Entwicklung und Funktionsprüfung eines oxygenierenden Hohlfaserreaktors zur Kultivierung von Leberzellen

Die Kultivierung differenzierter hepatischer Zellen gilt allgemein als schwierig, ist aber vielseitig für pharmakologische Zwecke, als bioartifizielle Leber oder zur Produktion von Gerinnungsfaktoren oder Serumproteinen einsetzbar. Darum wurde in dieser Arbeit ein Kultivierungssystem entwickelt, das den hohen Anforderungen der Erhaltung und Expansion von Leberzellen entspricht. Der dabei entwickelte oxygenierende Hohlfaserbioreaktor (Oxy-HFB) bietet eine vom Mediumfluss unabhängige Versorgung mit Sauerstoff über die Trägerstruktur und vermeidet durch einen homogenen Aufbau bei Abständen von etwa 200 µm zwischen den Hohlfasern lange Diffusionsstrecken. Die Zellen werden im extrakapillaren Raum kultiviert und kommen in direkten Kontakt mit dem Medium. Der Oxy-HFB wurde hinsichtlich des Verweilzeitverhaltens, der Mischzeit und der Sauerstoffversorgung charakterisiert. Anschließend wurden primäre porcine Leberzellen unter Variation des zur Verfügung stehenden Mediumvolumens, der Zelldichte und des Mediumflusses kultiviert. Dabei zeigten die Zellkulturen nicht nur ein stabiles Verhalten des Primärmetabolismus, sondern auch der Biotransformationsleistung über den untersuchten Zeitraum von zwei Wochen. Die Experimente zeigten, dass der Oxy-HFB zur Zeit für viele Anwendungen im Bereich der Leberzellkultivierung besser geeignet ist als andere veröffentlichte Kultivierungssysteme: Der Oxy-HFB erlaubt eine Vielzahl von Variationen im Versuchsaufbau und ist daher für die Untersuchung von zellulären oder pharmakologischen Mechanismen gut geeignet.The cultivation of differentiated hepatic cells is a difficult task, as is international well-known. However, they are of great value for pharmaceutical screening, as a bioartificial liver or for production of clotting factors or serum proteins. Therefore, a cultivation system with an optimal cell environment and a high flexibility in experimental design was developed in this project. The constructed oxygenating hollow fiber bioreactor (Oxy-HFB) offers oxygenation independent from medium supply and an extremely simple design consisting of only one fiber system for the oxygenation. The low distance of 200 µm between the hollow fibers prevents the appearance of diffusion barriers and limitations of substrates or oxygen for the cells. The cells are cultivated in the extrafibrous space and are therefore in direct contact to the medium. The system was characterized concerning residence time distribution, mixing time and oxygen supply. To prove the suitability for the cultivation of primary cells porcine hepatocytes were cultivated under variation of cell density, medium flow rate and medium volume. The cells showed not only a stable primary metabolism but also a stable phase I and II metabolism over the cultivation time of two weeks under variation of the cell density. The experiments showed that the bioreactor is highly suitable for cultivations of primary hepatocytes and superior to other published systems. The design of the bioreactor allows lots of variations in the experimental design and is therefore predestined for investigations on cellular and pharmacologically mechanisms

Untersuchungen zur digitalen Erfassung der Tieraktivität und der Haltungsumwelt im Kontext der Tierwohlbewertung von Mastschweinen

Die Gesundheit der Nutztiere, deren Lebensbedingungen und ihr Wohlergehen während der Aufzucht und Haltung gewinnen politisch wie auch gesellschaftlich immer mehr an Bedeutung und werden zwingende Qualitätsmerkmale tierischer Produkte für die Verbraucher/-innen. Das Tierwohl, die Tiergesundheit und ihre Haltungsbedingungen zu überprüfen und zu dokumentieren, erfordert jedoch bei steigenden Tierzahlen pro Betrieb bzw. pro Tierhalter/-in technische Hilfsmittel zur Umsetzung. Ziel dieser Dissertation war es, zwei solcher technischen Hilfsmittel vorzustellen und zu validieren. Diese dienen zum einen der digitalen Erfassung der Tieraktivität und zum anderen der Messung der Ammoniakkonzentration in der Haltungsumwelt von Mastschweinen. Sie sind Teil der Tierwohlbewertung im Projekt und im daraus entstandenen Prototypen des Beratungstools PigsAndMore. Studie 1 erläutert den Aufbau einer Gasmischanlage zur präzisen Dosierung von Gasen bei unterschiedlichen Luftfeuchtigkeiten auf Laborebene. Mit dieser Anlage erfolgte die Validierung zweier Gerätetypen zur Messung von Ammoniak. So konnte auf Laborebene kein signifikanter Einfluss der relativen Luftfeuchtigkeit auf die Messgenauigkeit der untersuchten Messgeräte nachgewiesen werden. Aus diesen und weiteren Ergebnissen ließen sich Schlussfolgerungen für den praktischen Einsatz der Messgeräte im Stall formulieren. Studie 2 befasst sich mit den Einsatzmöglichkeiten passiver Infrarotdetektoren zur Erfassung der Aktivität einer Gruppe von Mastschweinen innerhalb einer Bucht sowie in bestimmten Fokusbereichen wie dem Trog- oder dem Beschäftigungsbereich. Im Vergleich zu einer visuellen Bewertung der Tieraktivität als Referenzmethode zu den Daten der Detektoren konnten signifikante Korrelationen mit Werten von bis zu 0,87 nachgewiesen werden. Die Ergebnisse der Studie sowie Ergebnisse der Vorversuche, die im Konferenzbeitrag beschrieben werden, legten die Basis für die Nutzung der passiven Infrarotdetektoren im Rahmen einer sogenannten Screeningphase in PigsAndMore. Diese Dissertation liefert wichtige Erkenntnisse zur Validierung von Ammoniaksensoren und passiven Infrarotdetektoren zur Messung der Tieraktivität für den Einsatz in Schweineställen. Auf Basis der gewonnenen Informationen werden Lösungsansätze für ermittelte Probleme vorgestellt und weitere Anwendungsmöglichkeiten im praktischen Einsatz diskutiert. Darüber hinaus werden Möglichkeiten der Nutzung und Weiterentwicklung dieser wie auch anderer technischer Hilfsmittel im Rahmen einer vielschichtigen, digital unterstützten Tierwohlbewertung analysiert.The health of farm animals, their living conditions and their welfare during rearing and husbandry are becoming increasingly important, both politically and socially. They develop into indispensable quality characteristics of animal products for the consumer. However, the monitoring and documentation of animal welfare, health and their housing conditions requires technical tools for implementation as the number of animals per farm or per livestock owner increases. The aim of this dissertation was to present and validate two such technical tools. These are used for the digital recording of animal activity and for the measurement of ammonia concentration in the housing environment of fattening pigs. They are part of the animal welfare assessment in the project and the resulting prototype of the consulting tool PigsAndMore. Study 1 explains the development of a gas calibration unit for the precise dosing of gases at different humidities at the laboratory level. With this unit, the validation of two types of devices for measuring ammonia took place. Thus, at the laboratory level no significant differences in measuring accuracy as a function of relative humidity could be detected for the tested devices. From these and other results subsequent conclusions could be formulated for the practical use of the measuring devices in the barn. Study 2 deals with the possible applications of passive infrared detectors for recording the activity of a group of fattening pigs within a pen as well as in certain focus areas such as the trough or the exploration area. In comparison to a visual assessment of animal activity as a reference method to the detectors‘ data, significant correlations of up to 0.87 could be found. The results of the study, as well as results of preliminary experiments described in the conference paper, laid the foundation for the use of passive infrared detectors in a so-called screening phase in PigsAndMore. This dissertation provides important insights into the validation of ammonia sensors and passive infrared detectors for measuring animal activity for use in pig barns. Based on the information gained, approaches to solving identified problems are presented and further application possibilities in practical use are discussed. Furthermore, possibilities for the use and further development of these as well as other technical tools in the context of a multi-layered, digitally supported animal welfare assessment will be analyzed

Recording group and area-specific activity of fattening pigs by using Passive Infrared Detectors on farm

Animal activity in pigs can be a direct indicator of animal welfare. Passive infrared detectors (PID) provide one method of measuring animal activity on the pen level as a cost-effective and easy-to-use sensor technique. The study aimed to test PIDs on different commercial farms with fattening pigs. On each farm, a focus pen was selected and group activity, and activity in the feeding and exploration area was measured by using three PIDs. For data evaluation, three continuous 24h time periods were selected for each farm. Additionally, animal behavior was recorded by video cameras for visual scan sampling. To compare the PID outcome with the recorded behaviors, an ethogram was used to categorize active and inactive behaviors. Using scan sampling, the validation of the PID data was based on still frames at 10 min intervals. In addition, barn climate such as temperature, relative humidity, and ammonia concentration were measured. The analysis of seven farms showed a strong correlation between PID data and visual assessment for group activity from 0.67 - 0.91 (p < 0.001; n = 432). For the activity in the feeding area, medium to strong correlations between 0.44 - 0.65 (p < 0.001; n = 327) could be found. The PID data for the exploration area reached correlations with a smaller effect strength. Based on the activity data measured by PIDs, a typical diurnal rhythm for pigs could be found for all farms. Moreover, the PID data indicated different activity patterns depending on, e.g., feeding times and sex group composition. The results demonstrated that PIDs can also be used in different housing conditions for measuring animal activity. In combination with barn climate data, the PIDs can provide useful information for the farmer and also characterize farm-specific management

Origin and significance of clay‐coated fractures in mudrock fragments of the SAFOD borehole (Parkfield, California)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95148/1/grl21674.pd

Three-dimensional Numerical Modeling and Computational Fluid Dynamics Simulations to Analyze and Improve Oxygen Availability in the AMC Bioartificial Liver

A numerical model to investigate fluid flow and oxygen (O(2)) transport and consumption in the AMC-Bioartificial Liver (AMC-BAL) was developed and applied to two representative micro models of the AMC-BAL with two different gas capillary patterns, each combined with two proposed hepatocyte distributions. Parameter studies were performed on each configuration to gain insight in fluid flow, shear stress distribution and oxygen availability in the AMC-BAL. We assessed the function of the internal oxygenator, the effect of changes in hepatocyte oxygen consumption parameters in time and the effect of the change from an experimental to a clinical setting. In addition, different methodologies were studied to improve cellular oxygen availability, i.e. external oxygenation of culture medium, culture medium flow rate, culture gas oxygen content (pO(2)) and the number of oxygenation capillaries. Standard operating conditions did not adequately provide all hepatocytes in the AMC-BAL with sufficient oxygen to maintain O(2) consumption at minimally 90% of maximal uptake rate. Cellular oxygen availability was optimized by increasing the number of gas capillaries and pO(2) of the oxygenation gas by a factor two. Pressure drop over the AMC-BAL and maximal shear stresses were low and not considered to be harmful. This information can be used to increase cellular efficiency and may ultimately lead to a more productive AMC-BAL

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs) - integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications

Novel in vitro and mathematical models for the prediction of chemical toxicity

The focus of much scientific and medical research is directed towards understanding the disease process and defining therapeutic intervention strategies. The scientific basis of drug safety is very complex and currently remains poorly understood, despite the fact that adverse drug reactions (ADRs) are a major health concern and a serious impediment to development of new medicines. Toxicity issues account for ∼21% drug attrition during drug development and safety testing strategies require considerable animal use. Mechanistic relationships between drug plasma levels and molecular/cellular events that culminate in whole organ toxicity underpins development of novel safety assessment strategies. Current in vitro test systems are poorly predictive of toxicity of chemicals entering the systemic circulation, particularly to the liver. Such systems fall short because of (1) the physiological gap between cells currently used and human hepatocytes existing in their native state, (2) the lack of physiological integration with other cells/systems within organs, required to amplify the initial toxicological lesion into overt toxicity, (3) the inability to assess how low level cell damage induced by chemicals may develop into overt organ toxicity in a minority of patients, (4) lack of consideration of systemic effects. Reproduction of centrilobular and periportal hepatocyte phenotypes in in vitro culture is crucial for sensitive detection of cellular stress. Hepatocyte metabolism/phenotype is dependent on cell position along the liver lobule, with corresponding differences in exposure to substrate, oxygen and hormone gradients. Application of bioartificial liver (BAL) technology can encompass in vitro predictive toxicity testing with enhanced sensitivity and improved mechanistic understanding. Combining this technology with mechanistic mathematical models describing intracellular metabolism, fluid-flow, substrate, hormone and nutrient distribution provides the opportunity to design the BAL specifically to mimic the in vivo scenario. Such mathematical models enable theoretical hypothesis testing, will inform the design of in vitro experiments, and will enable both refinement and reduction of in vivo animal trials. In this way, development of novel mathematical modelling tools will help to focus and direct in vitro and in vivo research, and can be used as a framework for other areas of drug safety science