Development of polymer membranes with improved haemocompatibility for biohybrid organ technology.

Biomedical technology has opened up possibilities of treating the failure of internal organs like kidney and liver by artificial organ therapy. Most of these techniques are based on polymer membranes, which allow the removal of excess of water, salts and toxins from the circulation. However, haemodialysis for the replacement of kidney function results in an increased morbidity and mortality of patients after long-term application. Conventional therapy, such as haemofiltration for the treatment of acute liver failure does not improve significantly the survival rate of patients. Biohybrid organ support as a combination of the artificial organ therapy with the functional activity of immobilised cells seems to be a solution of the problem. Membranes applied in these devices have to face both tissue cells and blood. Organ cells in biohybrid organs have to make intimate contact with the surface of membrane but must also develop close cell-cell-connections as a prerequisite for their survival and high functional activity. Blood to be detoxified will contact the other side of membrane and may not become activated by the synthetic material. New polymer membranes based on acrylonitrile were developed to address these requirements by tailoring the composition of copolymers and to be applied in a specific hollow fibre bioreactor with an outer fibre for blood contact, and an inner fibre for tissue contact or vice versa.

Morphological studies on the culture of kidney epithelial cells in a fiber-in-fiber bioreactor design with hollow fiber membranes.

A hollow fiber-in-fiber-based bioreactor system was tested for the applicability to host kidney epithelial cells as a model system for a bioartificial kidney. Hollow fibers were prepared from polyacrylonitrile (PAN), polysulfone-polyvinylpyrollidinone (PVP) blend (PSU) and poly(acrylonitrile-N-vinylpyrollidinone) copolymer P(AN-NVP). Hollow fibers with smaller and larger diameters were prepared so that the smaller fitted into the larger, with a distance of 50-100 microm in between. The following material combinations as outer and inner fiber were applied: PAN-PAN; PSU-PSU, PSU-P(AN-NVP). Madin-Darby kidney epithelial cells (MDCK) were seeded in the interfiber space and cultured for a period up to 14 days. Light, scanning, and transmission electron microscopy were used to follow the adhesion and growth of cells, and to characterize their morphology. As a result, we found that MDCK cells were able to grow in the interfiber space in mono- and multilayers without signs of systemic degeneration. Comparison of the different materials showed that PAN and P(AN-NVP) provided the best growth conditions, indicated by a tight attachment of cells on hollow fiber membrane, and subsequent proliferation and development of structural elements of normal epithelia, such as tight junctions and microvilli. In conclusion, the fiber-in-fiber design seems to be an interesting system for the construction of a bioartificial kidney.

The role of surface wettability on hepatocyte adhesive interactions and function.

In this paper the effect of surface wettability on hepatocyte morphology and function was studied, using clean and octadecylsylane (ODS)-coated glass as a model for hydrophilic and hydrophobic surfaces, respectively. C3A cells--a hepatoblastoma cell line, and freshly obtained porcine hepatocytes were cultured for a short-time period of up to 4 days on the above substrata. Hepatocyte adhesive interactions were characterized monitoring the initial cell attachment, the overall cell morphology, the formation of focal adhesions, and actin filaments. Since hepatocytes showed a clear tendency for homotypic adhesion on ODS, specific E-cadherin staining was used to visualize the intercellular contacts by immunofluorescence microscopy. Additionally, functional assays were carried out to monitor proliferation, metabolic activity, and albumin synthesis of C3A cells. It could be shown that both C3A cells and normal porcine hepatocytes spread better on hydrophilic glass; spreading being accompanied by the development of pronounced actin stress fibers and focal adhesion contacts. In contrast, on hydrophobic substrata predominant cell-cell interactions took place which led to intense E-cadherin staining in the intercellular contacts of porcine hepatocytes but not in C3A cells. On the other hand, metabolic activity and growth of C3A cells were reduced on hydrophobic ODS, but albumin synthesis was similar on both surfaces. It was concluded that the wettability of materials has a strong influence on the attachment and morphology of hepatocytes while the influence of surface properties on the functional activity of hepatocytes still remains to be elucidated.

Development of membranes for the cultivation of kidney epithelial cells.

The development of biohybrid organs (BHO) will benefit from improved membranes regarding transport and cell contacting properties. Here we describe in a first study the development and testing of membranes made of polyacrylonitrile (PAN) and polysulfone (PSU) for the immobilisation of kidney epithelial cells. Comparative investigations on overall polymer toxicity tested with 3T3 fibroblasts, and morphology and proliferation of Madin-Darby canine kidney (MDCK) cells cultured on the membranes could show that these materials have comparable cell contacting properties like Millicell membranes. Since PAN and PSU have superior membrane forming properties with regard to membrane geometry, i.e. for the preparation of hollow fibres, and porosity, i.e. for immuno isolation, both materials or modifications thereof seem to be suitable for the application in BHO such as biohybrid kidney.

Functionality of MDCK kidney tubular cells on flat polymer membranes for biohybrid kidney.

The prerequisite for the development of a biohybrid artificial kidney, is a substrate for confluent growth of renal cells forming an epithelial monolayer without any leaks. Conventional cell culture supports cannot be adapted for this purpose, because they lack adequate mechanical properties and thermal stability. From two suitable materials, polysulfone and polyacrylonitrile, two permeable polymeric membranes have been produced that were, according to ISO 10993-5, not cytotoxic. Cloned Madin Darby Canine Kidney (MDCK) cells (an established renal cell line) were cultured on the surface of the plastic materials, and on conventional cell culture supports. With all materials, assays of mitochondrial and lactate dyhydrogenases exhibited similar proliferation and the viability of the MDCK cells. Transmission electron microscopy showed the expression of a normal morphology of kidney tubular cells. Perfect barrier function, consequent on the formation of intercellular junctions in a confluent tight epithelium, was visualized in electron micrographs, and quantified by measurement of the transepithelial resistance. The uniformity of the cells grown was demonstrated in samples by electron microscopy and in the whole epithelium by intravital impedance analysis. It was concluded that polymeric membranes produced from polysulfone or polyacrylonitrile are appropriate substrates in the design of biohybrid kidney devices.