Sascha Pahernik

Collagen gel immobilization: A useful cell culture technique for long-term metabolic studies on human hepatocytes

1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Neoadjuvant photodynamic therapy before curative resection of proximal bile duct carcinoma

BACKGROUND Hilar bile duct carcinoma has an 80% probability of local recurrence after curative resection, which might be reduced if neoadjuvant photodynamic therapy is feasible. CASE AND TREATMENT: After intravenous injection of sodium porfimer we treated an adenocarcinoma of the proximal common bile duct (T2 N0 M0, Bismuth type II) in a 72-year-old man with red laser light (applied from the lumen at a dose 250 Joules/cm2), and the adjacent right and left hepatic and common bile duct at a dose of 125 Joules/cm2. After 23 days the tumor was completely resected (adenocarcinoma pT2 pNO; G2). RESULTS In the lumenal, 4-mm-thick layer the bile duct specimen exhibited complete tumor necrosis with pigmentation of photodegraded porfimer and no viable tumor cells, while in the outer layer of the wall (at 5-8-mm depth) viable cancer cell nests without degraded porfimer were seen. The bile duct tissue showed little damage. Eighteen months after surgery, neither tumor recurrence nor stricture formation was found at the pretreated bilioenteric anastomoses. CONCLUSIONS a) Photodynamic therapy with sodium porfimer seems to be confined to the superficial 4-mm layer of bile duct cancer. b) Neoadjuvant photodynamic therapy is feasible for hilar bile duct carcinoma.

Pharmacokinetics and selectivity of aminolevulinic acid–induced porphyrin synthesis in patients with cervical intra-epithelial neoplasia

Photodynamic therapy (PDT), due to its tumor selectivity, represents an alternative approach to diagnose and treat cervical intra‐epithelial neoplasia (CIN) without altering normal surrounding tissue. Our aim was to investigate the pharmacokinetics and the selectivity of 5‐aminolevulinic acid (5‐ALA)–induced porphyrin fluorescence after topical administration, to obtain basic clinical data for future diagnostic fluorescence imaging and PDT protocols for CIN. Twenty‐eight non‐pregnant women with a cytological diagnosis of low‐grade or high‐grade squamous intra‐epithelial lesions were included. An aqueous solution containing 3% 5‐ALA was topically applied 1 to 6 hrs prior to conization using a cervical cap. After excision, porphyrin‐induced fluorescence was quantified in dysplastic (n = 14) and normal epithelium (n = 28) by means of quantitative fluorescence microscopy. High values of porphyrin fluorescence were found in squamous epithelium between 150 and 450 min, with a maximum at 300 min following administration of 5‐ALA. Ratios of porphyrin fluorescence of dysplastic vs. surrounding normal epithelium were 1.3 and 1.21 for CIN 1 (n = 3) and CIN 2 (n = 3), respectively. In CIN 3 patients (n = 8), this ratio was 2.35; the best selectivity of 5‐ALA‐induced porphyrin fluorescence in CIN 3 lesions (ratio 3) was observed with a topical administration time of between 150 and 250 min. Our results demonstrate that patients with CIN 3 show higher 5‐ALA‐induced fluorescence compared with normal epithelium. The optimal administration time of topically applied 5‐ALA was between 3 and 4 hr. Our data suggest that topical ALA‐PDT and photodynamic diagnosis might be suitable for detecting CIN. Int. J. Cancer 78:310–314, 1998.© 1998 Wiley‐Liss, Inc.

Hyperthermia induces T1 relaxation and blood flow changes in tumors. A MRI thermometry study in vivo.

Regional hyperthermia in combination with chemotherapy and/or radiotherapy has proven to be an effective treatment concept for locally advanced deep-seated tumors. Simultaneous MR-imaging could be a promising approach for therapy optimization. Purpose of this study was the in vivo investigation of temperature induced longitudinal relaxation time (T(1)) and blood flow changes in a tumor model. Using a 1.5 Tesla MR system, the T(1) sensitivity on temperature and the onset of tissue changes at temperatures >44 degrees C were investigated in muscle samples. Also, fourteen Syrian Golden Hamsters with amelanotic melanoma A-MEL-3 were examined during heating of the tumors. Temperature induced blood flow and T(1) changes were determined continuously during hyperthermia. Changes of T(1) correlated linearly with temperature over a wide range (27-44 degrees C) in the tissue sample. Tissue changes became notable above 44 degrees C. In the tumor model, relative changes of T(1) (unlike blood flow) showed linear correlation with temperature over the entire range of hyperthermia relevant temperatures (32-44 degrees C). For a low thermal dose, T(1) allows the assessment of temperature changes in tumors in vivo. At higher thermal doses, in addition to temperature changes, T(1) also shows tissue changes. Both temperature and tissue changes supply important information for hyperthermia.

Metabolism of pimobendan in long-term human hepatocyte culture: in vivo-in vitro comparison

The aim of this study was to investigate further the potential of a new hepatocyte culture based on the hypothesis that liver cells in an appropriate in vitro environment (immobilizing gel technique) maintain high metabolic activity comparable with that in vivo. Pimobendan (UD-CG 115), a pyridazinone derivative, is a cardiotonic vasodilator that increases myocardial contractility through calcium sensitization and relaxation of vascular smooth muscle, probably due to phosphodiesterase inhibition. In man, pimobendan is O-demethylated to UD-CG 212. This latter is metabolized to O- and N-glucuronides. Pimobendan itself is also glucuronidated to a N-glucuronide. Human hepatocytes immobilized in collagen gel were incubated with pimobendan to investigate their metabolic activity in the long-term and to compare the results to the data from clinical trials. 14C-labelled pimobendan was incubated at two concentrations (10 and 100 microM) at day 3, 11 and 22 of culture, and samples were analysed after 4, 24 and 48-h incubation. Metabolic patterns were evaluated by hplc with radioactivity-, diode array-, and mass spectral-detection. In vitro, pimobendan was O-demethylated and subsequently O-glucuronidated. The rate of metabolism of pimobendan could be maintained in this culture system for > 3 weeks. However, the relative amount of a putative N-glucuronide under in vitro conditions was lower than in vivo.

High Density Culturing of Porcine Hepatocytes Immobilized on Nonwoven Polyurethane-Based Biomatrices

Objective: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). Methods: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 × 106 cells/cm2 on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. Results: The optimal cell density in this three-dimensional culture configuration was 1 × 106 cells/cm2. The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 ± 5.6 ng/h/µg DNA and of 1.8 ± 0.14 µg/h/µg DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/µg DNA (28.7, 42.8) and 32.7 ng/h/µg DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively. Conclusion: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems.

Synthesis of insulin-like growth factor binding proteins and of the acid-labile subunit of the insulin-like growth factor ternary binding protein complex in primary cultures of human hepatocytes

BACKGROUND/AIMS The liver is the main source of circulating insulin-like growth factor binding proteins. In man, the cellular origin of insulin-like growth factor binding proteins has remained obscure. METHODS Human hepatocytes isolated from surgical specimens were purified and cultured using a collagen gel immobilization technique. Gene expression of individual insulin-like growth factor binding proteins and of the acid-labile subunit of the insulin-like growth factor binding proteins by Western ligand blotting and immunoblot analysis. Neutral size chromatography of medium samples was used to detect insulin-like growth factors binding protein complexes. RESULTS In cultured hepatocytes transcripts for insulin-like growth factor binding protein-1, -2, -3, -4 and for acid labile subunit could be demonstrated. Ligand blotting revealed the secretion of insulin-like growth factor binding proteins of molecular weights of 24 kD, 30 kD, 34 kD, 43 kD and 46 kD, respectively. Using polyclonal antisera, these proteins were identified as insulin-like growth factor binding protein-1, -2 and the insulin-like growth factor binding protein-3 doublet. Neural size chromatography of culture supernatants showed the presence of an insulin-like growth factor binding protein complex of approximately 40 kD, but absence of the high molecular weight ternary complex of 150 kD. CONCLUSIONS It is concluded that in man parenchymal liver cells have to be regarded as a source of acid-labile subunit and of circulating insulin-like growth factor binding proteins including insulin-like growth factor binding protein-3.

Porcine hepatocytes from slaughterhouse organs. An unlimited resource for bioartificial liver devices.

&NA; Most recent strategies for the development of hybrid artificial liver devices focus on the use of parenchymal liver cells (hepatocytes). For clinical application of these devices, a sufficient cell supply is mandatory. Because human liver tissue is rarely available, isolated porcine hepatocytes from laboratory animals have been suggested for use in bioartificial livers. The authors introduce a modified isolation protocol to yield large scale numbers of viable porcine hepatocytes from slaughterhouse organs. Perfusion and enzymatic digestion of the left medial liver lobe (n = 74) resulted in 1.0 ± 0.3 × 107 viable hepatocytes per gram of tissue, and an overall yield of 1.92 ± 0.5 × 109 viable cells per isolation (viability: 93 ± 2%). Collagen gel immobilization maintained morphologic integrity and functional activity of hepatocyte cultures over long‐term periods. Cell morphology, as assessed by light microscopic evaluation, was maintained for 2 weeks. Stable DNA content (51 ± 5 μg) and low values of alanine aminotransferase release (8 μU/hr/μg DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 μg/hr/μg DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7‐ethoxycoumarin (4.5 nmol/hr/μg DNA) indicated long‐term metabolic activity of cultured hepatocytes. Hepatocytes from livers of slaughtered pigs represent an unlimited resource of viable material for cell culture, and their usefulness as functional units of bioartificial liver support devices should be tested. ASAIO Journal 1995; 41:189‐193.

Orthogonal polarization spectral (OPS) imaging: a new technique for the visualization and study of microcirculation

OPS-imaging is a novel technique which can be used to obtain images of the microcirculation using reflected light. High contrast transillumination quality images can be collected not only from thin tissues, but from the surface of solid organs as well. In OPS-imaging the tissue is illuminated with light that has been linearly polarized in one plane. The light is then both scattered and reflected by the tissue. In front of the camera there is a second polarizer which is oriented in a plane precisely orthogonal to that of the illuminating light. This means that light which is directly reflected by the tissue, which maintains its polarization, is rejected by the polarizer in front of the camera. The only light which enters the camera and forms the image is light which has become depolarized, which typically requires at least 10 scattering events. Thus, the light which forms the image comes from deep (0.5 mm) within the tissue and effectively back-illuminates the absorbing material in the foreground. When the light has a wavelength within the hemoglobin absorption spectrum (548 nm), the scattered light is absorbed by the hemoglobin in the red cells, making it possible to visualize the blood vessels as in transillumination intravital microscopy. Thus, images of the microcirculation of solid organs can be obtained without the use of fluorescent dyes. OPS-imaging has been incorporated into a small, hand held device which is easily transportable (CYTOSCAN). Because of these two advantages, it is possible to not only use OPS-imaging in the laboratory, but also in the clinic on patients.

Isolation and culture of porcine hepatocytes

Isolated hepatocytes have increasingly become an important research tool in the medical, pharmacological, biological, and biochemical sciences so that the demand for viable cell material has increased. Cell-lines could produce sufficient numbers of hepatocytes; however, uncertainty exists with respect to their stability and how well their properties compare with those of primary cells. In bioartificial liver applications the use of cell lines has been advocated [1] as well as challenged [2]. In most cases, hepatocytes for various kinds of experiments have been isolated from small vertebrates, such as rats. Other animal species have been used as donors in order to achieve large-scale isolation of liver cells. The choice of animal reflects not only well-recognized biological differences between various animal species [3], but is also a consequence of practical considerations in individual laboratory settings.