Augustinus Bader

Paracetamol treatment increases telomerase activity in rat embryonic liver cells

Although paracetamol is known to have a damaging effect, this pharmaceutical is widely applied to pregnant and lactating women. Despite substantial progress in our understanding of its hepatotoxicity, some mechanisms, particularly of its embryonal and developmental toxicity, are still unknown. Thus, cell culture assays that investigate its toxicity are of particular interest. We assessed the effects of acute paracetamol treatment on cell viability (LDH assay, MTT assay), glutathione content (GSH assay), metabolic status (albumin and urea assays) and telomerase activity using rat embryonic liver cells (RLC-18 cells). Incubation with low (6 mmol/l) and high (15 mmol/l) concentrations of toxin for 24 h leads to 20% and 50% cytotoxicity, respectively. Paracetamol exerted its toxicity in a similar pathway (depletion of GSH stores) as in adult liver cells, producing damage at the cellular level. Interestingly, paracetamol treatment significantly enhanced telomerase activity. Mechanisms involved in paracetamol-induced inhibition of cell senescence should be further elucidated. Telomerase activity in RLC-18 cells offers unique opportunities for examining basic biologic mechanisms. Our findings should encourage further studies to investigate a link between telomerase activity and toxicity, implying a role of impaired telomerase activity in human pathology.

Tissue engineering of heart valves – human endothelial cell seeding of detergent acellularized porcine valves

OBJECTIVE Tissue engineering of heart valves represents a new experimental concept to improve current modes of therapy in valvular heart disease. Drawbacks of glutaraldehyde fixed tissue valves or mechanical valves include the short durability or the need for life-long anticoagulation, respectively. Both have in common the inability to grow, which makes valvular heart disease especially problematic in children. The aim of this study was to develop a new methodology for a tissue engineered heart valve combining human cells and a xenogenic acellularized matrix. METHODS Porcine aortic valves were acellularized by deterging cell extraction using Triton without tanning. Endothelial cells were isolated in parallel from human saphenous veins and expanded in vitro. Specimens of the surface of the acellular matrix were seeded with endothelial cells. Analysis of acellularity was performed by light microscopy and scanning electron microscopy. Cell viability following seeding was assayed by fluorescence staining of viable cells. RESULTS The acellularization procedure resulted in an almost complete removal of the original cells while the 3D matrix was loosened at interfibrillar zones. However the 3D arrangement of the matrix fibers was grossly maintained. The porcine matrix could be seeded with in vitro expanded human endothelial cells and was maintained in culture for up to 3 days to document the formation of confluent cultures. CONCLUSIONS Porcine aortic valves can be almost completely acellularized by a non-tanning detergent extraction procedure. The xenogenic matrix was reseeded with human endothelial cells. This approach may eventually lead to the engineering of tissue heart valves repopulated with the patients own autologous cells.

Stem-cell-based, tissue engineered tracheal replacement in a child: A 2-year follow-up study

BACKGROUND Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor β to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

New Hepatocyte In Vitro Systems for Drug Metabolism: Metabolic Capacity and Recommendations for Application in Basic Research and Drug Development, Standard Operation Procedures

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4 hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this system is that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used for the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes

Damage to and degeneration of articular cartilage is a major health issue in industrialized nations. Articular cartilage has a particularly limited capacity for auto regeneration. At present, there is no established therapy for a sufficiently reliable and durable replacement of damaged articular cartilage. In this, as well as in other areas of regenerative medicine, tissue engineering methods are considered to be a promising therapeutic component. Nevertheless, there remain obstacles to the establishment of tissue-engineered cartilage as a part of the routine therapy for cartilage defects. One necessary aspect of potential tissue engineering-based therapies for cartilage damage that requires both elucidation and progress toward practical solutions is the reliable, cost effective cultivation of suitable tissue. Bioreactors and associated methods and equipment are the tools with which it is hoped that such a supply of tissue-engineered cartilage can be provided. The fact that in vivo adaptive physical stimulation influences chondrocyte function by affecting mechanotransduction leads to the development of specifically designed bioreactor devices that transmit forces like shear, hydrostatic pressure, compression, and combinations thereof to articular and artificial cartilage in vitro. This review summarizes the basic knowledge of chondrocyte biology and cartilage dynamics together with the exploration of the various biophysical principles of cause and effect that have been integrated into bioreactor systems for the cultivation and stimulation of chondrocytes.

Multilineage differentiation potential of human dermal skin-derived fibroblasts.

Abstract:  Dermal skin‐derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal‐derived tissues: bone, cartilage and fat. Human dermal skin‐derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem‐cell‐like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi‐lineage differentiation potential. We could show similar expression patterns for CD14(−), CD29(+), CD31(−), CD34(−), CD44(+), CD45(−), CD71(+), CD73/SH3‐SH4(+), CD90/Thy‐1(+), CD105/SH2(+), CD133(−) and CD166/ALCAM(+) in well‐established adipose tissue derived‐stem cells and fibroblastic mesenchymal stem‐cell‐like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem‐cell‐like cells expressed vimentin, fibronectin and collagen; they were less positive for α‐smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem‐cell‐like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.

Drug metabolism in hepatocyte sandwich cultures of rats and humans

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.

A Novel Full‐Scale Flat Membrane Bioreactor Utilizing Porcine Hepatocytes: Cell Viability and Tissue‐Specific Functions

When designing an extracorporeal hybrid liver support device, special attention should be paid to providing the architectural basis for reconstructing a proper cellular microenvironment that ensures highest and prolonged functional activity of the liver cells. The common goal is to achieve high cell density culture and to design the bioreactor for full‐scale primary liver cell cultures under adequate mass transfer conditions. An important aim of this study was to evaluate the biochemical performance of a flat membrane bioreactor that permits high‐density hepatocyte culture and simultaneously to culture cells under sufficient oxygenation availability conditions comparable to the in vivo‐like microenvironment. In such a bioreactor pig liver cells were cultured within an extracellular matrix between oxygen‐permeable flat‐sheet membranes. In this investigation we used a novel scaled‐up prototype consisting of up to 20 modules in a parallel mode. Each module was seeded with 2 × 108 cells. Microscopic examination of the hepatocytes revealed morphological characteristics as found in vivo. Cell concentration increased in the first days of culture, as indicated by DNA measurements. The performance of the bioreactor was monitored for 18 days in terms of albumin synthesis, urea synthesis, ammonia elimination, and diazepam metabolism. The ability of the hepatocytes to synthesize albumin and urea increased during the first days of culture. Higher rates of albumin synthesis were obtained at day 9 and remained at a value of 1.41 pg/h/cell until day 18 of culture. The rate of urea synthesis increased from 23 ng/h/cell to 28 ng/h/cell and then remained constant. Cells eliminated ammonia at a rate of about 56 pg/h/cell, which was constant over the experimental period. Hepatocytes in the bioreactor metabolized diazepam and generated three different metabolites: nordiazepam, temazepam, and oxazepam. The production of such metabolites was sustained until 18 days of culture. These results demonstrated that the scale‐up of the bioreactor was assessed, and it could be demonstrated that the device design aimed at the reconstruction of the liver‐specific tissue architecture supported the expression of liver‐specific functions of primary pig liver cells.

Evaluation of cell behaviour related to physico-chemical properties of polymeric membranes to be used in bioartificial organs.

In bioartificial organs using isolated cells, polymeric semipermeable membranes are used as immunoselective barriers as a means for cell oxygenation and also as substrata for adhesion of anchorage-dependent cells. The capacity of the membrane to perform its functions and to provide a cytocompatible support for cell culture depends in particular on its surface properties. In this study we investigated the physico-chemical aspects of the interaction between the membrane and mammalian cells in order to provide guidelines to the selection of cytocompatible membranes. We evaluated the adhesion and metabolic behaviour of isolated liver cells cultured on various polymeric membranes such as those modified by protein adsorption. The physico-chemical properties of the membranes were characterised by contact angle measurements. The different parameters such as acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Osss model. The adsorption of protein modified markedly both contact angle and components of membrane surface tension. In particular, base parameter of surface tension decreased drastically with increased water contact angle. For each investigated membrane we observed that cell adhesion increased with increasing base parameter of membrane surface tension. The absolute value of cell adhesion is higher in the presence of serum proteins adsorbed on the membrane surface, which change the wettability by increasing the base parameter of surface tension. Also, the metabolic functions improve on hydrophilic membranes. Liver cells synthesised urea with a rate that increased with increasing base parameter value of membrane surface tension. The metabolic activity is particularly expressed at high levels when cells were cultured on polycarbonate and cellulose acetate membranes.

Both epithelial cells and mesenchymal stem cell-derived chondrocytes contribute to the survival of tissue-engineered airway transplants in pigs.

OBJECTIVE We sought to determine the relative contributions of epithelial cells and mesenchymal stem cell-derived chondrocytes to the survival of tissue-engineered airway transplants in pigs. METHODS Nonimmunogenic tracheal matrices were obtained by using a detergent-enzymatic method. Major histocompatibility complex-unmatched animals (weighing 65 +/- 4 kg) were divided into 4 groups (each n = 5), and 6 cm of their tracheas were orthotopically replaced with decellularized matrix only (group I), decellularized matrix with autologous mesenchymal stem cell-derived chondrocytes externally (group II), decellularized matrix with autologous epithelial cells internally (group III), or decellularized matrix with both cell types (group IV). Autologous cells were recovered, cultured, and expanded. Mesenchymal stem cells were differentiated into chondrocytes by using growth factors. Both cell types were seeded simultaneously with a dual-chamber bioreactor. Animals were not immunosuppressed during the entire study. Biopsy specimens and blood samples were taken from recipients continuously, and animals were observed for a maximum of 60 days. RESULTS Matrices were completely covered with both cell types within 72 hours. Survival of the pigs was significantly affected by group (P < .05; group I, 11 +/- 2 days; group II, 29 +/- 4 days; group III, 34 +/- 4 days; and group IV, 60 +/- 1 days). Cause of death was a combination of airway obstruction and infection (group I), mainly infection (group II), or primarily stenosis (group III). However, pigs in group IV were alive, with no signs of airway collapse or ischemia and healthy epithelium. There were no clinical, immunologic, or histologic signs of rejection despite the lack of immunosuppression. CONCLUSIONS We confirm the clinical potential of autologous cell- and tissue-engineered tracheal grafts, and suggest that the seeding of both epithelial and mesenchymal stem cell-derived chondrocytes is necessary for optimal graft survival.