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Biomedical subjects

K Funatsu

Publications and source records attributed to K Funatsu.

At least 19 recordsLinked to original sources
PubMed2003

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.

Albumins↗
PubMed2002

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.

Animals↗
PubMed2002

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.

Ammonia↗
PubMed2001

Hybrid artificial liver using hepatocyte organoid culture.

We developed 2 types of hybrid artificial liver modules using hepatocyte organoid culture. One was a polyurethane foam (PUF)/hepatocyte spheroid packed-bed module. Hepatocytes spontaneously formed spheroids in the PUF pores, and they maintained liver-specific functions well for at least 2 weeks in vitro. As a preclinical experiment, a hybrid artificial liver with 200 g porcine hepatocytes was applied to a pig (25 kg) with liver failure and showed that the hybrid artificial liver was effective in support of liver functions and stabilization of general conditions. We established a new technique of hepatocyte organoid formation using centrifugal force. A hepatocyte organoid formed by centrifugation in hollow fibers maintained functions for more than 4 months in vitro. We developed a new sinusoid-like structure module having hollow fibers arranged by spacers in a micro-regular arrangement. Inoculated hepatocytes in the extra-fiber space of the module formed the organoid by centrifugation, and they maintained the functions for at least 1 month in vitro. The results indicated that this module seems to be promising as a hybrid artificial liver.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Alanine Transaminase↗
PubMed2001

[Basic study about the development of the hybrid-artificial liver support system using human hepatoma cell lines (Hep G2, Huh 7): effects on liver functions by extracellular matrix (type I collagen) in monolayer culture].

The risk of xenozoonosis infections poses the greatest obstacles against the clinical application of hybrid-artificial liver support system (HALSS). To resolve this issue, we used human hepatoma cell lines (Hep G2, Huh 7) in a type I collagen-coated monolayer culture system, and analyzed liver specific functions such as ammonia removal and albumin synthesis capacity. Ammonia removal activity (nmol/10(6) nuclei/hour) and albumin synthesis activity (microgram/10(6) nuclei/day) were upregulated in both Hep G2 and Huh 7 by type I collagen-coated monolayer culture. In particular, Hep G2 cultured in type I collagen-coated monolayer demonstrated relatively high ammonia removal and albumin synthesis capacity. These results indicate the possibility of the application of human hepatocytes to HALSS.

Albumins↗
PubMed2001

Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial.

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 10(8) total cells with a viability of 69.2+/-25.3 %, but our combined method had a yield of 167+/-31 x 10(8) 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 10(8) 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 10(10) porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 - 500 microm 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 10(8) 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.

Animals↗
PubMed2001

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.

Albumins↗
PubMed2000

Novel canonical coding method for representation of three-dimensional structures

A new canonical coding method for representation of three-dimensional structures, CAST (CAnonical representation of STereochemistry), is described. CAST canonically codes stereochemistry around an atom in a molecule. The same CAST notations are given for atoms of molecules in the same conformation. The CAST code is based on the dihedral angles of four atoms that are uniquely defined by a molecular tree structure. CAST has successfully represented similarities and differences between several conformers.

Journal Article↗
PubMed2000

Partial least squares modeling and genetic algorithm optimization in quantitative structure-activity relationships.

Quantitative structure-activity relationship (QSAR) studies based on chemometric techniques are reviewed. Partial least squares (PLS) is introduced as a novel robust method to replace classical methods such as multiple linear regression (MLR). Advantages of PLS compared to MLR are illustrated with typical applications. Genetic algorithm (GA) is a novel optimization technique which can be used as a search engine in variable selection. A novel hybrid approach comprising GA and PLS for variable selection developed in our group (GAPLS) is described. The more advanced method for comparative molecular field analysis (CoMFA) modeling called GA-based region selection (GARGS) is described as well. Applications of GAPLS and GARGS to QSAR and 3D-QSAR problems are shown with some representative examples. GA can be hybridized with nonlinear modeling methods such as artificial neural networks (ANN) for providing useful tools in chemometric and QSAR.

Algorithms↗
PubMed2000

Intensive promotion of spheroid formation by soluble factors in a hepatocyte-conditioned medium.

We developed a hybrid artificial liver and a drug metabolism simulator using polyurethane foam (PUF) in which primary hepatocytes spontaneously form functional spheroids. Gel filtration liquid chromatography analysis of a hepatocyte-conditioned medium during spheroid formation showed that some substances secreted by primary rat hepatocytes accumulated advantageously inside the pores of PUF compared with outside. Similar substances were detected in a hepatocyte-conditioned medium from a positively-charged surface by concentrating the substances using an ultrafiltration membrane of a molecular weight-cutoff of 50 kD. These substances were shown to act as soluble factors on freshly isolated primary rat hepatocytes to promote spontaneous and rapid spheroid formation, depending on their concentration by preventing them from initially attaching and spreading on a positively-charged surface. In particular, using 50-fold concentrated substances, about 80% of total hepatocytes formed the floating spheroids within 72 h of culture. The resulting spheroids had a diameter distribution mainly ranging from 40 to 70 microm and expressed high-level liver-specific functions compared with a conventional monolayer.

Ammonia↗
PubMed2000

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.

Albumins↗
PubMed2000

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.

Animals↗
PubMed1999

Evaluation of a hybrid artificial liver using a polyurethane foam packed-Bed culture system in dogs.

BACKGROUND AND AIMS: We developed a polyurethane foam packed-bed culture system of hepatocyte spheroids as a hybrid artificial liver (PUF-HAL), which was effective for recovery from liver failure in rat experiments. In this report, the design of a scaled-up PUF-HAL for dogs is described and evaluated using a dog acute liver failure model. METHODS: Warm ischemic liver failure was induced with a portocaval shunt in each dog. The dogs were divided into two groups: (1) a control group (N = 4), in which each dog was attached to a PUF-HAL without hepatocytes for 9 h, and (2) a HAL group (N = 5), in which each dog was attached to a PUF-HAL with hepatocytes. Blood pressure, blood ammonia, blood glucose, serum creatinine, and other parameters related to liver function were compared between the two groups. RESULTS: In the HAL group, blood ammonia and serum creatinine levels were significantly lower, and blood pressure and blood glucose levels significantly higher, than those in the control group. CONCLUSIONS: The scaled-up PUF-HAL developed for large animals is useful as a liver support system in the dog acute liver failure model.

Ammonia↗
PubMed1998

Formation of a spherical multicellular aggregate (spheroid) of animal cells in the pores of polyurethane foam as a cell culture substratum and its application to a hybrid artificial liver.

Monkey kidney cells (Vero), human embryonic kidney cells (293), human liver cells (PLC/PRF/5), and primary rat, dog, and porcine hepatocytes formed spherical multicellular aggregates (spheroids) in the pores of polyurethane foam which was used as a cell culture substratum. These spheroids of various cell types express high cell activity for a long period. A practical hybrid artificial live support system composed of a multi-capillary polyurethane foam packed-bed type cell culture module including primary hepatocyte spheroids was developed. The success of the system is indicated by an 80% recovery rate in hepatic failure rats which died in control experiments.

Animals↗