Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.

The mode of action of cytochalasin B was examined in vitro using bile canaliculus-enriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member. These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.

Hepatic differentiation of mouse embryonic stem cells in a bioreactor using polyurethane/spheroid culture.

BACKGROUND: We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. METHODS: The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. RESULTS: mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 x 10(8) cells/cm3 PUF). Differentiating ES cells expressed endodermal-specific genes such as alpha-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. SUMMARY: mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.

Hepatic differentiation of embryonic stem cells in HF/organoid culture.

OBJECTIVE: The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. MATERIALS AND METHODS: ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. RESULTS: ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. CONCLUSIONS: ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Evaluation of a hybrid artificial liver module with liver lobule-like structure in rats with liver failure.

We studied the recovery of rats with fulminant hepatic failure (FHF) by treating them with our original hybrid artificial liver support system (HALSS). We developed an original artificial liver module having a liver lobule-like structure (LLS). This module consists of many hollow fibers regularly arranged in close proximity and hepatocyte aggregates (organoids) induced into the extra capillary space of the module by centrifugal force. The LLS module can express some liver specific functions at high levels and maintain them for several months in vitro. In this study, we evaluated the efficacy of our LLS-HALSS by using rats with FHF induced by a method that combined partial hepatectomy with hepatic ischemia. In the animal experiments, blood ammonia levels rapidly increased in the control group (sham-HALSS group). These rats died during or immediately after application of the sham-HALLS. On the other hand, in the LLS module application group (LLS-control group), the increase in blood ammonia was completely suppressed and all rats recovered. Blood constituents at 4 weeks after application were at normal levels, and the weight of the liver was the same as that of a normal rat. These results indicate that HALSS may be useful for treating liver failure patients until liver transplantation can be performed or until regeneration of the native liver occurs.

Formation of a sheet-shaped organoid using rat primary hepatocytes for long-term maintenance of liver-specific functions.

In recent years, use of hepatocyte aggregates has led to development of a hybrid artificial liver support system (HALSS) that has high performance. However, in general, their thickness is 100 microm or more, and generation of a dead cell layer due to oxygen exhaustion inside the aggregates has been a universal problem. The present study proposes a novel organoid culture method with better performance than previous organoid culture methods by forming a sheet-shaped organoid (organoid-sheet) with a thickness of approximately 100 microm. The cell number of the organoid-sheet was maintained at approximately 75% of the initial number at 4 days of culture. On the other hand, that of a cylindrical organoid (cylindroid), which formed inside of a plasma separation hollow fiber with 285 microm inner diameter in our previous study, decreased to approximately 50% within 2 days. The ammonia removal rate of the cells in the organoid-sheet was higher than that of the cells in the cylindroid on the first day, but it decreased during the culture time. At day 15, the rate was reduced by almost 50% with respect to the value on the first day. The cells in the cylindroid displayed a lower ammonia removal rate. A significant difference was not observed between the albumin synthesis rates of the two cultures on the first day. However, over a period of time the cells in the organoid-sheet showed a higher albumin synthesis rate than cells in the cylindroid. As this novel organoid maintains these functions for at least 1 month, it is expected to be applied for the development of a HALSS with higher performance.

Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): a possible cell source for hybrid artificial liver.

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase--a urea cycle enzyme--was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Evaluating the performance of a hybrid artificial liver support system with a recoverable hepatic failure rat model.

To evaluate the performance of an artificial liver, we created a recoverable hepatic failure rat model. This involves a 30-60 minute warm ischemia, via clamping, of one-third of the liver with a partial (two-thirds) hepatectomy. Variations on this method provide for the possibility of several modes of hepatic failure. Survival time of the rats was prolonged (35%) by applying our hybrid artificial liver. However, the extracorporeal circulation is a considerable burden to the rat. Therefore, we need to apply the hybrid artificial liver intermittently and repeatedly.

Efficacy of a polyurethane foam/spheroid artificial liver by using human hepatoblastoma cell line (Hep G2).

We invesigated the availability of human hepatoblastoma cell line (Hep G2), compared with human primary hepatocytes (HH) and porcine primary hepatocytes (PH), as a cell source for the hybrid artificial liver support system (HALSS) by using polyurethane foam (PUF). All three kinds of hepatocytes spontaneously formed spherical multicellular aggregates (spheroids) of 100-200 microm diameter in the pores of PUF within 3 days of culture. In a PUF stationary culture, Hep G2 spheroids recovered the ammonia removal activity that was lost in monolayer culture, although the removal for each unit cell number was about one tenth that of HH spheroids and about one eighth of PH spheroids. The synthesis activities of albumin and fibrinogen of each unit cell number of Hep G2 were also upregulated by PUF spheroid culture, and were about twice as high as in monolayer culture. The albumin secretion activity of Hep G2 spheroids was almost the same as that of PH spheroids. HH scarcely secreted these proteins in this experiment, probably because they were cultured in a serum-free medium. In the PUF module in a circulation culture, HH had high ammonia removal and low synthesis activities similar to stationary culture. Hep G2 proliferated to a high cell density, such as about 4.8 x 10(7) cells/cm3-module at 10 days of culture. Although Hep G2 spheroids had low ammonia removal activity in each cell, the removal rate in the PUF module was almost the same as for PH at 7 days of culture because of the high cell density culture by cell proliferation. The albumin secretion rate by Hep G2 in the PUF module also increased with cell proliferation and was about 10 times higher than the initial for the rate for PH at 7 days of culture. These results suggest that Hep G2 is a potential cell source PUF-HALSS.

Multilocational hepatocyte transplantation for treatment of congenital ascorbic acid deficiency rats.

We attempted multilocational hepatocyte transplantation (HCTx) including hepatocyte-bearing polyurethane foam (PUF) to treat congenitally ascorbic acid (AsA) biosynthetic enzyme-deficient (ODS-od/od) rats. Hepatocytes isolated from the liver of congeneic rats were transplanted into the portal vein (Pv), spleen (Sp), omentum (Om), and mesentery (Ms). Hepatocyte-bearing PUF was transplanted into the Om and Ms. Experimental groups were divided into four groups (group I; Pv + Sp, group II; Pv + Sp + Om + Ms, group III; Pv + Sp + hepatocyte-bearing PUF, group IV; control). The average serum AsA level of the surviving rats in group II and III was significantly higher than that in group I 3 mo after HCTx. Histological examination showed small foci of surviving hepatocytes in the Om and Ms tissues and in the connective tissue in the PUF. ODS-od/od rats survived for a long time by multilocational HCTx.

Polyurethane foam/spheroid culture system using human hepatoblastoma cell line (Hep G2) as a possible new hybrid artificial liver.

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we used human hepatocytes with replication capacity (fetal hepatocytes, Hep G2, and Huh 7) in a polyurethane foam (PUF)/spheroid culture system in vitro, and analyzed liver functions such as ammonia removal and albumin synthesis capacity; results were compared to those of porcine hepatocytes. Human fetal hepatocytes, Hep G2, and Huh 7 formed spheroids spontaneously within 24 h in a PUF/spheroid culture system; ammonia removal activity (micromol/10(6) nuclei/h) was upregulated, as was albumin synthesis activity (microg/10(6) nuclei/day). In particular, Hep G2 spheroids demonstrated high ammonia removal and albumin synthesis activities: 85% of the ammonia removal activity and 171.7% of the albumin synthesis activity of porcine hepatocytes in the monolayer culture. These results indicate the possibility of the development of a multicapillary PUF (MC-PUF) packed-bed culture system of hepatocyte spheroids as a HALSS using Hep G2.

Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain.

Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of [3H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system.

Thyrotropin releasing hormone increases 5-hydroxytryptamine1 receptors in the limbic brain of the rat.

The effect of TRH on 5-HT1 receptors in the rat brain was investigated. A crude membrane preparation was incubated at 37 degrees C for 15 min with or without TRH prior to [3H]5-HT binding assay. TRH at 100 nM increased the number of 5-HT1 receptors significantly (approximately 20%) in the limbic forebrain and the hippocampus without altering their affinity. As this concentration of TRH is close to its dissociation constant (2 nM and 51 nM in the limbic forebrain, 11 nM in the hippocampus), this effect is probably of physiological relevance. This finding seems to support a pharmacological finding of others that the anti-convulsion effect of TRH may be related to increased serotonergic transmission.

5-Hydroxytryptamine and (+)-lysergic acid diethylamide displace [3H]thyrotropin-releasing hormone at its binding sites in the rat limbic forebrain.

Binding of [3H]thyrotropin-releasing hormone (TRH) to its receptors in the rat limbic forebrain was partially displaced by 5-hydroxytryptamine (5-HT, ligand for 5-HT1 receptors) and (+)-lysergic acid diethylamide ((+)-LSD, ligand for 5-HT1 and 5-HT2 receptors) at nanomolar concentrations. Spiperone (ligand for 5-HT2 receptors) displaced [3H]TRH in a dose-dependent manner at micromolar concentrations. These results suggest that some TRH receptors are related to 5-HT1 receptors, probably adjoining them on the membrane. This type of TRH receptor is shown to be among the high-affinity receptors which we reported previously. The significance of the receptor-coexistence is such that TRH facilitates serotonergic transmission by increasing the density of 5-HT1 receptors. This finding seems to support a pharmacological observation of other investigators that TRH potentiates 5-HT-induced hyperactivity in mice, probably by affecting postsynaptic 5-HT receptors.

Modulation of immunologic reactions between cultured porcine hepatocytes and human sera.

In the clinical application of a hybrid artificial liver system using porcine hepatocytes, some immunologic reactions occur between human serum and porcine hepatocytes. In this study, we investigated the immunologic mechanisms of the cytotoxic reactions, and we tried to inactivate the human serum cytotoxicity by heating the serum or the addition of nafamostat mesilate (NM). Immunologic reaction between human serum and porcine hepatocytes by evaluating the immunochemical response against human IgM, IgG, and C3 was investigated. The immunochemical analysis of inactivation by heated human serum (56 degrees C, 30 min) and adding NM were performed. The evaluation of serum cytotoxicity was as follows: when porcine hepatocytes were cultured with heating the human serum or the addition of NM, the survival ratio was observed. Immunochemical reactions against human C3 were all positive, but positive reaction against human IgM occurred in only one case (5%); those against human IgG were all negative. Both heating the serum and adding NM inhibited the immunochemical reaction of human C3. The inhibition of human C3 with NM was dependent on that concentration. Both heating of the serum and adding NM to the medium decreased damage of porcine hepatocytes. An immunologic reaction between human serum and porcine hepatocytes in a porcine bioartificial liver clearly occurred, and this reaction was controlled by heating the serum and adding NM. We believe that NM is useful in the clinical application of our hybrid artificial liver system.

Hepatocyte organoid culture in elliptic hollow fibers to develop a hybrid artificial liver.

A novel organoid culture was developed in which hepatocytes maintain high liver functions for more than several weeks in vitro. The main disadvantage of tissue-engineered organoids is the lack of a blood vessel structure between the aggregated cells. Because of depletion of oxygen, the thickness from the surface of an organoid at which hepatocytes can survive is limited. This study showed that a rat hepatocyte organoid that forms by using centrifugal force in a hollow fiber (HF) had a survival limit thickness of about 80 - 100 microm from the surface of the organoid. Based on the value, we designed an elliptic HF having less than 150 microm minor diameter by using a simple annealing method. All hepatocytes were supplied with oxygen and formed an organoid without a dead cell layer in this HF A hepatocyte organoid in an elliptic HF maintained ammonia removal activity twice as high as in the original HF for at least one month during culture. Albumin secretion activity of an organoid in an elliptic HF was also maintained for at least one month and was the same level as that of liver in a living body. In conclusion, organoid culture by using an elliptic HF seems to be a promising technique to develop a hybrid artificial liver.

Recovery of rats with fulminant hepatic failure by using a hybrid artificial liver support system with polyurethane foam/rat hepatocyte spheroids.

We studied the recovery of rats with fulminant hepatic failure (FHF) by treating them with our original hybrid artificial liver support system (HALSS). FHF was induced by a two-thirds partial hepatectomy and 10 minutes of hepatic ischemia. Rats with FHF were treated with a polyurethane foam/spheroid HALSS including 2.0 x 10(8) hepatocytes for 1 hour (HALSS group, n = 5), and with the same system without hepatocytes in the artificial liver module as a control experiment (sham-HALSS group, n = 3). The level of blood constituents, ammonia, glucose and creatinine, showed no major difference between the two groups at the end of treatment. All rats in the sham-HALSS group died within 5 hours after treatment. However, the level of blood constituents of rats with FHF in the HALSS group improved with time, and all rats in the HALSS group recovered. Liver tissue of rats treated with HALSS showed cell mitosis and improvement from injury. These results indicated that our HALSS has a strong possibility to induce recovery from hepatic failure.

Conditions required for a hybrid artificial liver support system using a PUF/hepatocyte-spheroid packed-bed module and it's use in dogs with liver failure.

We studied the effects of a hybrid artificial liver support system we developed on dogs with hepatic failure. The system consisted of a multi-channel polyurethane foam packed-bed culture module, including primary dog hepatocyte spheroids. Blood ammonia was well metabolized by 20 g hepatocytes, but the other functions such as glucose concentration, total bile acid concentration, and survival time required 30 g hepatocytes to improve conditions. We found that we should use a culture substratum that easily forms spheroids, and that an artificial liver module should be used as soon as possible after spheroid formation by hepatocytes in the module.

Development of a hybrid artificial liver using a polyurethane foam/hepatocyte-spheroid packed-bed module.

Primary dog hepatocytes spontaneously formed spheroids in the pores of polyurethane foam (PUF) within 1-2 days of stationary culture. The spheroids, about 100-150 microm in diameter, partly attached to the surface and immobilized inside these pores. The lidocaine disappearance rate decreased to about 4 microg/10(5) viable cells/day for 10 days, while in the PUF/spheroid culture the rate was maintained at almost the initial level of 8 microg/10(5) viable cells/day for 10 days. Then, two scales of PUF packed-bed modules were designed. A small module (PUF volume; 14.5 cm3) was used for in vitro culture to investigate optimum culture conditions, and a large module (PUF volume; 300 cm3) was designed for dog experiments. Hepatocytes inoculated in these modules also formed spheroids and maintained almost the same activity of albumin secretion rate (111 microg/cm3 PUF/day in the small module and 87.7 microg/cm3 PUF/day in the large module). These results indicate that the PUF packed-bed module containing hepatocyte-spheroids is promising as a hybrid artificial liver.

The efficacy of nafamostat mesilate on the performance of a hybrid-artificial liver using a polyurethane foam/porcine hepatocyte spheroid culture system in human plasma.

Nafamostat mesilate (FUT) is a protease inhibitor of complement activation. The present study investigates whether FUT protects porcine hepatocytes from being injured by human plasma in a multi-capillary polyurethane foam packed-bed culture system (MC-PUF) such as the hybrid-artificial liver (PUF-HAL). Human plasmas with 1 mM of added ammonia were perfused using a small-scale PUF-HAL with porcine hepatocytes. FUT was continuously infused (10 microg/ml, 50 microg/ml). The ammonia detoxification was maintained in human plasma for 24 hours and for 48 hours with FUT which suppressed the rapid increase of asparaginic acid aminotransferase (AST) and alanine aminotransferase (ALT). After 60 hours of perfusion, hepatocyte spheroids completely collapsed in the human plasma, but a small amount of hepatocyte spheroid was maintained by FUT. The effect of FUT was slightly greater at 50 microg/ml than at 10 microg/ml. Our results suggest that FUT has protective effects against porcine hepatocytes in human plasma, and our PUF-HAL using porcine hepatocytes can function in human plasma for about 48 hours with FUT.

Development of a hybrid artificial liver using polyurethane foam/hepatocyte spheroid culture in a preclinical pig experiment.

We describe a preclinical study of our original hybrid artificial liver support system (HALSS) for a clinical trial. We designed a HALSS comprising a multi-capillary polyurethane foam packed-bed module (MC-PUF module) containing a total 200 g (2 x 10(10) cells) porcine hepatocytes, and an extracorporeal circulation device. Almost all porcine hepatocytes in the MC-PUF module formed many spherical multicellular aggregates (spheroids). This extracorporeal circulation device was improved to promote solute exchange between a living body and a MC-PUF module by including a plasma bypass line in the circulation loop. The efficacy of the HALSS was evaluated using a 25-kg pig with warm ischemic liver failure by portocaval shunt and ligation of hepatic artery (HALSS group, n=3). As a control experiment, the same system without hepatocytes in the module was used with the same kind of liver failure pig (Control group, n=3). The blood ammonia in the control group was 143 N-microg/dl at the start of circulation, and rapidly increased to 351 N-microg/dl at 2 hours and to 704 N-microg/dl at 6 hours. But the blood ammonia in the HALSS group was completely suppressed, and remained less than the hepatic coma level (over 200 N-microg/dl) during the circulation time. The blood glucose in the control group gradually decreased, and became less than 40 mg/dl within 6 hours of circulation. But the blood glucose in the HALSS group was maintained well, and remained the normal glucose level (50 - 105 mg/dl) for more than 20 hours of circulation. Improvement in blood creatinine and lactate, and the stabilization of vital signs and urinary excretion, were observed in the HALSS group. The survival time of the pigs in the HALSS group was 19.3 hours compared with 8.9 hours in the control group. In conclusion, our HALSS was effective to stabilize the general conditions of the body in addition to supporting various liver functions. These results suggest that our HALSS has a strong possibility to be used in treating liver failure patients. We have applied for approval of the clinical trial of our HALSS to our institutional ethics committee.