Liver-specific Functions Of Hepatocytes Research Articles

Creation of a novel lipid-trehalose derivative showing positive interaction with the cell membrane and verification of its cytoprotective effect during cryopreservation

Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6nM-16μM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.

Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes.

Background and objective. The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro.Methods. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells.Results. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes.Conclusions. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.

Polycaprolactone scaffold modified with galactosylated chitosan for hepatocyte culture

A suitable extracellular matrix that can maintain long-term proliferation and liver-specific functions of hepatocytes is essential for both extracorporeal bioartificial liver (BAL) assist systems and implantable cell-scaffold constructs. In this work, a polycaprolactone (PCL) scaffold was modified with galactosylated chitosan (GC) to achieve better bioactivity and mechanical stability. The PCL scaffold, prepared by a method of gelatin particle leaching, was hydrolyzed to produce carboxylic groups that were utilized to react with the amine groups of the GC, which was synthesized by grafting the galactose to chitosan. Results showed that the content of carboxylic groups was increased initially with the hydrolysis time until 10 min, at which a COOH density of 2.0×10−5 mol/mg scaffold was reached. The incorporated GC amount showed a positive relationship with the COOH density and finally reached a 100 μg/mg scaffold with a hydrolysis time of 15 min. The incorporated GC was rather stable against incubation in the medium, and could significantly enhance the compression strength of the PCL scaffold in a wet state. With the galactose ligands on the surface, the PCL scaffold could be better recognized by hepatocytes, and show better cell viability, spheroid formation and long-term maintenance of liver-specific functions such as albumin secretion. Open image in new window

Current development of bioreactors for extracorporeal bioartificial liver (Review)

The research and development of extracorporeal bioartificial liver is gaining pace in recent years with the introduction of a myriad of optimally designed bioreactors with the ability to maintain long-term viability and liver-specific functions of hepatocytes. The design considerations for bioartificial liver are not trivial; it needs to consider factors such as the types of cell to be cultured in the bioreactor, the bioreactor configuration, the magnitude of fluid-induced shear stress, nutrients' supply, and wastes' removal, and other relevant issues before the bioreactor is ready for testing. This review discusses the exciting development of bioartificial liver devices, particularly the various types of cell used in current reactor designs, the state-of-the-art culturing and cryopreservation techniques, and the comparison among many today's bioreactor configurations. This review will also discuss in depth the importance of maintaining optimal mass transfer of nutrients and oxygen partial pressure in the bioreactor system. Finally, this review will discuss the commercially available bioreactors that are currently undergoing preclinical and clinical trials.

Primary throughput screening of protectants for hypothermic preservation of bioartificial liver in gel entrapped hepatocytes

Hypothermic preservation of hepatocytes has been widely studied for potential storage and transportation of bioartificial liver (BAL), but the liver-specific functions of hepatocytes are severely impaired by hypothermic treatment. A miniaturized gel entrapment-based BAL without circulation system was used to screen protectants from Chinese herbal medicines in this paper. Although anisodamine (ANI), matrine (MAT) and schisandrin B (Sch B) individually enhanced, to some extent, cell viability and liver-specific functions of hypothermically preserved hepatocytes, glycyrrhizic acid (GA), performed superior to these three extracts. The multieffect of GA on enhancement of mitochondrial membrane potential and inhibition of oxidative stress as well as lipid accumulation might determine its protection on hepatocytes from hypothermia-induced cell death. Furthermore, cell viability and intracellular glutathione (GSH) content decreased more dramatically at 6 h of the rewarming compared to those immediately after hypothermic preservation, indicating the aggravated cell injury by rewarming treatment. Therefore, gel entrapped hepatocytes in this study could be proposed for the throughput screening of desired conditions for hypothermic preservation of BAL.

Effect of photo-immobilization of epidermal growth factor on the cellular behaviors

We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg 2+ although integrin-mediated cell adhesion to natural ECMs is dependent on Mg 2+. Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell–substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand–receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.