Synthesis of a chitosan derivative soluble at neutral pH and gellable by freeze-thawing, and its application in wound care.

Conventional chitosan hydrogels exhibit an acidic nature and contain unfavorable additives because (i) chitosan is soluble only in acidic solutions and (ii) toxic chemicals or proteins of non-human origin that serve as antigens are necessary for preparing chitosan hydrogels. These characteristics of the chitosan hydrogels limit their possibilities as wound dressings. In this study, a chitosan-gluconic acid conjugate is developed, soluble in an aqueous solution at neutral pH and gellable by freeze-thawing (cryogelation) without using additives. The viability of L929 fibroblasts cultured in the presence of the chitosan derivative for 24 h was >96%. The degradation rate of the corresponding chitosan cryogels by lysozyme was tunable via the derivative concentration in the gels. The gels had low cellular adhesiveness. The gels promoted the accumulation of inflammatory cells such as polymorphonuclear leukocytes, which have the potential to release chemical mediators effective for wound healing, in full-thickness skin wounds in rats and accelerated the healing of the wounds. These results demonstrate that cryogels are promising for wound care.

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.

Digoxin transport by renal proximal tubule cells is enhanced by adhesive synthetic RGD peptide.

INTRODUCTION: The dialyzer apparatus has been widely used as an artificial kidney in medical treatment. However, side effects such as amyloidosis have occurred during long-term treatment. Therefore, we focused on developing a hybrid artificial kidney with a filtration and reabsorption apparatus, but it was found that cells spread extensively and it is difficult to maintain a uniform monolayer with a regular cell shape on a collagen-coated substrate. The purpose of this study was to improve cell adhesion, uniform stable monolayer formation and active transport function by immobilization of arginine-glycine-aspartic acid (RGD) on the culture substratum. MATERIALS AND METHODS: Polycarbonate semipermeable membranes were coated with collagen, fibronectin, laminin and synthetic polypeptide, including RGD (Pronectin F). Cell adhesion and digoxin transport were estimated using a renal proximal tubule cell line that overexpressed the P-glycoprotein gene. RESULTS AND DISCUSSION: Under initial and confluent conditions, immobilized cell density in Pronectin F-coated wells was higher than that under other conditions. Transepithelial electrical resistance and digoxin transport activity on Pronectin F-coated membranes were the highest of all conditions. This might have been caused by uniform cell morphology and high cell density.

Effect of diglucosamine on the entrapment of protein into liposomes.

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.

Effects of vascular occlusion on maximal force, exercise-induced T2 changes, and EMG activities of quadriceps femoris muscles.

The purpose of our study was to determine the effect of vascular occlusion on neuromuscular activation and/or the energy metabolic characteristics of the quadriceps femoris (QF) muscles during muscle contractions. Seven men participated in the study. An occlusion cuff was attached to the proximal end of the right thigh, so that blood flow in the anterior medial malleolar artery was reduced to approximately 88 % of the non-occluded flow. Muscle functional magnetic resonance imaging and maximal voluntary contraction (MVC) were carried out before and immediately after 5 sets of 10 repetitions of knee extension exercises at 50 % of the 10 repetitions maximum, from which transverse relaxation times (T2) and maximal force were measured, respectively. Integrated electromyography (iEMG) activity was recorded from the belly of the rectus femoris, vastus lateralis, and vastus medialis muscles during MVC and repetitive exercises. The percentage change in T2 was significantly increased for individual QF muscles, and there was a significant increase in iEMG activity over the 5 sets of repetitive exercises under conditions of vascular occlusion, but there was no significant effect on isometric force and iEMG activity during MVC. These results are consistent with the idea that there is greater osmolite accumulation during exercise with occlusion, although increased neural activation cannot be ruled out.

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.

Development of co-culture system of hepatocytes with bone marrow cells for expression and maintenance of hepatic functions.

In this study, a co-culture system of hepatocytes and bone marrow cells (BMCs) was developed and characterized for the expression and maintenance of ammonia metabolism and albumin secretion activities. A culture medium supplemented with epidermal growth factor, insulin, L-proline, hydrocortisone and 20% (v/v) heat-inactivated fetal bovine serum was developed. In addition to adhesive bone marrow cells, the co-existence of non-adhesive bone marrow cells was effective in expressing liver-specific functions for at least 3 weeks. On the other hand, experiments with Transwell in which cultured cells were separated by a semi-permeable membrane, suggested that soluble factors secreted by BMCs are the key components in the functional enhancement of cells. Furthermore, direct contact between hepatocytes and BMCs enhanced the formation of spheroids and the expression of liver specific functions. These results indicate that this co-culture system is promising in, for example, bioartificial liver, regenerative medicine, and liver function simulator applications.

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.

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.

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.

[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.

Synthesis and transport characterization of alginate/aminopropyl-silicate/alginate microcapsule: application to bioartificial pancreas.

To develop a novel type of immunoisolation membrane for a microcapsule-shaped bioartificial pancreas, we attempted to use a sol-gel synthesized silicate. An aminopropyl-silicate membrane derived from 3-aminopropyltrimethoxysilane and tetramethoxysilane was formed on Ca-alginate gel beads via electrostatic interaction. The positively charged amino groups remaining on the surface of the resultant gel beads were neutralized by immersion in an aqueous Na-alginate solution. From measurements of the partition coefficients and effective diffusivities of different substances to the gel beads, it was found that the aminopropyl-silicate membrane prepared under optimized composition of silicon alkoxide precursors successfully rejected gamma-globulin, giving good permeability to substances having a low molecular weight. Islets could be encapsulated within the newly developed microcapsules while retaining their ability to secrete insulin.

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.

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.

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.

Newly developed aminopropyl-silicate immunoisolation membrane for a microcapsule-shaped bioartificial pancreas.

An aminopropyl-silicate membrane, synthesized from tetramethoxysilane (TMOS) and 3-aminopropyltrimethoxysilane (APTrMOS) by the sol-gel method, was formed onto Ca-alginate gel beads via electrostatic interactions. The permeability of the membrane could be controlled easily by changing the molar ratio of both the precursors ([APTrMOS]/[TMOS]). The aminopropyl-silicate membrane prepared at a molar ratio of 2.40 rejected gamma-globulin and BSA successfully, whereas it permeated ovalbumin. This result indicates that the molecular weight cutoff point of this newly developed aminopropyl-silicate membrane is approximately 60 kDa.

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.

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.

Control of molecular weight cut-off for immunoisolation by multilayering glycol chitosan-alginate polyion complex on alginate-based microcapsules.

Glycol chitosan is a positively charged polysaccharide which is water-soluble at pH 7.4, and is able to form a polyion complex (PIC) with anionic polymers, such as alginate. The authors attempt to develop a novel type of alginate-based microcapsule using this glycol chitosan for a islets-encapsulated bioartificial pancreas. The number of layers composed of glycol chitosan-alginate (GC-Alg) PIC were optimized, in order to cut off immunoglobulin transport and to protect encapsulated islets from the host immune reaction, and the transport characteristics were evaluated of glucose, bovine serum albumin (BSA) and gamma-globulin. To add mechanical stability to the microcapsule, calcium ions, which crosslinked the alginate polymers close to the interface between core Ca-alginate and multilayered membrane, were partially substituted with barium ions after the formation of multilayered Ca-alginate gel beads. The partition coefficients of BSA and gamma-globulin were decreased with the increasing number of layers. The immunoisolation was achieved against gamma-globulin with four layers of the GC-Alg PIC membrane, while BSA could permeate the membrane. The four-layered Ba-alginate gel bead had a good permeability for glucose, giving a diffusion coefficient corresponding to 80% of that in pure water. Insulin secretion from the islets in the four-layered Ba-alginate microcapsule was satisfactorily observed with the fractional stimulation ratio of 2.17. This result indicates that the encapsulated islets maintained their viability even after encapsulation. It was, thus, shown that the Ba-alginate microcapsule with four layers of the GC-Alg PIC membrane is promising as the microencapsulation material for a bioartificial pancreas.