)pandemic has a tremendous impact on the health of millions of people worldwide.Unfortunately,those suffering from previous pathological conditions are more vulnerable and tend to develop more severe disease upon infection with the new SARS-CoV-2.This coronavirus interacts with the angiotensin-converting enzyme 2 receptor to invade the cells.Recently,another receptor,neuropilin-1(NRP-1),has been reported to amplify the viral infection.Interestingly,NRP-1 is expressed in nonparenchymal liver cells and is related to and upregulated in a wide variety of liver-related pathologies.It has been observed that SARS-CoV-2 infection promotes liver injury through several pathways that may be influenced by the previous pathological status of the patient and liver expression of NRP-1.Moreover,coronavirus disease 2019 causes an inflammatory cascade called cytokine storm in patients with severe disease.This cytokine storm may influence liver sinusoidal-cell phenotype,facilitating viral invasion.In this review,the shreds of evidence linking NRP-1 with liver pathologies such as hepatocellular carcinoma,liver fibrosis,nonalcoholic fatty liver disease and inflammatory disorders are discussed in the context of SARS-CoV-2 infection.In addition,the involvement of the infection-related cytokine storm in NRP-1 overexpression and the subsequent increased risk of SARS-CoV-2 infection are also analyzed.This review aims to shed some light on the involvement of liver NRP-1 during SARSCoV-2 infection and emphasizes the possible involvement this receptor with the observed liver damage.

Effect of Qishen decoction on dedifferentiation of sinusoidal endothelial cells by autophagy

Objective:To observe the effect of Qishen decoction on dedifferentiation and autophagy of liver sinusoid endothelial cells(LSEC);Methods:LSEC were randomly divided into the control group,model group,Qishen Decoction low,medium,high dose group,and inhibitor group.The model was induced by 100μg/ml oxidized low-density lipoprotein(oxLDL)for 24 hours,and the corresponding drugs or medicated serum were given for intervention.The expression levels of VEGFR2 and ET1 were detected by RT-qPCR and immunofluorescence staining,the ultrastructure of LSEC was detected by transmission electron microscopy,the content of NO was detected by ELISA,the expression levels of autophagy related proteins(LC3BI,LC3BⅡand p62)and endothelial function related proteins(eNOS and p-eNOS)were detected by western blot;Results:The results of transmission electron microscopy showed that Qishen decoction medicated serum could increase the number of fenestra and autophagy in LSEC cells,and inhibit the formation of basement membrane under endothelium.Compared with the model group,Qishen decoction medicated serum could significantly up-regulate the expression level of VEGFR2 mRNA and protein in LSEC,down regulate the expression level of ET1 mRNA and protein,the difference was statistically significant(P<0.05).In addition,Qishen decoction medicated serum could significantly increase the expression of LC3BII,p-eNOS,eNOS protein and the ratio of LC3BII/LC3BI,p-eNOS/eNOS,and reduce the expression of LC3BI and p62 protein in LSEC,which is statistically significant compared with the model group(P<0.05).Conclusion:Qishen decoction can inhibit the dedifferentiation of LSEC by promoting the autophagy level of LSEC,and then play an anti-fibrosis role.

Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo

The hepatic sinusoids are lined by a unique population of hepatic sinusoidal endothelial cells (HSEC), which is one of the first hepatic cell populations to come into contact with blood components. However, HSEC are not simply barrier cells that restrict the access of blood- borne compounds to the parenchyma. They are func- tionally specialised endothelial cells that have complex roles, including not only receptor-mediated clearance of endotoxin, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Thus understand- ing the differentiation and function of HSEC is critical for the elucidation of liver biology and pathophysiology. This article reviews methods for isolating and studying human hepatic endothelial cell populations using in vitro models. We also discuss the expression and functions of phe- notypic markers, such as the presence of fenestrations and expression of VAP-1, Stabilin-1, L-SIGN, which can be used to identify sinusoidal endothelium and to permit discrimination from vascular and lymphatic endothelial cells.

Alcohol metabolites and lipopolysaccharide: Roles in the development and/or progression of alcoholic liver disease

The onset of alcoholic liver disease (ALD) is initiated by different cell types in the liver and a number of different factors including: products derived from ethanol-induced inflammation, ethanol metabolites, and the indirect reactions from those metabolites. Ethanol oxidation results in the production of metabolites that have been shown to bind and form protein adducts, and to increase inflammatory, fibrotic and cirrhotic responses. Lipopolysaccharide (LPS) has many deleterious effects and plays a significant role in a number of disease processes by increasing inflammatory cytokine release. In ALD, LPS is thought to be derived from a breakdown in the intestinal wall enabling LPS from resident gut bacterial cell walls to leak into the blood stream. The ability of adducts and LPS to independently stimulate the various cells of the liver provides for a two-hit mechanism by which various biological responses are induced and result in liver injury. Therefore, the purpose of this article is to evaluate the effects of a two-hit combination of ethanol metabolites and LPS on the cells of the liver to increase inflamma-tion and fi brosis, and play a role in the development and/or progression of ALD.

Excessive portal flow causes graft failure in extremely small-for-size liver transplantation in pigs

AIM: To evaluate the effects of a portocaval shunt on the decrease of excessive portal flow for the prevention of sinusoidal microcirculatory injury in extremely smallfor-size liver transplantation in pigs.METHODS: The right lateral lobe of pigs, i.e. the 25%of the liver, was transplanted orthotopically. The pigs were divided into two groups: graft without portocaval shunt (n = 11) and graft with portocaval shunt (n=11).Survival rate, portal flow, hepatic arterial flow, and histological findings were investigated.RESULTS: In the group without portocaval shunt, all pigs except one died of liver dysfunction within 24 h after transplantation. In the group with portocaval shunt,eight pigs survived for more than 4 d. The portal flow volumes before and after transplantation in the group without portocaval shunt were 118.2±26.9 mL/min/100 g liver tissue and 270.5±72.9 mL/min/100 g liver tissue,respectively. On the other hand, in the group with portocaval shunt, those volumes were 124.2±27.8 mL/min/100 g liver tissue and 42.7±32.3 mL/min/100 g liver tissue, respectively (P＜0.01). As for histological findings in the group without portocaval shunt, destruction of the sinusoidal lining and bleeding in the peri-portal areas were observed after reperfusion, but these findings were not recognized in the group with portocaval shunt.CONCLUSION: These results suggest that excessive portal flow is attributed to post transplant liver dysfunction after extreme small-for-size liver transplantation caused by sinusoidal microcirculatory injury.

Reconstruction of liver organoid using a bioreactor

瞄准：用一个简历人工的肝为肝机关在试管内的重建开发有效技术。方法：我们以前报导一个光线流动的生物反应器(RFB ) 能提供一个三维的高密度的文化系统。我们目前重建了和老鼠把一根功能的人的肝细胞癌房间线(FLC-5 ) 用作 hepatocytes 的肝或硬鳞鱼使房间(秒) 线 M1 和老鼠使不朽的正弦曲线内皮不朽肝的星形的房间(HSC ) 在 RFB 作为非实质的的房间衬里 A7。二 x 10 (7 ) FLC-5 房间在 RFB 被孵化。在 5 d 以后， 2 x 10 (7 ) A7 房间以 10 的另一增加(7 ) 跟随的一种类似的方式被增加 M1 房间 5 d 以后。在三天灌注以后，有支持者房间的一些纤维素祷告被收获。最后潜伏期与支持者房间一起为 2 h 然后留下的纤维素祷告与 200 nmol/L swinholide A 包括了灌注从 RFB 被收获。房间形态学被传播电子显微镜学(TEM ) 和扫描电子显微镜学(SEM ) 观察。估计 hepatocyte 功能，我们在 RFB 单个类型的文化和 cocultures 与那些相比在单层文化由 FLC-5 为脲周期酶以及白朊合成比较了 mRNA 表示。结果：由传播电子显微镜学， FLC-5， M1，和 A7 在一个像肝的组织在与灌注方面的关系被安排。类似于胆汁 canaliculi 的结构在 FCL-5 房间之间被看见。扫描电子显微镜学在秒表面上表明了窗。当我们介绍了肌动朊绑定代理人 swinholide 时， vesiculo-vacuolar 细胞器(VVO ) 和窗的数字增加了 -- 在为 2h 的 RFB 的 A。关于肝功能，脲在编码 arginosuccinate 合成酶的 mRNAs 的媒介，和表达式被发现，当三种房间类型是在 RFB 的 cocultured 时，精氨酸酶增加了。然而，白朊合成减少了。结论：在 RFB 系统罐头的合作文化戏剧性地改变所有房间类型的结构和功能，包括 hepatocytes 的功能的特征。我们的系统用一个简历人工的肝为一个肝或硬鳞鱼的重建证明有效。