Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Diffusivity of Cu2+ in calcium alginate gel beads

The diffusivity of Cu(2+) in calcium alginate beads calculated by the shrinking core model (SCM) was reevaluated in this work. The results obtained in this work were significantly different than those by the original authors. There were excellent agreements between the results obtained by the SCM in this work and those by the more rigorous linear absorption model (LAM) by the original authors. (c) 1994 John Wiley & Sons, Inc.     

Isolated hepatocytes in a bioartificial liver: A single group view and experience

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte-based artificial liver (BAL). It is based on plasma perfusion through a hollow-fiber module seeded with matrix-anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 +/- 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. (c) 1994 John Wiley & Sons, Inc.     

Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.     

Diffusivity of Cu2+ in calcium alginate gel beads: recalculation

Calculations of the diffusivity of Cu(2+) in calcium alginate gel beads using the shrinking core model were checked by us. Corrected results are reported here. Diffusivity was still found to increase with increasing alginate concentration, but at a lower rate than reported in the cited paper. The diffusivity increased by a factor of 2 over the range of alginate concentrations studied rather than 10. The original data is included with sample calculations. (c) 1994 John Wiley & Sons, Inc.     

Diffusivity of Cu(2+) in calcium alginate gel beads

A linear absorption model (LAM) is used to describe the process of metal binding to spherically shaped biopolymers particles. The LAM was solved using a numerical algorithm which calculates diffusivities of metal ion in biopolymer gels. It assumes attainment of rapid metal-biopolymer binding equilibrium accompanied by rate limiting diffusion of the metal ions through the gel. The model was tested using batch experiments in which copper (Cu(2+)) binding with calcium alginate beads was investigated. Biopolymer density in the beads was varied between 2% and 5%. The diffusion coefficient of Cu(2+) calculated from the LAM ranged from 1.19 x 10(-9) to 1.48 x 10(-9) m(2) s(-1) (average 1.31 +/- 0.21 x 10(-9) m(2) s(-1)), independent of biopolymer density. The LAM has theoretical advantages over the shrinking core model (shell progressive model). The latter calculated an unreasonable exponential increase in the diffusion coefficient as density of alginate polymer in the bead increased. (c) 1993 John Wiley & Sons, Inc.     

Diffusion of Cu2+ in calcium alginate gel beads: further analyses

The diffusivity of Cu(2+), as determined by previous authors from analysis of experimental data in terms of the shrinking core (SCM) and linear absorption (LAM) models, is examined in light of the ability of the models to curve fit all the data. It is concluded from this further analysis that previous conclusions depicting the LAM to have an advantage over the SCM for predictive value are not justified. It is also shown that equally good curve fits can be obtained with a recent absorption/desorption model of diffusion which considers directly, through distribution theory, the effect of heterogeneity of material properties on the rate of diffusion. (c) 1995 John Wiley & Sons, Inc.     

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.     

Oxygen is a factor determining in vitro tissue assembly: Effects on attachment and spreading of hepatocytes

Many recent studies related to the development of bioartificial liver devices have utilized hepatocytes cultured within devices of various geometries. Because hepatocytes are anchorage-dependent cells, they need to attach and spread onto the extracellular matrix to be able to function, a process that requires energy. Thus, it is important to deliver enough oxygen to hepatocytes contained within bioartificial liver devices during the early phase of cellular organization while the cells interact with the extracellular matrix. In this study, we investigated the effect of oxygen on the attachment and spreading of hepatocytes. Increasing the gas phase oxygen from 0 to 160 mmHg resulted in an increase in the percentage of cells attaching from 43.0 +/- 5.8% to 103.6 +/- 29%, 1 h after seeding. In a similar manner, increasing the gas phase oxygen from 0 to 160 mmHg resulted in an increase of the projected surface area from 310 +/- 35 to 827 +/- 127 mum(2), 24 h after seeding. Furthermore, the partial pressure of oxygen at the cell level was estimated using a diffusion-reaction model. The model indicated that a cell surface oxygen partial pressure of 0.064 mmHg was required for the half-maximal (K(m) (a)) attachment of hepatocytes to collagen-based substrate. On the other hand, the K(m) (s) value of the spreading process was predicted to be 0.13 mmHg. The results of this study demonstrate the importance of oxygen during the initial stages of attachment and spreading of hepatocytes, and it has important implications in the design of hepatocyte-based bioartificial liver devices. (c) 1994 John Wiley & Sons, Inc.     

Stimulation of liver functions in hierarchical co-culture of bone marrow cells and hepatocytes

A hierarchial co-culture, in which rat hepatocytes and non-parenchymal liver cells (NPLCs) were separated by a collagen layer and which was designed to mimic the in vivo microenvironment, was carried out with the aim of developing a module for bio-artificial liver support. Compared with a monolayer co-culture and hepatocytes cultured alone in a monolayer, higher urea synthesis activity was maintained for 6 d in the hierarchical co-culture. When a rat hepatoma cell line H4-II-E-C3, which retains the induction of tyrosine aminotransferase (TAT), was co-cultured in a monolayer with NPLCs, dose-dependent stimulation of TAT induction was observed. In a hierarchical co-culture, NPLCs further stimulated TAT induction in H4-II-E-C3 cells. Since peritoneal macrophages could stimulate TAT induction in hepatocytes in both monolayer and hierarchical co-cultures, bone marrow cells, which can proliferate and differentiate into macrophages in vitro, were investigated as a possible substitute for NPLCs. Bone marrow cells isolated from rat femurs were cultivated in the presence of IL-3 and macrophage colony-stimulating factor (M-CSF), and co-cultured with hepatocytes. Urea synthesis and TAT induction of hepatocytes were stimulated in the co-culture. The co-culture of bone marrow and H4-II-E-C3 cells, both of which have proliferation ability in vitro, was also shown to be effective in stimulating liver functions. The hierarchical configuration, in which two cell types can communicate with the soluble factor(s) through a collagen layer, was found to be more effective than a monolayer in long-term co-culture.     

Energy metabolism and re-establishment of intercellularadhesion complexes of gel entrapped hepatocytes

We studied the effect of continuous medium flow on the viabilityand structural organization of hepatocytes high density entrapped inalginate gel beads in the first few hours after isolation.The metabolic energy status of the entrapped cells, monitored invivo by ³¹P NMR spectroscopy, was stable during theexperimental time and a physiological redox ratio was reachedafter the first three hours of culture. The morphologicalanalysis revealed that the entrapped hepatocytes placed in a fixed-bed bioreactor under continuous flow showed a polyhedricalshape with numerous microvilli on cell surface and reconstitutedtight junctions as well as bile canalicular structures, closelyresembling those present in the liver.These results suggest that continuous flow allows the culture ofhepatocytes at very high cell density within a matrix withoutloss of viability and accelerates cellular tissue reconstructionat very short times after isolation. This type of culture couldrepresent a very useful model for physiological andtoxicological studies as well as a promising approach toward thedevelopment of a bioartificial hybrid support device in acuteliver failure.     

Immobilization of Cryopreserved Primary Rat Hepatocytes for the Development of a Bioartificial Liver System

Primary rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing 40% (v/v) fetal bovine serum (FBS) and 10% (v/v) dimethyl sulfoxide (DMSO) in liquid N₂ for 6 months. After thawing, the cells were immobilized using 2% (w/v) alginate and 0.5% (w/v) chitosan solutions. The capacities of ammonia removal and urea synthesis of the immobilized-thawed hepatocytes were similar to those of immobilized hepatocytes without cryopreservation. This result shows that immobilized hepatocytes after cryopreservation are useful for the development of a bioartificial liver system.     

Production of erythropoietin by BHK cells growing on the microcarriers trapped in alginate gel beads

Baby hamster kidney (BHK) cells engineered to produce recombinant human erythropoietin (EPO) were cultured at high density on microcarriers entrapped by calcium alginate gel particles. In this system, the BHK cells proliferated not only on the microcarriers but also in vacant spaces in the alginate gel particles. These spaces contributed greatly to high-density cultivation of the cells and a high productivity of EPO.Abbreviations BHK Baby Hamster Kidney - EPO Erythropoietin     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

Preparation and characterization of calcium pectinate gel beads entrapping catechin-loaded liposomes

Calcium pectinate gel (CPG) beads entrapping catechin-loaded liposomes were prepared with or without hydroxypropylmethylcellulose (HPMC) (denoted as CPG-LH and CPG-L beads, respectively) and characterized in comparison with the CPG beads prepared without liposome and HPMC (denoted as CPG-C beads). For all types of beads, the catechin entrapment efficiency decreased by about 40-50% as the concentration of CaCl2 in gelling media increased from 2 to 6%. At a constant CaCl2 level, the entrapment efficiency was higher in the order of CPG-LH, CPG-L, and CPG-C beads. The in vitro release test showed that in simulated intestinal fluid the rate of catechin release was higher in the order of CPG-C, CPG-L, and CPG-LH beads, indicating that the catechin release was slowed by liposome and further retarded when HPMC was used simultaneously, whereas not in simulated gastric fluid. The addition of cholesterol in liposome could not retard but accelerated the catechin release. The results suggest that the CPG beads reinforced with liposome and HPMC could be employed for a sustained oral delivery of catechins, although further improvements are necessary.     

Unexpected distribution of immobilized microorganisms within alginate beads

Immobilization refers to the prevention of free cell movement by natural or artificial means. It has always been assumed that immediately after an immobilization procedure is performed, cells are distributed homogeneously in the beads that entrap them. However, in this study, Escherichia coli and Trichoderma asperellum distribution in alginate-gel beads was found to be nonhomogeneous. In fact, there was a greater presence of cells on the surface of the alginate beads than in their cores.     

Cell-based therapy of acute liver failure: The extracorporeal bioartificial liver

The need for an alternative ttreatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporalily providing liver functions are being actively tested. Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function. An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation. Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines. Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We have isolated normal porcine hepatocytes by collagenase perfusion of the liver. Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor. Porcine hepatocytes expreessed liver-specific functions at high levels, particularly protein neosysnthesis and enzymatic activities involved in detoxication and biotransformation processes. In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins. This system represents a promising tool for the design of anoartificial liver in human beings.Abbreviations ALF acute liver failure - EBAL extracorporeal bioartificial liver - OLT orthotopic liver transplantation     

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [³⁵S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [³⁵S]Na₂SO₄ was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.     

包埋法固定化真菌漆酶及其应用研究

采用海藻酸钠包埋法固定真菌漆酶,海藻酸钠和CaCl2的最佳浓度分别为3%和4%,最佳给酶量为30U,最大回收率为48.0%.与游离漆酶相比,固定化漆酶的热稳定性有明显改善,最适反应pH向酸性方向漂移0.5,最适反应温度提高了5℃.使用固定化酶处理低浓度造纸废水,运行8批次后残留酶活为64%.