Albert C.W.A. van Wijk

Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver

The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)‐BAL during a 7‐day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2‐fold more ammonia and produced 3‐fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5‐fold higher than MPHs and 6.6‐fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue‐like structures inside the AMC‐BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading. Liver Transpl 13:589–598, 2007.

Liver Regeneration After Portal Vein Embolization Using Absorbable and Permanent Embolization Materials in a Rabbit Model

Objective: To compare the safety and hypertrophy response after portal vein embolization (PVE) using 2 absorbable and 3 permanent embolization materials. Background: Portal vein embolization is used to increase future remnant liver volume preoperatively. Application of temporary, absorbable embolization materials could be advantageous in some situations, provided sufficient hypertrophy is achieved from the nonembolized lobe. Methods: Six groups of rabbits (n = 5) underwent PVE of 80% of the total liver volume using saline (sham), gelatin sponge, fibrin glue, polyvinyl alcohol particles with coils, n-butyl cyanoacrylate, or polidocanol. The rabbits were killed after 7 days. Portography, computed tomographic volumetry, Doppler ultrasonography, laboratory liver function and damage parameters (nonembolized) liver-to-body weight ratio, immunohistochemistry, and cytokine and growth factor tissue levels were assessed to examine the differences in the liver regeneration response. Results: Polidocanol was discontinued because of toxic reactions in 3 rabbits. Gelatin sponge was the only material that was absorbed after 7 days and resulted in less hypertrophy of the nonembolized lobe than the other 3 materials. There were no significant differences in hypertrophy response between the other 3 embolization groups. Volumetric data obtained from computed tomography were supported by liver-to-body weight ratio and the amount of proliferating hepatocytes. The volume gain of the nonembolized lobe was proportional to the volume loss of the embolized liver lobes. The number of Kupffer cells in the embolized liver lobe was significantly higher in the fibrin glue, polyvinyl alcohol particles with coils, and n-butyl cyanoacrylate groups than in the sham and gelatin sponge groups. However, the levels of interleukin-6, tumor necrosis factor-&agr;, hepatocyte growth factor, and transforming growth factor-&bgr;1 were significantly lower. Conclusions: Temporary occlusion using gelatin sponge for PVE resulted in significantly less hypertrophy response than the use of permanent embolization materials. Except for polidocanol, none of the embolization materials exhibited evident hepatotoxicity.

In vitro functionality of human fetal liver cells and clonal derivatives under proliferative conditions.

Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we evaluated the hepatic function of HFLCs under proliferative conditions, to determine whether HFLCs can replace mature hepatocytes for in vitro applications. HFLCs were isolated from fetal livers of 16 weeks gestation. Hepatic functions of HFLCs were determined in primary culture and after expansion in vitro. Clonal derivatives were selected and tested for hepatic functionality. Results were compared to primary mature human hepatocytes in vitro. No differences were observed between primary HFLCs and mature human hepatocytes in albumin production and mRNA levels of various liver-specific genes. Ureagenesis was 4.4-fold lower and ammonia elimination was absent in HFLCs. Expanding HFLCs decreased hepatic functions and increased cell stretching. In contrast, clonal derivatives had stable functionality and morphology and responded to differentiation stimuli. Although their hepatic functions were higher than in passaged HFLCs, functionality was at least 20 times lower compared to mature human hepatocytes. HFLCs cannot replace mature human hepatocytes in in vitro applications requiring extensive in vitro expansion, because this is associated with decreased hepatic functionality. Selecting functional subpopulations can, at least partly, prevent this. In addition, defining conditions that support hepatic differentiation is necessary to obtain HFLC cultures suitable for in vitro hepatic applications.

Assessment of in vitro applicability of reversibly immortalized NKNT-3 cells and clonal derivatives

In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4–20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.

Inhibition of hypoxia-inducible factor 1 with acriflavine sensitizes hypoxic tumor cells to photodynamic therapy with zinc phthalocyanine-encapsulating cationic liposomes

Photodynamic therapy (PDT) is a tumor treatment modality in which a tumorlocalized photosensitizer is excited with light, which results in local production of reactive oxygen species, destruction of tumor vasculature, tumor hypoxia, tumor cell death, and induction of an anti-tumor immune response. However, pre-existing tumor hypoxia may desensitize tumors to PDT by activating the hypoxia-inducible factor 1 (HIF-1) survival pathway. Therefore, we hypothesized that inhibition of HIF-1 with acriflavine (ACF) would exacerbate cell death in human epidermoid carcinoma (A431) cells. PDT of A431 tumor cells was performed using newly developed and optimized PEGylated cationic liposomes containing the photosensitizer zinc phthalocyanine (ZnPC). Molecular docking revealed that ACF binds to the dimerization domain of HIF-1α, and confocal microscopy confirmed translocation of ACF from the cytosol to the nucleus under hypoxia. HIF-1 was stabilized in hypoxic, but not normoxic, A431 cells following PDT. Inhibition of HIF-1 with ACF increased the extent of PDT-induced cell death under hypoxic conditions and reduced the expression of the HIF-1 target genes VEGF, PTGS2, and EDN1. Moreover, co-encapsulation of ACF in the aqueous core of ZnPC-containing liposomes yielded an adjuvant effect on PDT efficacy that was comparable to non-encapsulated ACF. In conclusion, HIF-1 contributes to A431 tumor cell survival following PDT with liposomal ZnPC. Inhibition of HIF-1 with free or liposomal ACF improves PDT efficacy.

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

Background: Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL). Methods: Distribution and organization of porcine hepatocytes inside the AMC-BAL as well as GS and CPS expression were analyzed (immuno-)histochemically in time. Ten zonally expressed proteins were analyzed by RT-PCR on cell isolate and bioreactor samples. General metabolic and hepatocyte-specific functions were determined as well. Results: Viable hepatocyte layers of approximately 150 µm were observed around gas capillaries, whereas inside the matrix, single cells or small aggregates were present. GS protein and mRNA levels were upregulated in time. GS protein was preferentially expressed in hepatocytes adjacent to oxygen-supplying capillaries and in previously CPS-positive hepatocytes. No shift towards a periportal or pericentral phenotype was observed from RT-PCR analysis. Conclusion: Induction of GS expression inside the AMC-BAL is not dependent of (low) oxygen tensions and hepatic nuclear factor 4α transcript levels. GS expression might be related to (1) low substrate levels and/or autocrine soluble factors, or (2) to cytoskeleton interactions, putatively associated with the β-catenin signaling pathway.

Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF-1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3) determine whether HIF-1 inhibition reduces survival signaling and enhances PDT efficacy. Results: Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and topoisomerases I and II. Methods: The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was determined by immunohistochemistry in human perihilar cholangiocarcinomas. In addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT with liposome-delivered zinc phthalocyanine was investigated under both normoxic and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1β dimerization inhibitor and a potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on PDT efficacy. Conclusions: HIF-1, which is activated in human hilar cholangiocarcinomas, contributes to tumor cell survival following PDT in vitro. Combining PDT with acriflavine pretreatment improves PDT efficacy in cultured cells and therefore warrants further preclinical validation for therapy-recalcitrant perihilar cholangiocarcinomas.

Mild hypothermic preservation for transport purposes of the AMC bioartificial liver charged with porcine hepatocytes.

Background. Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. Methods. Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37°C. Bioreactors in the transport group were kept at 37°C during day 1, at 15°C during day 2, and again at 37°C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15°C and 26°C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. Results. A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. Conclusions. Mild hypothermic preservation at temperatures as low as 15°C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.

Characterization and quantification of porcine circulating endothelial cells

Endothelial damage is a critical step in the development of (xeno) transplantation‐related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker.