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H G Koebe

Publications and source records attributed to H G Koebe.

34 records · Page 2Linked to original sources
PubMed1996

Extracorporeal artificial liver: the influence of a second cell layer on the morphology and function of immobilized human hepatocytes.

Hepatocytes in long-term cultures represent a promising approach to preserve liver function under standard culture conditions. Hepatocyte cultures as the key components in an extracorporeal artificial liver (EAL) in the treatment of hepatic insufficiency, would be a great advantage. However, one of the numerous unsolved problems is the limitation of the surface area of a future EAL. To decrease the dimensions of same, we modified the cell immobilization technique by placing a second layer of immobilized human hepatocytes onto a layer of pre-immobilized hepatocytes creating a "sandwich immobilization" (SI) system. Immobilization and sandwich immobilization were compared over an investigation period of 30 days: functional performance mirrored by cholinesterase (CHE) and albumin secretion showed remarkable differences only in the course of the first week, whereas we found almost no differences from day 8 on. The total DNA-values on days 0, 1, 7, 14, 21 and 30 varied strongly after the first week but were very similar up to day 30. Finally, it appears disadvantageous to enlarge number/cm2 of (human) hepatocytes in long-term cultures or for application in an EAL by means of sandwich immobilization.

Albumins↗
PubMed1995

Surgical treatment of tumor metastases: general considerations and results.

The prerequisite for a curative resection of metastases is their restriction to the key organs, the liver and lungs, in the sense of a limited dissemination. For long-term prognosis, the type of primary tumor as well as the radical resection of lung and liver metastases is essential. To improve the process of surgical indication and therapy of tumors, clear definitions for the terms "tumor recurrence" and "metastases" have been agreed upon. Research and clinical investigation have led to a better understanding of tumor-regulating factors, some of which are briefly described: Metastasis promoting factors include the lack of E-cadherin, which leads to a local penetration of basal membranes by tumor cells; CD44 seems to play an important role in cell-cell and cell-matrix interactions, apparently increasing the metastatic potential of tumors and reducing the long-term survival of patients. High levels of urokinase in primary tumors are also associated with a poorer prognosis, as well as plasminogen inactivator inhibitor PAI II, which plays a crucial role in tumor growth. Positive findings in bone marrow aspirates of patients with different malignancies, stained for cytokeratin 18, either are associated with higher recurrence rates in colon and breast cancer or can be correlated to the prognosis of patients with gastric cancer. Technical aspects of surgery for hepatic, pulmonary and skeletal metastases are presented and discussed with respect to curative and palliative indications.

Bone Neoplasms↗
PubMed1995

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.

Blotting, Northern↗
PubMed1995

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.

Biotransformation↗
PubMed1995

Viable porcine hepatocytes from slaughterhouse organs.

A modified enzymatic isolation technique for the successful harvesting of porcine liver cells from slaughterhouse organs is introduced. Digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10E7 viable hepatocytes per gram tissue and an overall yield of 1.92 +/- 0.5 x 10E9 cells per isolation (viability: 93 +/- 2%).

Abattoirs↗
PubMed1995

[An artificial liver on its way to clinical reality?].

Temporary extracorporeal liver assist devices based on liver cell cultures represent a promising approach for the treatment of liver failure as well as for perioperative care in liver surgery and transplantation. Primary hepatocytes from donor animals as well as human cell lines have been discussed as cell sources. A viable cell mass of 110-220 g (= 2.5-5 x 10(10) liver cells) has to be functionally active in bioreactors over a time period of several days. This cell number correlates to 10-20% of liver cells in an adult human liver. Neither in animal experiments, nor in clinical trials a complete liver replacement could yet be shown for cell-based bioreactors, however, beneficial effects have been demonstrated: partial detoxification and specific synthetic functions have been reported in animal experiments. Two authors have shown first preliminary clinical data on different cell-based liver assist devices with influences on blood parameters and the neurological status of treated patients. Nevertheless, clinical improvements also have been achieved by using non-biological support, like charcoal-hemoperfusion and modified hemodialysis. In order to assess the influence of liver support devices on better results in therapy of liver failure, randomized clinical trials have to be performed. Prior to this, artificial liver support systems have to demonstrate their biocompatibility and biosafety for clinical use as well as their efficacy and availability.

Adult↗
PubMed1994

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.

Acetaminophen↗
PubMed1994

Collagen gel immobilisation provides a suitable cell matrix for long term human hepatocyte cultures in hybrid reactors.

An easy to apply culture technique is presented that protects a monolayer configuration of liver cells within an extracellular matrix. The Immobilising Gel (IG)-Technique not only preserves hepatocyte morphology and supports a variety of differentiated cell functions over long term periods, but also offers higher resistance of IG-culture systems against shear forces of fluids in a hybrid reactor device, as compared to other culture techniques. Human hepatocyte cultures in IG-Technique: DNA-normalised levels for the total production of cholinesterase, albumin, urea and lactate remained high throughout the investigational period (50 days). Glutamic-Pyruvic-Transaminase (GPT) release decreased after peak values during early culture adaptation. Electron Microscopic (EM) findings after the shear forces experiment revealed undisturbed subcellular structures and a preserved intercellular morphology, including bile canaliculi and desmosomes. We conclude that the IG-technique is of considerable advantage as compared to other culture systems, especially in the field of dynamic applications, e.g. hybrid reactors for artificial organ development.

Alanine Transaminase↗
PubMed1993

[Surgery of metastases--can surgery of metastases be radical?].

Resection of metastases gains more and more importance in day to day surgical work task. Mainly only liver- and pulmonary metastases can be resected with a curative aim, depending on the primary tumor and the metastasing cascade. According to tumor surgery a R0-resection is warranted. Whether a multimodal approach may improve long-term results by eliminating occult micrometastases should be answered by ongoing studies. Even in palliative situations complete resections of metastasis are preferred and in selected situations a multimodal approach may be chosen.

Bone Neoplasms↗
PubMed1990

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration.

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.

Animals↗
PubMed1989

Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration.

Adult rat hepatocytes cultured in a collagen sandwich system maintained normal morphology and a physiological rate of albumin secretion for at least 42 days. Hepatocytes cultured on a single layer of collagen gel essentially ceased albumin secretion within 1 wk but could recover function with the overlay of a second layer of collagen gel. This culture configuration more closely mimics the hepatocytes' in vivo environment and provides a simple method for their long-term maintenance.

Albumins↗
PubMed1986

Transplantation of cryopreserved hepatocytes or liver cytosol injection in the treatment of acute liver failure in rats.

Intraperitoneally injected cryopreserved hepatocytes or liver cytosol were used for the treatment of acute galactosamine-induced liver failure in rats. Both methods were similarly capable to improve the survival rates as compared to control groups. Furthermore, a new viability test for transplanted hepatocytes, using a nuclearmedical tracer technique, was evaluated in this study. This test demonstrated viability of intraperitoneally transplanted hepatocytes for at least 6 days after operation. Cytosol injection should be preferred to hepatocyte transplantation in the treatment of acute liver failure, avoiding the problems of cell storage and post-transplant immunosuppression.

Acute Disease↗
PubMedDate not supplied

Analysis of laser Doppler flux motion in man: comparison of autoregressive modelling and fast Fourier transformation.

In order to investigate laser Doppler (LD) flux motion in healthy subjects and patients with peripheral arterial occlusive disease (PAOD), spectrum analysis of LD signals is needed. Autoregressive analysis (AR) is presented as an alternative method of power spectrum estimation. This procedure is compared to the commonly used fast Fourier transform algorithm (FFT) by describing the analytical power of both spectra in the analysis of flux motion waves. LD signals were recorded from the forefoot of 8 healthy volunteers and 11 patients with different degrees of PAOD. The flux, concentration of moving blood cells and velocity signal was digitized and stored for off-line analysis. Special software was designed to calculate AR and FFT spectra of the LD signals and to compare the suitability of both methods for the spectral analysis of LD recordings. Additionally, three-dimensional spectrum diagrams were calculated to demonstrate time-dependent flux changes during standardized provocation maneuvers. AR facilitates the determination of frequency and amplitude of flux motion waves as compared to the FFT. Low frequency-large amplitude waves (LF waves) were detected in both groups. High frequency-small amplitude waves (HF waves), which predominantly appear in severe ischemia, were observed in 7 of the 11 patients and in 2 of the 8 controls. The spectra revealed pulsatile waves in all healthy controls, but only in 1 of the 11 patients. AR modelling allows a reliable description of important flux motion components and has considerable advantages in spectral estimation of LD signals as compared to the FFT.

Algorithms↗
PubMedDate not supplied

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

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 x 10(7) viable hepatocytes per gram of tissue, and an overall yield of 1.92 +/- 0.5 x 10(9) 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 micrograms) and low values of alanine aminotransferase release (8 microU/hr/micrograms DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 micrograms/hr/micrograms DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7-ethoxycoumarin (4.5 nmol/hr/micrograms 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.

7-Alkoxycoumarin O-Dealkylase↗