Karl-Friedrich Weibezahn

Microstructured scaffolds for liver tissue cultures of high cell density: Morphological and biochemical characterization of tissue aggregates

Very high cell densities and optimal vascularization characterize among others organs and tissues in vivo. In order to study organ‐specific functions in vitro or to make use of them in medical devices/treatments in the future, this natural architecture should be rebuilt. An important aspect in this context is the appropriate ratio of medium to cell volume being so far not optimally reestablished in most of the currently available in vitro systems. To improve such culture conditions, we constructed a microstructure to culture hepatocytes and (without any addition of extracellular matrix material) characterized liver tissue in the form of evenly sized aggregates. The liver‐specific differentiation status of such aggregates was monitored by their ability to perform CYP450 dependent xenobiotic metabolism along with the measurement of albumin secretion. Freshly isolated adult rat hepatocytes show an initial loss of total CYP450 content and of associated activities (mixed function oxidases). However, in the aggregate system, this level did not decrease further but remained stable or even increased throughout the culture period of 10–13 days. The CYP450 dependent metabolism of the hepatocytes is able to respond to classic inducing agents. The described culture efficiently supports liver‐specific functions of adult rat hepatocytes and seems to be suited not only for use in an extracorporeal liver device but also for the formation of evenly sized small aggregates to be of use in transplantation of differentiated liver tissue. Moreover, after design variations, the microstructure can be applied for functional analysis of metabolically active hepatocytes as well as for toxicological and pharmacological validation.

Microbioreactor design for 3-D cell cultivation to create a pharmacological screening system

A biocompatible cell culture environment that enables continued existence of three dimensionally aggregated cells in a polycarbonate‐based scaffold structure was developed. A micro structured polymeric scaffold allows perfusion of cells due to a microporous structure generated by ion track etching and micro thermoforming. Biocompatibility and sterilizability was approved for the whole system. As oxygenation and mass transport within a closed system is most relevant for 3‐D cell culture, two approaches of pumping systems were tested. The human hepatocarcinoma cell line HepG2 was used to examine basic cytological parameters in response to the enviroment. Our data indicate that an actively perfused 3‐D cell culture induces a more differentiated phenotype in HepG2 cells than the 2‐D setup. Thus, our results provide further support to the theory that 3‐D‐cultivated cells display a non‐proliferative behavior. In this respect, 3‐D cultures resemble in vivo conditions more closely. Microreactors are widely applied for organic syntheses, but can also be used for screening applications in drug discovery and medical research. The bioreactor versions presented here were equipped with active fluidic components.

Chip-based three-dimensional cell culture in perfused micro-bioreactors.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermo-forming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 micron (h x w x l) or round recesses with diameters of 300 micron and a depth of 300 micron (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed sterile circulation loop that, in the simplest configuration, is additionally comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

Effects of Interferential Current in Cellular Systems

Electrotherapy with so-called Interferential Current (IFC) represents a well established type of medical treatment of pain, chronic inflammations and various other diseases (1). IFC is a modulated wave form generated by superposition of 2 alternating currents with slightly different frequencies usually close to 4000 Hz. It is applied via skin electrodes and can readily penetrate into the body. Some of the beneficial effects of IFC are based on direct stimulation of excitable cells. Until now there is not enough knowledge about possibly diverse effects of IFC upon the various types of non-excitable cells in the body.

Bildverarbeitung für ein motorisiertes Lichtmikroskop zur automatischen Lymphozytenidentifikation

Zur Diagnose potentiell maligner Erkrankungen konnen die Grosenverhaltnisse von Lymphozyten aus arteriellem und venosem Blut herangezogen werden. Die Bestimmung dieser Grosen wird bisher manuell durchgefuhrt. Es wird ein Detektionssystem weiterentwickelt, das die Selektion der Lymphozyten und ihre Auswertung objektiv gewahrleisten soll. Dafur wird ein motorisiertes Lichtmikroskop zusammen mit einer CCD-Kamera benutzt. Bei 20facher Vergroserung wird in einem Blutausstrich zunachst nach angefarbten Zellen gesucht, die danach mit 100facher Vergroserung angefahren und genau vermessen werden. Zuletzt werden diese Zellen in Lymphozyten und NichtLymphozyten klassifiziert.

INTERFERENTIAL ELECTRIC FIELDS REVEAL NONUNIFORM EFFECTS ON PROLIFERATION AND hCG SECRETION IN JAr CELLS

The effect of low frequency electromagnetic fields on changes in intracellular cAMP concentrations was investigated in the frequency range 10–100 Hz using a choriocarcinoma cell line. JAr cells significantly reduce proliferation and increase β-hCG secretion upon dibutyryl cAMP and forskolin treatment after 10 days of culturing. Choriocarcinoma cells exposed to a modulation frequency of 10 Hz for 5 min change their intracellular cAMP level significantly to higher as well as to lower concentrations in half of the experimental series, respectively. At frequencies of 70 and 100 Hz levels, half of the experimental series revealed a significant decrease in cAMP levels. Long term exposure at 100 Hz for 10 days leads to a significant reduction in proliferation but not in β-hCG secretion. These results point to a modulatory effect of low frequency electromagnetic fields on intracellular cAMP levels which are dependent on the frequency window. The reduced proliferation after long term exposure at the frequency of 100 Hz, which lowers cAMP levels, is discussed.

Polymeric Tissue Culture Substrates patterned by UV Irradiation

We studied the physico/chemical effects of deep UV irradiation of polystyrene, PMMA and polycarbonate with respect to cell adhesion and protein immobilization. Photochemical modifications of the polymer surfaces yielded unstable peroxides and carboxylic acid groups. Patterned protein adsorbates were realized by coupling via carbodiimid activation of the COOH moieties. We have immobilized enzymes and antibodies. It was observed that hepatoma cells (HepG2) and fibroblasts (L929) adhered in the presence of serum proteins in the culture medium on the irradiated regions of the substrate without any further treatment.