Hybrid liver support systems (LSS) for the use of the detoxifying, metabolic synthetic and regulatory capabilities of liver cells are under development for extracorporeal therapy of acute liver failure and for bridging to liver transplantation. A summary of our development is discussed. A five-step technique for primary liver cell isolation has been introduced in order to address larger scale procurement of hepatocytes. Immobilisation of the cells after isolation appears to be one of the main factors in maintaining hepatocyte function in vitro. Different techniques have been investigated. Using the cell-cell adhesion technique, a culture model was developed for the immobilisation of hepatocytes between capillary membranes. Four separate capillary membrane systems, each forming independent compartments are woven in order to create a three dimensional network. A bioreactor design has been developed. The construction provides different functions, including decentralised cell perfusion. The bioreactor enables 3 dimensional reorganisation of cells, integral oxygenation and decentralised metabolite exchange. The bioreactor has been scaled-up to allow hepatocytes and sinusoidal endothelial cells to be cultured in quantities sufficient for therapeutic application. In a healthy pig model, possible limiting side effects of therapy with this device were excluded. The efficacy of the system has been demonstrated in a hepatectomised pig model. Subsequently, a complete hybrid liver support system for human studies was introduced and applied clinically.
Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy. Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery. Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells. The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density. Treatment started after complete recovery from anesthesia and was performed continuously. A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group. Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.
Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.
A comparison of nonenzymatic and enzymatic hepatocyte isolation was performed on pig livers. The collagenase perfusion showed superior results: mean viability 72 +/- 10% versus a maximum viability of 21% using EDTA-perfusion. A five-step collagenase perfusion technique, developed for pig livers enables larger scale investigations, in order to develop methods for hepatocyte cultures in therapeutical liver cell perfusion systems.
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