Osmotic fragility (OF) on RBC was studied for hemodialysis (HD) and hemofiltration (HF). Cellulose acetate membrane (CA) and cuprophane membrane (CP) were used for HD and CA was used for HF. The lowest OF was observed after HD using CA and secondly after HF using CA. 15 minutes after start of operation, however, the lowest OF was seen in CP. This time was equal when hemodialysis leukopenia occured, and some relationship was suggested between OF and hemodialysis leukopenia during hemopurification. No correlation was observed between OF and BUN or serum creatinine for each membrane, and clearance was lower in HF than HD. As for OF on RBC, HD using CA was seemed the best hemopurification.

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We observed complement activation during hemodialysis in cases with acute renal failure and discussed its role in disdialysis. Levels of anaphylatoxin except C5a were already elevated at the beginning of the procedure in most of cases. C3a were markedly increased in both atrial and venous side samples. Elevation of C5a were noted only in venous side. This result indicate that anaphylatoxin has a significant role in disdialysis.

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Influence of two different alkalizers in dialysate to acid-base balance and plasma concentration of electrolyte in chronic dialyzed patients were studied. Blood PH, HCO₃^- and base excess were examined before and after hemodialysis (HD) in order to evaluate the improvement of metabolic acidosis. After HD using bicarbonate, PH, HCO₃^- and base excess were remarkablly elevated. However, these values were not so much incresed using acetate as bicarbonate. On HD using bicarbonate concentration of sodium and patassium in red bolld cell did not change significantly, although abnormalities in plasma concentration of Na and K were obviously corrected.

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The plasma Mg, erythrocyte Mg and PTH levels were determined in 16 uremic patients on chronic hemodialysis with different Mg concentrations in dialysate. In 9 patients, Mg concentration in dialysate was reudced from 1.5mEq/l to 1.0mEq/1 for a period of 3 months and in the remaining 7 patients it was maintained at the same level. At the end of the 3 months period, the decrease of the Mg concentration in the dialysate resulted in a reduction of plasma Mg and erythrocyte Mg. And, no significant change were observed in the PTH levels. The results of this study indicate that the 1.0mEq/l of Mg concentration of dialysate is useful in the normalization of hypermagnesaemia and magnesium excess of erythrocyte without PTH elevation.

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Zinc behavior during dialysis. (Zinc Dialysance) Zinc movement during dialysis and serum zinc concentration in fifteen chronic hemodialysis patients were examined. The serum zinc concentration and movement were discussed from the point of zinc dialysance and free diffusible zinc. We measured serum zinc concentration before and after hemodialysis. Also, we examined zinc in the arterial blood and in the dialysate at the inflow and outflow sites of the dialyzer. Zinc levels of ultrafiltrate fluid obtained with ECUM method were measured at the fifteen minutes after the beginning of hemodialysis. We calculated zinc dialysance and examined the relationship zinc dialysance between free diffusible zinc. Hematocrit and total serum protein were measured before and after hemodialysis to examine hemoconcentration. We obtained the following results: serum zinc concentration before dialysis 74.0±7.8, after dialysis 88.1±9.7μg/dl, serum zinc levels of the blood at the inflow site of the dialyzer 74.7±8.1, outflow site 80.2±6.5μg/dl, dialysate zinc concentration inflow site 10.6±2.5μg/dl, outflow site 9.5±5.9μg/dl. There was no significant difference between (Zn levels after HD)/(Zn levels before HD) and (T. P levels after HD)/(T. P levels before HD). From these facts, we reached the following conclusion that serum zinc concentration during dialysis can increase by hemoconcentration as water is lost and by diffusion of zinc from the dialysate to the blood. Zinc dialysance was negative in 13 of 15 hemodialysis patients.

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We studied symptomatic types and therapeutic effects of the dialysis disequilibrium syndrome (DDS) in hemodialysis (HD) with acetate dialysate using two group: 10 cases of the unexperienced (within three months after initiation of HD) and 20 cases of the experienced (over three months). In the former, nausea, vomiting and discomfort were seen during HD. Headache and head discomfort were obserbed in the later and after HD, and 10% glycerine was more effective than analgesics for these symptoms. Arterial blood (AB) and cerebro-spinal fluid (CSF) were drawn in three patients and analyzed. The difference in osmotic pressure between AB and CSF were seen during HD and also that of pH after HD. Hypotension was most common symptom in the experienced which was seen in later part of HD. 64 cases were newly treated with HD using acetate dialysate in 1983. 13 cases complained of venous symptom and their dialysate was changed to bicarbonate solusion.

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We made and examined new extracorporeal livar perfusion set that is easy to control QB and temperature. ELP set contained ELP box (ELF-1), Blood warmer (BEW-II), Heat exchanger (NEX-3) and 2 pulsatile pumps. In 5 jaundiced dogs perfusion, livar temperature (38.0°C) and QB (0.7ml/g·liver/min) were well controlled during 180min. Bile output was about 2-3ml/hr and oxgen consumption was 0.0332±0.0031ml/g·liver/min. Microscopic findings of thee perfused liver after 180min was well preserved.

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DHP through the IONEX column was performed safely with PUT, but the marked decrease in platelet counts and PMNs were not avoided, as they are with heparin. PUT inhibited complement systems assessed by CH50. PUT is suspected to be a useful regional anticoagulant. But, in order to perform DHP of IONEX, the prevention of thrombocytopenia must be insured.

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Extracellular matrices of various organs were prepared and utilized for the culture substrate of the hepatocytes in the primary culture, with an aim of establishing biological artificial-liver support. Isolated rat hepatocytes attached the extracellular matrix of the liver “liver biomatrix” more rapidly than those of spleen and lung. Cells attached liver biomatrix started spreading immediately after attachment under cooperation with some serum factors, but cells with no contact to the matrix did not during the first 30 minutes of cell seeding. Cell attachment and spreading did not show any differences in between intra- and interlobular matrices of the liver also in blockade of fibronectin and laminin with the respective antibodies. These results indicated that the extracellular matrix of the liver had an organ specific ability of hepatocyte attachment and spreading.

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For the basical research of hybrid artificial liver using hepatocytes, the activities and the maintenance of hepatocytes on several collagenous substrata were examined. The maintenance of hepatocytes on collagenous surfaces were better than non-coated PSt surface. Hepatocytes cultured on succinylated collagen surface had the most highest activities. Moreover, hepatocytes were cultured on hollow fiber in Artificial Capillary Culture Module.

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Metabolic support device using isolated hepatocytes was made on an experimental basis. PMMA membrane was chosen for the separation of hepatocyte suspension and patient blood. Cut-off MW of the membranes were 4×10⁴ and 10⁵ daltons. The difference of metabolic ability was evaluated experimentally. In in-vitro perfusion tests, the changes of ammonia, BUN and glucose were almost same in both membranes. However, in in-vivo perfusion test the survival time was rather longer in 10⁵ daltons group, and the blood chemical analysis showed some improvements in the group.

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Porta-Caval shunt and 70% hepatectomy were performed on 59 SD rats, and they were devided into two groups, AST-group and control group. In the AST-group, 0.5-2g/day of the oral adsorbent was administered postoperatively. On the 5th, 10th and 20th P. O. D., DNA and protein synthesis of the liver were measured with the administration of ³H-thymidine and ³H-amino acids mixture intraperitoneally, and they were significantly increased in AST-group.
It was considered that liver regeneration after P-C shunt and hepatectomy was improved by administration of the oral adsorbent.

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We developed of a new porous type anion exchange resin (DIAION) for removal of bilirubin. In present study, in vitro adsorption of bilirubin was tested with this material. In vivo studies were performed to investigate its adsorptive capacities for bilirubin in connection with direct hemoperfusion (DHP) and plasmapheresis using jaundiced dogs. As results of in vitro and vivo tests, adsorbability of its material for bilirubin was about 5mg/gDIAION. It was concluded that the porous type anion exchange resin had selective adsorbability for bilirubin and had to effect in high hyperbilirubinemia.

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Performance of a hybrid-type artificial liver utilyzing isolated hepatocytes is inevitably limited by the hepatocyte viability. Thus, for the purpose of improving culture conditions of hepatocytes, the use of ‘conditioned’ medium commonly used in cell line cultures was attempted in the suspension culture of the rat hepatocytes. Conditioned medium was prepared by mixing 4 volume of the first culture of the isolated hepatocyte with 6 volume of the fresh medium. It was found that death rate constants of hepatocytes in the conditioned culture were one order lower than those in the conventional culture conditions, while the metabolic rate constants remained unaffected.

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Heterogeneous hepatocytes were isolated, entrapped in bead shaped calcium alginate hydrogel, which contains serum-free modified Williams' medium E to avoid immunological contaminations, and encapsulated with poly (L-Lys)/poly (ethylene imine) membrane. These double encapsulated hepatocytes showed about the same level of metabolic activity for ammonium and caproic acid, and lower death rate constant by the factor of 1/100 in comparison with free cells. These result suggest the possibility of application to a hybrid-type, artificial liver support.

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Hepatocytes were isolated from adult rat liver by perfusing with collagenase. Isolated hepatocytes were entrapped within various materials and entrapped hepatocytes were examined on abilities of ammonia removal and urea synthesis. Collagen, fibrin, Ca-alginate, K-carrageenan, agar, low gelling point agarose, urethanprepolymer (PU-3, PU-6), and photopolymerizing resin (ENT-4000) were used for entrapping materials. Hepatocytes entrapped within collagen, fibrin, and Ca-alginate showed high activities of ammonia removal and urea synthesis. Among these three materials, Ca-alginate was chosen as the best material for entrapment, since it can be gelled under very gentle conditions and resulting gel has good intensity. Besides ammonia removal and urea synthesis, hepatocytes entrapped within Ca-alginate could remove indole and phenol, synthesize the corresponding conjugants, and remove short chain fatty acids. The entrapped hepatocytes maintained these abilities of detoxication during seven days. These results suggest that hepatocytes entrapped within Ca-alginate could be applied to a biological artificial liver support.

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The canine liver pieces (3 to 5mm cubic) were force frozen with helium gas. After long preservation of FLP, they were thawed, The thawed FLP has a good viability as glucose systhesis (3.65mg/g. FLP/hr), production of urea nitrogen (0.2mg/g FLP/hr) and prothrombin (363% activity). The system consisted of plasma separator and column with thawed FLP. In vivo study, blood coagulation in the system and immunological reaction were not observed. In total hepatectomized dogs, the direct haemoperfusion was continued during 36 hours. BUN and creatinine remained at the normal limit. In the near future, one will be able to apply the system clinically.

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New artificial liver system composed of isolated hepatocytes, polyethylene hollow fiber plasma separator and membrane oxygenator was developed. The system was applied to anhepatic rats as a model of acute hepatic failure and was evaluated by the difference of survival time and the change of blood ammonia concentration. Significant prolongation of survival time and marked reduction of ammonia concentration were shown compared to the control group. These results indicate the possibility of this new system replacing the deteriorated liver function.

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A direct hemoperfusion (DHP) system has been developed for use in an artificial liver support system using the polyetherurethane sheet embedded with powdered charcoal (UPC) that had been prepared mainly for the purpose of effectively adsorbing protein-bound substances. Powdered charcoal with specially selected pore size distribution and particle size was used for UPC. A polypropylene container was used to prepare a column containing 80g of powdered charcoal by filling it with a 45m×15cm roll of the 150μm thick UPC. Adsorption capacity of UPC-column is about 350mg of bilirubin. DHP was also performed on jaundiced patients. A 3-hour hemoperfusion using one UPC column obtained the removal amounts of 30% of bilirubin and 42% of bile acid.

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Plasma exchange (PE) for acute hepatic failure (AHF) was evaluated from 6 patients. 2 patients showed poor prognosis. Before the initiation of PE, plasma concentration of bilirubin was ever 20mg/dl during 2 weeks in one patient, and prothrombin time was below 40% during 3 weeks in another patient. The early initiation of PE for AHF was applied to 4 patients. The survival rate was 75%. We concluded that the early initiation of PE for AHF was effective.

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We think that Direct hemoperfusion (Charcoal hemoperfusion) for liver failure after the operation. After the operation, we treated seven patients of liver failure with CHP and other treatment. We thought there was an interrelation between Molar ratio and coma grade, but the result of aminogram was not easy to get, and there was an interrelation between the total value of bilirubine and coma grade so that the index could be made out of it. As the function of the remaining hepatic cells was most important, it was important to consider appropriate operation for the remaining hepatic cells function in the resection with liver cirrhosis, and for the long time it was necessary to develop the safe treatment to hosten the remaining hepatic cells revival.

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The function of reticuloendtherial system decrease in the hepatic failure. And it get sometimes the ARDS in the lung catching of toxic substances that be spilled over liver. So that it is easy to develop to the MOF. Those hepatic failure appears as the end stage or the complication of the various diseases related to liver. Those of groups are liver cirrhosis, occlusive icterus, septic icterus, severe hepatitis, fuluminant hepatitis. We tried the establishment of the therapy by studing differences of clinical features in those diseases.

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The artificial liversupport system using charcoal hemoperfusion, cuprophan membrane hemodialysis and plasma exchange using cellulose acetate hollow fiber (CAHF) has been developed and applied to patients with acute hepatic failure. 8 out of 43 patients with fulminant hepatic failure and all 8 patients with acute fatty liver of pregnancy were survived. Even in fatal cases of fulminant hepatic failure, the prolongation of survival time was shown to be possible by daily repeated charcoal hemoperfusion and plasma exchange. These clinical results indicated that this system was effective in treatment of acute. hepatic failure. Elevation of AFP in serum indicated to be cured.

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Basal insulin secretion in low glucose was appearently increased in monolayer cultures but not in free-floating cultures. High glucose increased the insulin secretion in both. At the presence of 1.0mM IBMX with high glucose, insulin secretion significantly increased in monolayer cultures but not in free-floating. Moreover, the monolayer islets had remarkably higher insulin content. It seems the most favorable conditions for long term survival of isolated islets in cultured when the islets form monolayer.

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This study compares hamster islets encapsulated by a semipermeable membrane composed of alginate and polysacharids with those by copolymer of acrylonitrile-methoxypolyethelene glycol. In vitro studies on insulin released from enapsuiated hamster islets by the two different materials was attempted incubating in the presence of various concentrations of glucose. The mean total insulin released from the islets encapsulated by a semipermeable membrane incubated in medium containing 500mg/dl glucose was 572±26μU/islet/90min. In contrast, no insulin release from encapsulated islets by copolymer of acrylonitrile-methoxypolyethelene glycol was observed. In vivo experiments were performed to determine whether encapsulated islets could have function without being disturbed by immunological responses. Transplantation of the hamster islets encapsulated only by a semipermeable membrane composed of alginine and polysacharids resulted in xenograft survival in all animals of 5 to 11 days. Average survival time was 7.8±2.0 days.

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Artery-to-vein shunts made of highly biocompatible synthetic materials with an inner diameter of approximately 5mm consistently remained patent in diabetic dogs for periods of at least 10weeks without any anticoagulants, provided the shunt flow rate ranging between 150 and 230ml/min. Biohybrid endocrine pancreas (BHEP) which consists of a single artificial hollow tube with a large inner diameter (2.7mm) was used. Patency periods of BHEP without inoculation of pancreatic islets exceeding 5 weeks without episodes of clot formation were achieved in diabetic animals. Blood flow rates varying between 100 and 170ml/min were observed after insertion of BHEP into the shunt through the course of this experiment. These results indicate that patency of the hollow tube for BHEP and of the blood access to BHEP was remarkably improved using a large bore tube and expanded polytetrafluoroethylene shunt tube, respectively.

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pancreas-Development of subcutaneous glucose sensor--The subcutaneous glucose sensors were prepared by using some glucose semipermeable membranes and tested in vitro under various oxygen tensions of 5 to 21(%). They responded within 2 minites at 95% of steady state and almost linearly up to 1500mg/dl of glucose concentration. Although these sensors responded only up to 200mg/dl under 5% of oxygen tension, they did linearly up to 500mg/dl by using the glucose semipermiable membranes which restrict glucose penetration more seriously.

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The new glucose sensor for subcutaneous tissue is being developed by our group. The shape of this sensor which has glucose electrode and reference oxygen electrode is stick-like, and it is implanted into subcutaneous tissue. The sensor detects tissue fluid glucose which corresponds to blood glucose, and responses to tissue fluid glucose immediately after loading glucose intravenously. However, it takes about 2 minutes to detect the peak of tissue fluid glucose. There is good correlation between tissue fluid glucose value by the sensor and blood glucose value by ordinary glucose autoanalyzer.

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Ambulant insulin-dependent diabetics were controlled with the wearable artificial endocrine pancreas to evaluate its feasibility. When the needle-type glucose sensors were kept in subcutaneous tissue for 72hrs, “relative” sensor outputs were depressed (73.5%), and “relative” sensors' response times were elongated from 5.1 to 13.5min. Perfect glycemic controls for up to 6 days were obtained with the system by replacing a sensor at the 4th day. These data indicated the feasibility of the long-term control with the system.

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For long-term clinical application of the wearable artificial endocrine pancreas, closed-loop subcutaneous insulin infusion algorithm has been developed from simulation study on subcutaneous insulin absorption kinetics. With this algorithm, glycemic levels were controlled without hyperglycemia nor hypoglycemia after oral glucose loads to pancreatectomized dogs or after meal loads to insulin-dependent diabetics. These data indicated feasibility of long-term glycemic control in diabetics with closed-loop subcutaneous insulin infusion by the wearable artificial endocrine pancreas.

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Ex vivo evaluations in dogs were performed to measure maximum filtration rates (Q_Fmax) with 7 different types of modules. The dependency of Q_Fmax measured in the ex vivo evaluations on hematocrit (Ht) was normalized using the relations between Q_Fmax and Ht, which was derived from in vitro studies with bovine blood. Q_Fmax measured at various cappillary length (L), membrane area (A) and mean wall shear rate (γw) were found to be well correlated by a single linear function of A1.3·γw/L1/3. Based upon the linear relation, capillary plasma filters for use in therapeutic and donor plasmapheresis were designed.

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Filtration properties of PVA plasma separators with various geometric parameters were studied in vitro test. Plasma flux is directly related to wall shear rate, and good relationship of plasma flux to wall shear rate with high correlation was obtained for various types of module with surface area ranged from 0.07 to 1.65m². Smaller number of fibers and thus surface area at a given length of module gave higher wall shear rate leading increase of plasma flux, and also higher differential pressure drop across blood channel leading decline of plasma flux. The module with smaller diameter of fiber gave higher wall shear rate and lower priming volume compared to that with larger one at a given surface area. Optimization of module design is achieved by consideration of wall shear rate and pressure drop effects.

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A newly improved method using radioisotope-labeled urea, glucose, sucrose, and PEG 4000 was employed to measure solute permeability for nine tubular membranes. Both solute and pure water permeability allow determination of membrane pore radius by pore theory, for example, tested membranes made of regenerated cellulose have a pore radius from 25 to 45Å. Further consideration was given to the effect of ultrafiltration on the performance of a dialyzer in an in vitro experiment using bovine blood and to the effect of pore radius distribution on solute and pure water permeability.

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Normally, the filtrate space of a plasma separator is not fully charged with plasma, with the result that filtrate pressure is measured at the upper aperture of the filtrate space. In a previous study filtrate pressure was measured considering the static pressure of the filtrte which filled the filtrate space. In membrane plasma separation, TMP is usually vanishingly small, so that an exact method for measuring filtrate pressure needs to be developed. This method recommends itself for filtrate pressure measurement. Correct filtrate pressure makes it possible to obtain pure water permeability Lp and TMPc (TMP at commencement of hemolysis).

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Effects of temperature and flow-rates on the sieving coefficient and transmembrane pressure were investigated using human preserved blood with four modules. The structure of membrane pore was not influenced by temperature from the result of filtration of pure water. In case of constant temperature at various flow-rates, SC slightly decreased with rising of TMP indicating that the fouling of pores depended on invasion of cells. In case of constant flow-rate at various temperature, SC greatly decreased with rising of TMP suggesting that the fouling of pores depended on adhesion of proteins.

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Second filters for DFPP were evaluated in vitro using albumin and IgG solutions and human plasma separated with 1st filter. The effects of operating conditions, such as plasma flow rate at inlet, filtration flow rate and temperature, on separating performance of membranes were investigated. The results showed that the sieving coefficient of IgG was strongly affected by temperature. This was due to the facts that the amounts of oligomers and polymers of IgG changed with temperature and the thickness of resistant layer of IgG polymers was influenced by temperature. The performance of membranes in mixed solution was different from that in one component solution. Thus, an evaluation method for the performance of 2nd filters was proposed.

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Using immunofluorescent (IF) technique, we evaluated the protein trapping and complement activation (CA) of the membrane plasma separators and filters. Among the plasma separators, cellulosic membrane activated alternative complement pathway (ACP) on the interface between blood and membrane surface. As to polyvinylalcohol, alternative and classical complement pathway activation was thought to occur intramembranously. CA was not observed about the other synthetic polymers, such as polypropylene (PP) and polyethylene. Although membrane plasma filter of PP did not show the findings of CA, the possibility of ACP was suggested in the one type of plasma filter consist of cellulose diacetate. These results indicate that evaluation by IF technique clearly demonstrates the CA and clarifies the pathway and portion of that on membranes for plasmapheresis.

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Activated complement levels of C3a, C4a, and C5a were examined during plasmapheresis with six kinds of plasmaseparator and two types of plasmafilter composed of CDA. While increase in venous C3a and C5a were higher in celluosic membrane than those in synthetic ones, the sieving coefficients (SC) of C3a and C5a were extremely high in PVA and PMMA compared to those in cellulose. This finding indicated that these two membranes stronghy activated complement intra-membranously. High SC of C4a in PVA demonstrated that PVA might activate complement via alternative (AP) and classical pathways (CP). PP and PE showed little increase in complements. In double filtration plasmapheresis, one type of filter activated complements via AP but the other one reduced complement levels in filtrated plasma.
These results indicate that plasma separators activate complements at the membrane surface and intra-membranously via mainly AP and partially CP, and membrane material is not the sole factor which determines the complement activation of plasmafilter.

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C3a, C4a and C5a were evaluated in three patients treated by PE using membrane plasma separator (MPS). C3a and C4a continuously increased in blood. Filtered plasma by MPS contained significantly higher concentration of C3a, C4a and C5a than blood. The continuous increase of C4a means the activation through the classical pathway in MPS. The activation by MPS differed from that by dialyzer.

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The improvement of biocompatibility was attempted with using nafamstat mesilate (FUT) as an anticoagulant for DHP. Complement activation was estimated by the values of C3a and C4a during the experimental DHP. FUT of 5μg/ml only inhibited classical pathway. PUT of 50μg/ml inhibited both pathways.

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Immunological changes during DFPP were discussed in 7 patients with rheumatoid arthritis. reduction of immunoglobulins were observed at post treatment, and after 4th treatment. Leukocytosis were observed at post treatment. A increase of OKT4 and OKT4/OKT8 ratio were observed at post treatment. OKT4 and OKT8 decreased slightly after 4th treatment. Thease phenomena suggest that changes of cell mediated immunological processes occurs during DFPP therapy.

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