Biomedical subjects

Arno W Tilles

Publications and source records attributed to Arno W Tilles.

10 recordsLinked to original sources

PubMed2007

Oxygen uptake rates and liver-specific functions of hepatocyte and 3T3 fibroblast co-cultures.

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibroblasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte function. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea production) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (V(m)) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions.

3T3 Cells↗

PubMed2006

A bioartificial liver device secreting interleukin-1 receptor antagonist for the treatment of hepatic failure in rats.

BACKGROUND: Liver transplantation is the treatment of choice for many patients with fulminant hepatic failure (FHF). A major limitation of this treatment is the lack of available donors. An optimally functioning bio-artificial liver (BAL) device has the potential to provide critical hepatic support to patients with FHF. In this study, we examined the efficacy of combining interleukin-1 (IL-1) receptor blockade with the synthetic function of hepatocytes in a BAL device for the treatment of FHF. MATERIALS AND METHODS: We injected an adenoviral vector encoding human IL-1 receptor antagonist (AdIL-1Ra) into the liver of D-galactosamine (GalN) intoxicated rats via the portal vein. We also transfected primary rat hepatocytes and reversibly immortalized human hepatocytes (TTNT cells) with AdIL-1Ra, and incorporated these transfected hepatocytes into our flat-plate BAL device and evaluated their efficacy in our GalN-induced FHF rat model after 10 h of extracorporeal perfusion. RESULTS: Rats injected with AdIL-1Ra showed significant reductions in the plasma levels of hepatic enzymes. Primary rat hepatocytes transfected with AdIL-1Ra secreted IL-1Ra without losing their original synthetic function. Incorporating these cells into the BAL device and testing in a GalN-induced FHF rat model resulted in significant reductions in plasma IL-6 levels and significantly improved animal survival. Incorporating the AdIL-1Ra transfected TTNT cells in the BAL device and testing in the GalN-induced FHF rat model resulted in significantly reduced plasma IL-6 levels, and a trend toward improved survival was seen. CONCLUSION: Hepatocytes producing IL-1Ra are a promising cell source for BAL devices in the treatment of GalN-induced FHF.

Adenoviridae↗

PubMed2006

Treatment of fulminant hepatic failure in rats using a bioartificial liver device containing porcine hepatocytes producing interleukin-1 receptor antagonist.

Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with high mortality. There is evidence that FHF is an inflammatory disease, which is supported clinically by elevated serum levels of cytokines. In an effort to develop hepatocytes with additional functions for use in our bioartificial liver (BAL) device, we focused on interleukin-1 (IL-1) blockade as a therapeutic modality. Primary porcine hepatocytes were isolated from the livers of miniature swine and then transfected with an adenoviral vector encoding human interleukin-1 receptor antagonist (AdIL-1Ra). The transfected hepatocytes secreted human IL-1Ra. These transfected hepatocytes were incorporated into a flat-plate BAL device to evaluate their efficacy in treating D-galactosamine (GalN)- induced FHF in a rat model. After extracorporeal perfusion with the BAL device containing the transfected hepatocytes, there were significant reductions in the plasma levels of hepatic enzymes (aspartate aminotransferase and alanine aminotransferase) and cytokines (IL-1 and IL-6), indicating a beneficial effect. Animal survival was significantly improved in the treated group compared to the control group. These experiments demonstrate that combining inflammatory cytokine blockade with a functional BAL device may be an effective therapeutic option in the treatment of FHF.

Adenoviridae↗

PubMed2006

Polyelectrolyte nano-scaffolds for the design of layered cellular architectures.

The design of in vitro multilayered cellular architectures that resemble the stratified, lattice-like structure in tissues poses a significant challenge for tissue engineering. There is currently no generally applicable methodology to design multilayered cellular constructs that mimic the structure of tissues in vivo. We report a novel and generalizable approach to create multilayered cellular constructs that addresses these issues. These in vitro constructs comprise alternating layers of cells and nano-scale biocompatible polyelectrolyte (PE) scaffolds. We apply this methodology to address two specific problems in hepatic tissue engineering: the design of in vitro liver sinusoidal structures and the critical need to increase viable cell mass in extracorporeal liver-assist devices. We assembled ultrathin polymer scaffolds on the top of a confluent monolayer of cells by the sequential deposition of oppositely charged PEs. The thickness of the PE scaffold lies in the nanometer range. The PE scaffold plays a dual role. First, it is a technique to culture hepatocytes in vitro that maintains their morphology, cytoskeletal structure, and liver-specific functions. Second, the nano-scaffold provides a cell-adhesive surface on which a second layer of cells can be cultured, resulting in layered architectures. We have used this approach to design layered three-dimensional hepatocyte-PE-hepatocyte constructs, hepatocyte-PE-endothelial cell constructs, and hepatocyte-PE-fibroblast constructs. As a result of its versatility, this approach can, in principle, be used to design layered cellular constructs of any tissue type, and therefore has potentially wide applications in tissue engineering, bioreactor devices, and in drug delivery. This methodology has the potential to generate realistic in vitro constructs of any tissue type.

3T3 Cells↗

PubMed2005

Microfabricated grooved substrates as platforms for bioartificial liver reactors.

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.

3T3 Cells↗

PubMed2004

Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography.

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.

3T3 Cells↗

PubMed2004

Hepatic tissue engineering for adjunct and temporary liver support: critical technologies.

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist.

Biomedical Technology↗

PubMed2003

Efficacy of an extracorporeal flat-plate bioartificial liver in treating fulminant hepatic failure.

BACKGROUND: Fulminant hepatic failure is associated with a high mortality rate. Orthotopic liver transplantation is the only established treatment for patients who do not respond to medical management. A major limitation of this treatment is a shortage of donor organs, resulting in many patients dying while waiting for a transplant. An extracorporeal bioartificial liver (BAL) has the potential to provide temporary support for patients with fulminant hepatic failure (FHF) and for patients awaiting orthotopic liver transplantation. We developed a flat-plate BAL with an internal membrane oxygenator in which porcine hepatocytes were cultured as a monolayer. MATERIALS AND METHODS: Twenty-four hours after cannulation of the left carotid artery and right jugular vein, FHF was induced in rats by administering 2 intraperitoneal injections of D-galactosamine (GalN) (1.2 g/kg) at a 12-h interval. The rats were connected to a BAL device 24 h after the first GalN injection and underwent extracorporeal perfusion for a duration of 10 h. Liver histology, liver-specific markers, and animal survival up to 168 h (7 days) were examined. RESULTS: Histologically, liver damage was reduced in the animal group treated with the hepatocyte-based BAL device. Significant reductions occurred in the plasma ammonia levels and prothrombin times in the group treated with the seeded BAL device. Animal survival in the group treated with the seeded BAL device was significantly higher (50.0%) than in the control animal group treated with an unseeded BAL device (11.1%). CONCLUSIONS: This flat-plate BAL with an internal membrane oxygenator and cultured porcine hepatocytes has yielded encouraging results in the treatment of rats with GalN-induced FHF.

Animals↗

PubMed2002

Bioengineering of liver assist devices.

Over 30 000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver device could provide vital support to a liver failure patient until a donor liver was available or until the patient's own liver regenerated. Although it is unclear which liver-specific functions must be provided by such a device to be effective, a constant challenge has been to obtain stable, well-differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased device efficiency. In this overview, the authors describe the critical issues involved in bioartificial liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat-plate bioartificial liver device with an internal membrane oxygenator.

Animals↗

PubMedDate not supplied

Selective enhancement of cytochrome p-450 activity in rat hepatocytes by in vitro heat shock.

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.

Albumins↗