Growth factor/heparin-immobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis

Hepatocyte transplantation is being explored as a treatment strategy for end-stage liver disease; however, the main limitation is the insufficient vascularization of transplanted hepatocytes. To overcome this problem, a suitable 3D microenvironment and the types of transplanted cells must be considered for hepatocyte transplantation. In this study, a growth factor (GF)/heparin-immobilized collagen gel-filled polyurethane foam (PUF) scaffold was developed for angiogenesis induction and hepatocyte transplantation. First, a vascular endothelial growth factor (VEGF)/heparin-immobilized, collagen-gel-filled PUF scaffold was developed to establish a prevascularized cavity in the subcutaneous space in rats. Second, accompanied by 70% partial hepatectomy (PH), hepatocytes were embedded inside heparin-immobilized, collagen-gel-filled PUF scaffolds, and were transplanted into the VEGF-induced prevascularized cavity. The benefits of using this system were confirmed by using three types of hepatocytes, namely single hepatocyte, hepatocyte spheroids, and fetal hepatocytes. The normalized hemoglobin content and live nucleus numbers were determined separately to evaluate the angiogenesis and viability of transplanted hepatocytes. In summary, after PH pretreatment, transplantation of fetal hepatocyte-embedded, heparin-immobilized, collagen-gel-filled PUF scaffold into a VEGF-induced prevascularized cavity appears to be a promising strategy for future liver tissue engineering.     

Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system

A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. The micro-space cell culture facilitated the formation of 3D HepG2 cell architecture. HepG2 cells cultured in a micro-space culture plate exhibited increased albumin secretion and enhanced mRNA expression levels of cytochrome P450 (CYP) enzyme compared to those cultured in a monolayer culture. When the cells were exposed to acetaminophen, a hepatotoxic drug, the damage to the HepG2 cells grown in micro-space culture was greater than the damage to the HepG2 cells grown in monolayer culture. In addition, human primary hepatocytes grown in micro-space culture also exhibited increased albumin secretion, enhanced CYP mRNA expression levels and increased sensitivity to acetaminophen compared to those grown in monolayer culture. These results suggest that this micro-space culture method enhances the hepatocyte-specific functions of hepatocytes, including drug-metabolizing enzyme activities, making hepatocytes grown in the micro-space culture system a useful tool for evaluating drug toxicity in vitro.     

Growth induction of rat primary hepatocytes using antisense oligonucleotides

We examined growth control of adult and fetal hepatocytes by regulating the expression of cell-cycle-related proteins using antisense S-oligonucleotides to tumor suppressors retinoblastoma (RB) protein and p53, and cyclin-dependent kinase (CDK) inhibitors p21 and p27. The protein expression in both adult and fetal hepatocytes was significantly suppressed with the addition of corresponding antisense oligonucleotides at a concentration of 2.5 microM. For the evaluation of growth, 3H-thymidine incorporation and DNA content were measured and the results demonstrated that all the antisense oligonucleotides had growth-promoting effects and the promoting potential was equivalent or slightly greater than that with the addition of hepatocyte growth factor (HGF) (10 ng/ml). The growth-promoting effect of the antisense oligonucleotides was enhanced by HGF in both adult and fetal hepatocyte cultures, and the effects on hepatocyte growth were also observed in a suspension culture.     

Albumin production activity of primary rat hepatocytes is improved on type V collagen

Primary rat hepatocytes were cultured on type V collagen. Hepatocyte spreading on type V collagen was inferior compared with that on type I collagen. However, the albumin production rates of hepatocytes cultured on type V collagen were approximately twice as high as those of hepatocytes cultured on type I collagen.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.     

The protective role of natural phytoalexin resveratrol on inflammation, fibrosis and regeneration in cholestatic liver injury

Liver injuries can trigger a cascade of inflammatory responses and as a result, initiate the process of hepatic regeneration and fibrogenesis. Resveratrol (RSV) has multiple health‐promoting benefits. This study evaluated the potential protective effects and mechanism of RSV as related to cholestatic liver injury. RSV was given (4 mg/kg/day, i.p.) for either 3 days or 7 days after bile duct ligation (BDL) injury. RSV significantly reduced serum ALT, AST but not T‐bil on Day 3. At this early stage of injury, RSV significantly reduced TNF‐α and IL‐6 mRNA and decreased the number of Kupffer cells (CD68⁺) recruited in the injured liver. RSV decreased hepatic fibrosis and reduced collagen Iα1 and TIMP‐1 mRNA on Day 7. At the later stages of injury, RSV increased the number of Ki67⁺ hepatocytes indicating that RSV promoted hepatocyte proliferation. Additionally, it resulted in decreased expression of 4‐hydroxynonenal and increased expression of the hepatocyte growth factor protein and mRNA in the RSV‐treated BDL group. Meanwhile, RSV reduced the mortality rate of BDL mice. In conclusion, RSV attenuated inflammation and reduced Kupffer cells activation. RSV decreased fibrosis and promoted hepatocyte regeneration, which increased the survival of BDL mice. RSV was beneficial for the treatment of cholestatic liver injury.     

Cultivation of rat hepatocytes in a medium based on commercially available infusate solutions

The possibility of using commercially available infusate solutions as a culture medium for hepatocytes was investigated in primary monolayer cultures of rat hepatocytes. The addition of Ca2+ to the infusate medium was necessary for hepatocytes to express their albumin secreting ability. The infusate medium supplemented with hormones (10(-7) M insulin and 10(-7) M dexamethasone) and Ca2+ (72.5 mg/l) allowed hepatocytes to produce albumin of an amount comparable to that produced in Williams' E medium. The activity of released lactate dehydrogenase (LDH) was kept at a low level throughout the cultivation in the infusate medium.     

Maintenance of hepatocyte functions by coculture with bone marrow stromal cells

Rat hepatocytes and bone marrow stromal cells (BMSCs) were cocultured with the aim of maintaining differentiated hepatocyte functions. After BMSCs were expanded to a confluent monolayer, freshly isolated hepatocytes were cultured with them, separated by a semipermeable membrane. The BMSCs significantly increased the urea synthesis and albumin secretion activities of the hepatocytes. Conditioned medium prepared from a BMSC monoculture had the same effect. Further study showed that interleukin-6 was involved in the maintenance of urea synthesis and another factor in the maintenance of albumin secretion.     

Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture

Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.     

自组装胶原蛋白复合膜对鱿鱼冷藏品质的影响

以草鱼鱼鳔为原料,酶法提取胶原蛋白后进行自组装反应,与壳聚糖、甘油制备复合涂膜,分析该涂膜性能,研究复合膜对鱿鱼冷藏保鲜效果的影响.结果表明:草鱼鱼鳔胶原蛋白属于I型胶原蛋白,具有三螺旋结构.胶原蛋白自组装后制备的复合膜拉伸强度增加,断裂伸长率降低,水蒸气透过率降低.未经涂膜处理的鱿鱼在冷藏第8天已腐败,而此时用自组装...     

Direct measurement of oxygen concentration inside cultured cartilage for relating to spatial growth of rabbit chondrocytes

In static culture of rabbit chondrocytes in collagen gel, the direct measurement of dissolved oxygen (DO) concentration revealed that the DO level at the top surface of gel decreased due to an increase in overall cell density with elapsed time. The local cell density at the top surface on day 21 was 5.7 × 10⁷ cells/cm³, being 11 times that at the bottom of gel. This heterogeneity of cell distribution in the gel was considered to occur by limitation of oxygen supply into a deeper part of the gel. In shaking culture using a dish with gas-permeable film, the DO level was enhanced inside the gel and the overall cell density in the gel was achieved to be 2.9 times that in the static culture.     

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.     

Modulation of biotransformation and elimination systems by BM-21, an aqueous ethanolic extract from Thalassia testudinum,and thalassiolin B on human hepatocytes

BM-21 is an extract obtained from Thalassia testudinum marine plant with pharmacological properties. The effects of BM-21 and thalassiolin B (TB), its main component, on enzyme and transport proteins involved in drug metabolism and excretion in human cultured hepatocytes were evaluated. Cells were exposed for 48 h to sub-cytotoxic concentrations of BM-21 or TB. Effects on P450 isoforms revealed significant reductions of CYP1A2, 3A4 and 2D6 activities (up to 56%, 66% and 44% inhibition, respectively) after exposition to BM-21, no changes on CYP2A6 and 2C9 activities. TB produced a concentration-dependent reduction of all P450 activities. In addition, a decrease in total UGT and UGT2B7 activities was found at 250 μg/mL BM-21, while UGT1A1 and 1A9 were significantly reduced (50 μg/mL). TB only inhibited significantly UGT1A9 activity. Both products were able to reduce P-gp activity in treated hepatocytes. Quantification of specific mRNAs revealed a reduction in CYP3A4 and 3A5 mRNAs content and an increase in CYP1A1 and 1A2 mRNAs. No appreciable effects in the levels of CYP2A6, 2B6, 2C9, 2C19, 2D6, 2E1, UGT1A1, UGT1A9 and ABCB1 (P-gp) were found. BM-21 inhibited P450, UGTs and P-gp activities in human hepatocytes; therefore, it should be examined for potential pharmacokinetic drug interactions in vivo.     

Characterization of in vitro anthocyanin-producing sour cherry (Prunus cerasus L.) callus cultures

Anthocyanin-producing callus cultures from in vitro sour cherry (Prunus cerasus L.) leaf tissues were established. As in the parent leaf tissues, the calli extracts showed the synthesis of a prevalent anthocyanin, cyanidin 3-glucoside. When the dark grown callus cultures were exposed to the light, cyanidin 3-glucoside content was increased from 0.1 to 4.5 mg 100 g⁻¹ fresh weight. Thus, of the available strategies for the enhancement of pigment production light resulted the triggering factor in this cell system. The addition of 50 μM jasmonic acid to the culture medium stimulated cyanidin 3-glucoside synthesis which resulted in an earlier appearance of pigment on the calli.     

Characteristics of ruminant mammary epithelial cells grown in primary culture in serum-free medium.

Cells were obtained from the mammary glands of sheep and cows by collagenase-hyaluronidase digestion. Characterization of cells as epithelial was by reaction with a monoclonal antibody to cytokeratin. A subpopulation of spindle-shaped or stellate cells reacted with a monoclonal antibody to desmin and may be related to myoepithelial cells. The development is described of a simple serum-free culture system for these cells on gels of rat tail (type 1) collagen. A commercial medium (M199) was used, buffered with Hepes and with bovine serum albumin as the sole protein supplement, plus fibronectin for the first 18 h only as an attachment factor. The cell cultures showed stimulated DNA synthesis in response to mitogens on attached gels and also responded as floating cultures to lactogenic hormones with production of alpha-lactalbumin.     

In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells

A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.     

Isolation and identification of an antiproliferative substance from fructose–tyrosine Maillard reaction products

We isolated a substance from fructose–tyrosine Maillard reaction products (MRPs) and investigated its antiproliferative effect on six human cancer cell lines. The ethyl acetate fraction of fructose–tyrosine MRPs showed a strong antiproliferative effect; this fraction was isolated and purified using silica gel column chromatography, semipreparative RP-HPLC, and recycling HPLC. The structure of the purified compound was determined using spectroscopic methods. The isolated compound was identified as 2,4-bis(p-hydroxyphenyl)-2-butenal (C₁₆H₁₄O₃, HPB242). HPB242 inhibited cell growth in a dose-dependent manner (10–80 μg/ml) on the six human cancer cell lines. The IC₅₀ values of HPB242 on the six human cancer cell lines were 17.34 μg/ml (MCF-7), 29.21 μg/ml (HCT-116), 34.57 μg/ml (H-460), 34.87 μg/ml (HepG2), 48.77 μg/ml (PC-3), and 55.83 μg/ml (MKN-45).     

Effect of microwell chip structure on cell microsphere production of various animal cells

The formation of three-dimensional cell microspheres such as spheroids, embryoid bodies, and neurospheres has attracted attention as a useful culture technique. In this study, we investigated a technique for effective cell microsphere production by using specially prepared microchip. The basic chip design was a multimicrowell structure in triangular arrangement within a 100-mm² region in the center of a polymethylmethacrylate (PMMA) plate (24 × 24 mm²), the surface of which was modified with polyethylene glycol (PEG) to render it nonadhesive to cells. We also designed six similar chips with microwell diameters of 200, 300, 400, 600, 800, and 1000 μm to investigate the effect of the microwell diameter on the cell microsphere diameter. Rat hepatocytes, HepG2 cells, mouse embryonic stem (ES) cells, and mouse neural progenitor/stem (NPS) cells formed hepatocyte spheroids, HepG2 spheroids, embryoid bodies, and neurospheres, respectively, in the microwells within 5 days of culture. For all the cells, a single microsphere was formed in each microwell under all the chip conditions, and such microsphere configurations remained throughout the culture period. Furthermore, the microsphere diameters of each type of cell were strongly positively correlated with the microwell diameters of the chips, suggesting that microsphere diameter can be factitiously controlled by using different chip conditions. Thus, this chip technique is a promising cellular platform for tissue engineering or regenerative medicine research, pharmacological and toxicological studies, and fundamental studies in cell biology.     

Effect of Monosaccharides Composing Glycosaminoglycans on Type 2 Collagen Accumulation in a Three-Dimensional Culture of Chondrocytes

The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes.