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To determine the most efficient design of a hollow fiber-based bioreactor device for a bioartificial liver support system through comparative bioengineering evaluations. We compared two types of hollow fiber-based bioreactors, the interwoven-type bioreactor (IWBAL) and the dialyzer-type bioreactor (DBAL), by evaluating the overall mass transfer coefficient (K) and the convective coefficient (X). The creatinine and albumin mass transfer coefficients and convective coefficients were calculated using our mathematical model based on the homoporous theory and the modified Powell method. Additionally, using our model, we simulated the mass transport efficiency in clinical-scale BALs. The results of this experiment demonstrate that the mass transfer coefficients for creatinine and albumin increased proportionally with velocity with the IWBAL, and were consistently greater than that found with the DBAL. These differences were further enhanced in the simulation of the large-scale model. Our findings indicate that the IWBAL with its unique 30° cross hollow fiber design can provide greater solute removal and more efficient metabolism when compared to the conventional DBAL design.

There is currently a pressing need for alternative therapies to liver transplantation. The number of patients waiting for a liver transplant is substantially higher than the number of transplantable donor livers, resulting in a long waiting time and a high waiting list mortality. An extracorporeal liver support system is one possible approach to overcome this problem. However, the ideal cell source for developing bioartificial liver (BAL) support systems has yet to be determined. Recent advancements in stem cell technology allow researchers to generate highly functional hepatocyte-like cells from human pluripotent stem cells (hPSCs). In this mini-review, we summarize previous clinical trials with different BAL systems, and discuss advantages of and potential obstacles to utilizing hPSC-derived hepatic cells in clinical-scale BAL systems.

ABH-F and ABH-P have been developed for hemodiafiltration (HDF) therapy. In this study, we evaluated the solute removal characteristics of the hemodiafilters in a bovine blood in vitro study. The hemodiafilters were examined for 120 min at various filtration flow rates (Q F) (31.2–250 mL/min) under a constant blood flow rate of 250 mL/min and constant dialysate flow rates of 500/250 mL/min in pre-dilution HDF (pre-HDF) and post-dilution HDF (post-HDF). Creatinine clearance in pre-HDF was approximately 85 % of that in post-HDF because it was removed by molecular diffusion dominantly. The initial clearances of β2-microglobulin and α1-microglobulin increased with Q F and these values slightly and steeply decreased with time due to membrane fouling. Under a same Q F of 62.5 mL/min, higher clearance values in post-HDF were obtained compared with those in pre-HDF. All clearance values of ABH-P were higher than those of ABH-F under the same Q F. It seems that the ABH-P has a larger pore size of membrane than that in ABH-F. The creatinine and α1-microglobulin clearance values were obtained as highest at post-Q F62.5, the β2-microglobulin clearance values and transmembrane pressure were obtained as highest at pre-Q F250. Large solute clearances such as α1-microglobulin and albumin decreased with time in all HDF experiments. Time decay of large solute clearance values was observed in the HDF modality that had a higher clearance of the solute at 5 min later after the start of experiment.

Objectives: To ascertain the positions of sulfated groups for HGF-inducing activity using differently sulfated heparin disaccharides and to investigate whether the heparin disaccharide elevates HGF levels in plasma in vivo and exerts protective effects on acute liver injury. Materials and Methods: The heparin disaccharides ΔUA-GlcNS, ΔUA (2S)-GlcN, ΔUA-GlcNAc (6S), ΔUA-GlcNS (6S), ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S), ΔUA-GlcNAc and ΔUA (2S)-GlcNS (6S) were added to MRC-9 fibroblasts and HGF concentrations in culture media were determined by enzyme-linked immunosorbent assay. Furthermore, ΔUA-GlcNS (100 μg/head) was injected into C57BL/6 mice and plasma levels of HGF measured at 12 h. After acute hepatitis was induced by CCl 4 (15 mg/kg) in mice, liver specimens were stained with hematoxylin and eosin (H and E). Levels of aspartate aminotransferase and alanine aminotransferase were measured at 24 h. Results: Among the disaccharides investigated, ΔUA-GlcNS, ΔUA-GlcNAc (6S) and ΔUA-GlcNS (6S) stimulated HGF production in MRC-9 fibroblasts. However, none of the 2- O -sulfated disaccharides [ΔUA (2S)-GlcNS, ΔUA (2S)-GlcNAc (6S) and ΔUA (2S)-GlcNS (6S)] showed any activity despite the presence of N -sulfated and/or 6- O -sulfated disaccharides. Thus, 2- O -sulfation of hexuronic acid has an inhibitory effect. Moreover, ΔUA-GlcNS administration increased plasma levels of HGF in normal mice and prevented CCl 4 -induced liver injury in mice. Conclusions: N -sulfation and/or 6- O -sulfation of glucosamine with nonsulfated hexuronic acid provides a structural basis for the HGF-inducing activity of disaccharides. ΔUA-GlcNS increases plasma levels of HGF and protects against CCl 4 -induced acute liver injury.

Internal filtration/backfiltration (IF/BF) of a dialyzer depends on several parameters. This study evaluated the effect of the blood flow rate (Q (B)) on the internal filtration flow rate (Q (IF)) measured using Doppler ultrasonography for a high-flux dialyzer with a polysulfone membrane, APS-15E. In an in vitro study, bovine blood was circulated through the dialyzer, at a Q (B) of 100-350 mL/min. The clearances (CL) of creatinine, β(2)-microglobulin, and α(1)-microglobulin were then investigated. Q (IF) increased with the Q (B) value. A good correlation was obtained between Q (IF) and the pressure difference between the pressures at the inlet of the blood compartment and the pressure at the outlet of the dialysate compartment. The creatinine CL values strongly depended on Q (B) because molecular diffusion was dominant. The β(2)-microglobulin CL also depended on Q (B), because its removal rate seemed to be affected by both diffusive and convective transport caused by the IF/BF. An extremely low CL value was obtained for α(1)-microglobulin because of its low diffusivity and membrane fouling induced by proteins plugging the membrane. In conclusion, the IF/BF in the dialyzer strongly depends on Q (B). Furthermore, the dependence of the solute clearance on Q (B) decreased with increasing molecular size of the solute because of the decrease in diffusivity through the membrane.

In order to improve solute removal efficiency, several types of dialyzers with enhanced internal filtration (IF) were introduced for clinical applications. In these dialyzers, enhanced IF increased convective transport of the solute in addition to diffusive transport. Internal filtration-enhanced hemodialysis (IFEHD) defined as hemodialysis therapy using the enhanced IF dialyzer seems to be more convenient in comparison with conventional hemodiafiltration therapies because of no additional equipments such as a roller pump. In this paper, the validity of IFEHD was examined during several types of experimental studies. As a result, the experimental study with several types of enhanced IF dialyzers indicated values of relative solute clearance in an aqueous solution that were higher than unity compared to the conventional dialyzer. The K values obtained for myoglobin as a larger molecule were more than twice as high. The value of IF flow rate (QIF) was evaluated by pulsed Doppler ultrasonography. The blood flow velocity at a cross-sectional plane of the dialyzer was directly measured along the dialyzer during a bovine in vitro study using a newly developed probe slider. Doppler ultrasonography is a useful method as a bedside monitoring of the QIF value in a dialyzer because it is noninvasive for the patient. Time course of the QIF value was examined for 6 h during a bovine in vitro study. The QIF decreased within 45 min after the start of the experiment and reached constant values after that. Although creatinine and β2-microglobulin K values remained constant with time during the experiment, α1-microglobulin K values gradually and albumin K value steeply decreased with time. This is because these solutes transfer through the membrane strongly affected by fouling. The validity of IFEHD was clarified during the experimental studies. Development of a dialyzer with enhanced IF, however, should take account of the patient’s safety. Contaminations such as endotoxin invasion from the dialysate to the patient should be avoided.

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 T 108 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.

To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63 ± 78 x 108 total cells with a viability of 69.2 ± 25.3 %, but our combined method had a yield of 167 ± 31 x 108 total cells with a viability of 87.9 ± 4.4 % (mean ± SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387 ± 89 x 108 hepatocytes with a viability of 87.1 ± 6.9 % and a purity of 93.6 ± 2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 1010 porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 – 500 μm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 108 porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.

SUMMARY: Computer simulation techniques may be useful for considering an appropriate treatment schedule for peritoneal dialysis by using a mathematical model. The authors derived a new approximated analytical solution for Pyle-Popovich’s peritoneal dialysis model that is known to simulate peritoneal transport quantitatively. In the present study, the validity of the new model was demonstrated by comparing it with another approximated analytical solution derived by other researchers using rabbit in vivo experimental data taken under normal continuous ambulatory peritoneal dialysis (CAPD) mode. The new model showed much better agreement with the experimental data no matter how the plasma concentration changed during the course of the experiment. The new analytical solution can simulate the change of metabolite concentration in the plasma using clinical data at an arbitrary treatment time.

Continuous Ambulatory Peritoneal Dialysis (CAPD) is one of the standard treatments for kidney disease patients. A washing solution, called dialysate, is put into the peritoneal cavity to remove waste products and excess amounts of water in CAPD. The dialysate is exchanged four to five times a day by the patient. However, it is not easy to prescribe CAPD therapy, which may have precluded popularization of CAPD therapy. Popovich et al. constructed a mathematical model (P-P model) that applies to the prescription of the treatment schedule. It requires, however, a number of iterative calculations to obtain an exact numerical solution because the model is a set of nonlinear simultaneous ordinary differential equations. In this paper, the authors derived a new approximated analytical solution by employing a time-discrete technique, assuming all the parameters to be constant within each piecewise period of time for the P-P model. We have also described an algorithm of a numerical calculation with the new solution for determining a set of unknown parameters in the P-P model. We compare the validation of the new solution for clinical use with another analytical solution (Vonesh’s solution).The new analytical solution consists of a forward solution (FW solution), that is the solution for the plasma and dialysate concentrations from ti to ti+1 (ti ti+1), and a backward solution (BW solution) from ti to ti–1 (ti–1 ti). The unknown parameters were determined by employing the Newton–Raphson method, a trial-and-error method and the modified Powell method in combination with FW and BW solutions.The new analytical solution show an excellent agreement with the exact numerical solution for entire dwelling time. Moreover, optimized parameters with the new analytical solution show much smaller discrepancy than those with Vonesh’s solution.Although the proposed method requires a slightly longer calculation time than Vonesh’s, it can simulate concentrations in plasma and dialysate for an entire single exchange in CAPD using the clinical data measured at arbitrary time. The proposed method may be useful for determining unknown parameters as well as for prescribing CAPD treatment.

Ablood purification module was developed with edible agar or Gelrite, in which activated charcoal was dispersed. Tap water (or normal saline) was boiled, and agar (or Gelrite) powder was dissolved in it. The solution was hardened with no additive or activated charcoal in a plastic cylindrical hard shell with a perforated bottom plate to assemble module A (agar only), module AC (agar with various amounts of charcoal), or module GC (Gelrite with various amounts of charcoal), respectively. The hardened gel was thrust with a plastic straw 21 times in the flow direction. Aqueous test solution was prepared for device evaluation. Bromophenol blue (BPB) concentration decreased only by 10% with module A, whereas it gradually decreased according to the amount of activated charcoal dispersed in the agar with module AC; moreover, it reached its lower limit found by direct use of the same amount of intact activated charcoal. Module GC was shown to remove creatinine continuously from bovine whole blood. A portable artificial kidney system may be constructed by combining these modules with a small hemofilter for removing excess water.

Polyurethane foam (PUF)/ spheroid-culture can improve liver-specific functions of hepatoma cell line, Hep G2. Therefore, gene expression profile in the PUF/spheroid culture is hypothesized to be different from that in the monolayer culture. The aim of this study is to clarify the characteristic gene expression in PUF/spheroid-cultured Hep G2 cells, as a cell source for bioartificial liver (BAL), using microarray analysis.Morphological change and liver specific functions of ammonia removal rate and albumin synthesis rate of Hep G2 were compared between in a monolayer or PUF/spheroid culture. Microarray analysis was performed using cDNA microarrays made in Hitachi Software Engineering Co., Ltd., (Yokohama, Japan), which contains a total of 1,281 cDNA clones.The ammonia removal rate of Hep G2 spheroids increased to 369%, and the albumin synthesis rate of Hep G2 spheroids also increased 311% when compared with monolayer culture. In addition, the ammonia removal capacity of primary human hepatocytes in the PUF/spheroid culture was superior to that in the monolayer culture. The microarray analysis demonstrated that the PUF/spheroid-cultured Hep G2 cells expressed 39 up-regulated (more than 3.0-fold) and 31 down-regulated (less than 0.333-fold) genes. Among the 70 genes differentially expressed in PUF/spheroid cultured Hep G2 cells, subsets of glutathione S-transferase- and angio-tensin-related genes were drastically up-regulated, on the other hand, subsets of assigned for growth factor, glucocorticoid, and stress response, were down-regulated.Hepatoma cell line, Hep G2 cells in the PUF/spheroid culture is a promising hepatocyte source for BAL. Microarray analysis revealed a number of characteristic genes altered by the PUF/spheroid.

We previously reported that heparin post-transcriptionally stimulates the production of hepatocyte growth factor (HGF). In this study, we addressed the size-dependency of heparin fragments on the HGF-inducing activity aiming to obtain fragments without antiblood coagulant activity. Heparin fragments, produced by digestion with heparinase, were size-fractionated and tested for HGF-inducing activity in cultured human fibroblasts. The HGF-inducing activity deceased with the reduction in oligosaccharide size. Decasaccharides exerted an activity comparable with undigested heparin, while smaller oligosaccharides showed lesser activities. The anticoagulant activity of heparin fragments also decreased with size and anticoagulant activity of decasaccharides was

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 × 1010 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-μg/dl at the start of circulation, and rapidly increased to 351 N-μg/dl at 2 hours and to 704 N-μg/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-μg/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.

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 (μmol/106 nuclei/h) was upregulated, as was albumin synthesis activity (μg/106 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.

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 μ g/ml, 50 μ g/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 μ g/ml than at 10 μ g/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.