Albumin Secretion Rate Research Articles

Preparation and HL-7702 cell functionality of titania/chitosan composite scaffolds

Titania/chitosan composite scaffolds were prepared through a freeze-drying technique. The composite scaffolds were highly porous with the average pore size of 120-300 microm, and the titania (TiO(2)) powders were uniformly dispersed on the surface of the pore walls. The compressive strength of the composite scaffolds was significantly improved compared to that of pure chitosan scaffolds. Composite scaffold with 0.3 of TiO(2)/chitosan weight ratio showed the maximum compressive strength of 159.7 +/- 21 kPa. Hepatic immortal cell line HL-7702 was used as seeding cells on the scaffolds, and after different culture periods, cell attachment and function was analyzed. HL-7702 cells attached on the pore walls of the scaffolds with the spheroid shape after 1 day of culture, but more cell aggregations formed within the TiO(2)/chitosan composite scaffolds as compared to pure chitosan scaffolds. Liver-specific functions, albumin secretion and urea synthesis were detected using a spectrometric method. The results showed that albumin secretion and urea synthesis rate of HL-7702 cells slightly decreased with the culture time, and there was no significant difference between composite scaffolds and pure chitosan scaffolds. In conclusion, the TiO(2)/chitosan composite scaffolds possessed an improved mechanical strength compared to pure chitosan scaffolds and supported the attachment and functional expression of hepatocyte, implying their potential application in liver tissue engineering.

Loofa Sponge as a Scaffold for Culture of Rat Hepatocytes

The dried fruit from Luffa cylindrica (loofa sponge, LS), which represents a new chitinous source material, was used as a 3-D scaffold for the culture of rat hepatocytes. With the macroporous structure and large pore size (ca. 800 microm) of LS, cell loading to the scaffold should be carried out by dynamic seeding with continuous shaking throughout the seeding period. Hepatocytes attach well to the surface of loofa fibers after seeding and maintain their round shapes. The initial ammonia removal and urea-N synthesis rates of hepatocytes immobilized within LS slightly decreased with increasing cell densities, but their metabolic activities were comparable to or better than those in monolayer culture on tissue culture polystyrene control surfaces. Both urea-N synthesis and albumin secretion rates could be maintained up to 7 days for cells immobilized within LS and spheroid-like cell aggregates could be found after the second day.

Silk Fibroin Microfluidic Devices.

Silk Fibroin Microfluidic Devices.

Effects of Retinoic Acid on Proliferation and Differentiation of HepG2 Cells

Hepatoma cell lines have characteristics derived from both their hepatic lineage and their transformation. We examined the potential of retinoic acid to enhance the function of a hepatoma cell line, HepG2, under conditions of reduced serum as are desired for bioreactor cultivation. We found that retinoic acid drastically slows the growth of HepG2 cells and induces a more spread morphology. Retinoic acid also augments the differentiated function of the cells, as measured by albumin and urea secretion rates. Expression levels of a panel of liver-enriched transcription factor were increased from retinoic acid exposure. Overall, we demonstrate that retinoic acid has significant effects on HepG2 growth and differentiated function. These results have implications for the use of retinoic acid both as a chemotherapeutic agent and as a medium component for cells of hepatic origin.

The role of vascular endothelial growth factor, tumor necrosis factor alpha and interleukin-6 in pathogenesis of diabetic retinopathy

The aim of the study was to analyze the relation between early diabetic retinopathy and the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) in children with diabetes mellitus type 1. Two hundred and two children with diabetes mellitus type 1 aged 13.2 ± 3.83 years and 85 healthy controls were analyzed. Patients were divided into two subgroups: children with retinopathy (Group 1, n = 39) and children without retinopathy (Group 2, n = 163). All the children had 24 h urine albumin secretion rate, glycosylated hemoglobin HbA1c level, and C-reactive protein level measured, underwent 24 h blood pressure monitoring and had ophthalmologic examination performed. Additionally, all the children had serum TNF-α, IL-6 and VEGF level measured using an ELISA test (Quantikine High Sensitivity Human). Statistically significant higher blood serum levels of HbA1c, VEGF, TNF-α and IL-6 were found in the Group 1 in comparison with the Group 2. Additionally, the children of the Group 1 showed statistically significant correlation between serum VEGF and serum TNF-α ( R = 0.35, p = 0.000), CRP level ( R = 0.23, p = 0.006), 24 h albumin urine secretion rate ( R = 0.45, p = 0.000) and duration of the disease ( R = 0.26, p = 0.002). The results of the current study suggest that there is a relationship between VEGF, TNF-α, IL-6 and the development of the diabetic retinopathy in children with diabetes mellitus type 1.

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDP-glucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP-U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.

A microfluidic platform for 3-dimensional cell culture and cell-based assays

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.

Endothelium-Mediated Hepatocyte Recruitment in the Establishment of Liver-like TissueIn Vitro

A major goal of liver tissue engineering is to understand how the constituent cell types interact to achieve liver-specific structure and function. Here we show that hepatocytes migrate toward and adhere to endothelial vascular structures formed on Matrigel in vitro, and that hepatocyte recruitment is dependent on endothelium-derived hepatocyte growth factor. The hepatocyte-decorated endothelial vascular structures resemble In vivo sinusoids containing plate-like structures, bile canaliculi, and a lumen. The sinusoid-like structures retained cytochrome P450 expression and activity, in addition to stable albumin expression and secretion rate for over 2 months in vitro. The stability of the sinusoid-like structures was dependent on the presence of vimentin-positive fibroblasts in culture. The sinusoid-like structures formed by hepatocytes and pure populations of endothelial cells collapsed after 10 days in culture. In contrast, culture of hepatocytes with fibroblast-contaminated human dermal microvascular endothelial cells or a combination of human umbilical vein endothelial cells and normal human dermal fibroblasts resulted in stable sinusoid-like structures surrounded by a fibroblastic capsule that maintained liver specific functions for several months in vitro. These results demonstrate that specification of endothelial cell position ultimately determines hepatocyte position in vitro, suggesting that similar interactions might occur In vivo. The novelty of the culture's sinusoid-like organization and long-term function suggest a new model for the study of liver toxicity, ischemia/reperfusion injury, and fibrosis.

Effects of hyperglycemia and iron deficiency on kidney and heart function in type 2 diabetes disease.

Untreated anemia can caused significant cardiac and kidney damage. The aim of this study was to investigate the efficiency of anemia and hyperglycemia treatment in type 2 diabetes and their impact on kidney and heart impairment. The study is clinical retrospective and prospective and it was conducted in Clinic of Endocrinology, Diabetes Mellitus and Metabolic Diseases, University Clinical Center of Sarajevo. Prior to the study all patients were taking oral hypoglycemic drugs included sulfonylureas and biguanides. These subjects were put on 2 times daily fix mix insulin and biguanides after lunch. Each day, subjects received Iron tab 1 x 100 mg/ day, and C vitamin 1 x 100 mg/day. The results of our study are showing that effective treatment of glycaemia and anemia in patients with diabetes, reduces blood pressure, urine albumin secretion and pulse rate, diminishing cardiovascular damage and improving kidney function.

Alginate/Galactosylated Chitosan/Heparin Scaffold As a New Synthetic Extracellular Matrix for Hepatocytes

Formation of multicellular hepatocyte spheroids in the three-dimensional culture is a potential approach for enhancing liver-specific functions in bioartificial liver (BAL) devices. In this study, as a synthetic extracellular matrix (ECM) for hepatocytes, a highly porous hydrogel (sponge-like) scaffold, 150-200 microm pore size in diameter, was fabricated with alginate (AL), galactosylated chitosan (GC), and heparin through electrostatic interaction. We attempt to select the best condition of AL/GC/heparin sponges for coculture with NIH3T3, as well as compare the liver-specific functions with monoculture. Cell adhesion to GC based on AL film was significantly increased with increasing GC concentration, but not to chitosan regardless of its concentration. The optimal concentration of GC and heparin in AL/GC/heparin sponges to perform the best liver-specific function was 1 and 6 wt% to AL contents, respectively, where albumin secretion were maintained with maximal rates. The mechanical properties in tensile strength of three types of sponges were very slightly different from one another. Cell viabilities performed on AL, AL/GC, and AL/GC/heparin sponges were 68.5, 83.3, and 90.4 % of control, respectively, after 15 days of incubation. Hepatocyte spheroids were more rapidly formed in the AL/GC and AL/GC/heparin sponges, with diameter enlarged to about 100 microm, than in AL sponges. Connexin32 and E-cadherin genes correlated with cell-to-cell adhesion were expressed in hepatocytes within AL/GC and AL/GC/heparin sponges at 36 h after incubation, but not in AL sponges. Treatment of a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, indicates that cell aggregation without GJIC does not perform the liver-specific functions for long periods. In the presence of HGF, the level of albumin secretion in AL/GC/heparin sponges was markedly elevated compared to that in AL/GC sponges. Coculture of hepatocytes in AL/GC/heparin sponges with NIH3T3 in a transwell insert resulted in significant increase of liver-specific functions, such as improved albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1 compared to those in hepatocyte monoculture. The results suggest that hepatocytes as stable spheroids enhance liver-specific functions in AL/GC/heparin sponges, providing a new synthetic ECM to design BAL devices.

Enhanced liver functions of hepatocytes cocultured with NIH 3T3 in the alginate/galactosylated chitosan scaffold

Formation of primary hepatocyte spheroids in the hydrogel scaffold is a promising approach for enhancing liver-specific functions in liver tissue engineering as well as for developing bioartificial liver (BAL) devices. In the present study, a highly porous hydrogel scaffold composed of alginate (AL) and galactosylated chitosan (GC) as a synthetic extracellular matrix (ECM) for hepatocytes was fabricated with 150–200 μm pore size in diameter. Cell adhesion onto AL/GC and AL/chitosan film was 72.7 and 45% at 1 wt% of GC (or chitosan) to AL content whereas cell adhesion onto AL film was 28.5%. The optimal concentration of GC in AL/GC sponge was 1 wt% to AL content by the measurement of albumin secretion. Cell viabilities performed on AL and AL/GC sponges were 72.2±3.6 and 81.3±3.5% of control, respectively, after 10 days incubation. Hepatocytes were aggregated to form multicellular spheroids in AL/GC sponge with diameter enlarged up to about 100 μm, 36 h postseeding, whereas most of them in the AL sponge remained as single cells and only a few cells began to form aggregates. Intercellular molecules such as connexin32 and E-cadherin genes related with cell–cell contact were expressed in hepatocytes within AL/GC sponge at 36 h after incubation, but not in AL sponge. Treatment with a gap junctional intercellular communication (GJIC) inhibitor, 18 β-glycyrrhetinic acid, resulted in a 1.5-fold marked decrease in albumin secretion levels in AL/GC sponge. Specially, coculture of hepatocytes in AL and AL/GC sponges with NIH3T3 in a transwell insert resulted in enhanced increase of liver-specific functions, such as albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1, compared to those in hepatocyte monoculture. The results suggest that formation of hepatocyte spheroids in coculture system enhances liver-specific functions for the AL/GC sponge as a new synthetic ECM to design developed BAL devices.

Culture and characterization of human hepatocytes isolated from hepatitis C virus infected liver: Effect of collagen-coated surface and FBS-supplemented medium

The aim of this study is to maintain and prolong the viability and metabolic functions of hepatocytes isolated from hepatitis C virus (HCV)-infected human livers. Collagen-coated surface and 10% fetal bovine serum (FBS) supplemented culture media were studied. Metabolic functions and attachment ability of human hepatocytes in these conditions were compared with normalized conditions (Petri dish, medium without FBS). Our results showed that collagen-coated dishes with 10% FBS-supplemented culture medium increased urea production and albumin synthesis. Urea concentration remained stable (30 pg/cell/h) during the first 3-days when human hepatocytes were cultured in collagen-coated dishes. These hepatocytes showed higher urea production when compared with hepatocytes cultured in normal Petri dishes producing 36.4 and 31.7 pg/cell/h concentrations of urea, respectively. Albumin secretion rate was maximal on day 3 after plating (0.86 pg/cell/h). When using culture medium supplemented with 10% FBS, the metabolic functions of hepatocytes were higher compared to when using normal culture medium. This study demonstrates that human hepatocytes can be isolated from HCV-infected livers via surgical biopsies with high cell yield rate, and their metabolic functions remain stable when cultured in collagen-coated dishes with FBS-rich medium for several days. Base on these results, HCV-infected human hepatocytes can be used in the study of clinical diseases and in the performance of physiological and pharmacological liver experiments in vitro.

Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment

In this study, xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsules. The rates of hepatocytes adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface were 89.1%, 91.1% and 25.5%, respectively, at 4 h after incubation at 37 °C. From the inhibition study in a cell adhesion assay, the adhesion rates of freshly isolated hepatocytes and preincubated hepatocytes with 20 m m galactose onto the XG-coated surface were 55.7 and 17.3%, respectively, after 30 min incubation at 37 °C. Flow cytometric analysis showed that the internalization of XG by freshly isolated hepatocytes was stronger than preincubated hepatocytes with 20 m m galactose. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained. The albumin secretion, ammonia elimination rates and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL capsules. More than 70% of the seeded hepatocytes in AL/XG capsules participated in spheroid formation after 2 days, whereas most hepatocytes in AL capsules remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase–polymerase chain reaction. Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three-dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and asialoglycoprotein receptors of hepatocytes.

Influence of pancreatic islets on spheroid formation and functions of hepatocytes in hepatocyte-pancreatic islet spheroid culture.

Hepatotrophic stimulation of hepatocytes is necessary to preserve long-term function of hepatocytes in hepatocyte transplantation and bioartificial liver system. The main source of hepatotrophic factors in portal venous blood seems to be the pancreatic islets. It was also reported that hepatocyte spheroids, tightly packed multicellular aggregates, showed enhanced liver-specific activities and a prolonged differentiated state compared with cells that were maintained as a monolayer. On the basis of these two facts, the authors tried to form hepatocyte-pancreatic islet spheroids and to evaluate the influence of pancreatic islets on spheroid formation and functions of hepatocytes in spheroid culture. Hepatocytes and pancreatic islet cells were harvested from adult male Sprague-Dawley rats weighing 200-250 g. Hepatocytes were cultured in spinner flasks with either basic nonstimulated medium (hepatocytes only [group BH] and cocultures with islet cells [group BI]) or hormone-stimulated medium (hepatocytes only [group HH] and cocultures with islet cells [group HI]). The size and morphology of spheroids, as determined by phase-contrast microscopy, and liver-specific functions, such as albumin secretion, urea synthesis, and ammonia removal, were compared among groups. The results were as follows: the size of spheroids, 66 +/- 53.4 microm, in group BH on day 2 was smaller than in group BI (179 +/- 66.2 microm on day 2, p < 0.05). In group BI, group HH, and group HI, smooth spheroids were observed on culture day 2. However, in group BH rugged incomplete aggregates were observed on the same day. In groups with basal medium, group BI showed better results in terms of hepatocyte-specific function such as albumin secretion, urea synthesis, and ammonia removal compared with group BH on days 2 and 3 (p < 0.05). In groups with hormone-defined medium, cocultures had no impact on albumin secretion rate, urea synthetic rate, and ammonia removal rate. In conclusion, we made a new type of hepatocyte-pancreatic islet spheroid, using a rotational culture method. Pancreatic islets in a spheroid culture system stimulated hepatocyte spheroid formation and some hepatocyte-specific functions in vitro.

Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions

Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.

Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.

Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

Behavior of isolated rat oval cells in porous collagen scaffold.

The oval cell is regarded as a compensatory cell in liver injury, and is thought to be equivalent to liver stem/progenitor cells. Oval cells were induced by the 2-AAF/CCl(4) dietary method in Fischer 344 rats, and were isolated from excised liver by the collagenase perfusion, enzyme treatment, and cell cloning method. Transmission electron microscopy observation and double immunofluorescence methods were used to characterize the cells. We have developed an in vitro system consisting of three-dimensional collagen and hormonal and cytokine factors. Over 3 weeks, albumin secretion and urea detoxification rate were estimated to assess the biological function of the oval cells cultured in a scaffold. Oval cells cultured in the scaffold demonstrated higher biological functions than did those in a two-dimensional tissue culture plate. The pore structure and collagen in a scaffold may play an important role in fostering the biochemical functions of oval cells. The three-dimensional culture of oval cells could be considered in designing a cell-delivering tool for hepatic disease.

Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor.

We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures. Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture. Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period. Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells. Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures. Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation. These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions. The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture.

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.

Perfusion culture of hepatocytes within galactose-derivatized biodegradable poly(lactide-co-glycolide) scaffolds prepared by gas foaming of effervescent salts.

Galactose, a specific ligand for asialoglycoprotein receptor in hepatocytes, was immobilized onto the internal surface of highly porous biodegradable poly(D,L-lactic-co-glycolic acid) scaffolds prepared by gas foaming of effervescent salts. Rat hepatocytes seeded within the scaffolds were cultivated by using a continuous flow and perfusion reactor system. Flow rate of medium circulating through the closed loop bioreactor system was optimized to minimize the extent of cell washout from the scaffold/cell construct while satisfying the oxygen transport rate to the seeded hepatocytes. Using the flow culture system, the scaffolds immobilized with galactose onto its internal surface retained a greater number of hepatocytes than those with unmodified or immobilized with glucose due to specific interactions between seeded hepatocytes and galactose moieties exposed onto the surface of the scaffolds. The perfusion culture system based on galactose-modified macroporous scaffolds, under optimal flow conditions, resulted in much higher albumin secretion rate, approximately 70 pg/cell/day for 7 days, compared to that with glucose modified scaffolds used as a negative control. The enhanced functional activity of hepatocytes seeded within the galactose modified scaffolds was likely caused by the formation of aggregated hepatocytes within the scaffolds.