J. Vienken

COMPARISON OF HOLLOW FIBRE MEMBRANES FOR HEPATOCYTE IMMOBILISATION IN BIOREACTORS

Various hollow fibre membranes of polyamide, cellulose and polypropylene were investigated as potential substrata for hepatocyte immobilisation in bioreactors for hybrid liver support systems. Membranes were subjected to a cytocompatibility test in which the attachment and morphology of primary hepatocytes were evaluated. The effect of coating with collagen and fibronectin was also studied. Adequate cell immobilisation was possible on polypropylene and polyamide membranes even without coating. The flattening process of the cells was dependent on the material and the coating. The incorporation of porous polypropylene and polyamide hollow fibres in hybrid liver cell bioreactors and their specific permeability properties could also offer means for cell oxygenation, metabolite distribution and immuno-isolation purposes.

Biocompatibility of membranes used in the treatment of renal failure

Haemodialysis membranes with a wide range of solute and hydraulic permeabilities are used clinically. Such membranes are manufactured from either cellulose or synthetic co-polymers and their biocompatibility is commonly characterized by the complement activation and white cell changes observed during their use. The cellobiosic unit may be modified by the partial or total replacement of the hydroxyl groups by diethylaminoethyl (Hemophan), acetate (cellulose acetate), triacetate (cellulose triacetate) or 2,5-acetate (Diaphan). We have undertaken a prospective study in which such renal membranes have been studied in terms of the complement activation and neutropenia produced with the aim of investigating the relationship between modification of the cellobiosic unit and the magnitude of neutropenia and complement activation, and the extent to which membrane base material influences these parameters, by comparing the changes observed in modified cellulose membranes with that for a synthetic membrane (polysulphone). Our findings show that, while the degree of substitution varies between < 1% and total substitution, there is no correlation between the numbers of hydroxyl groups replaced and alteration of complement activation and neutropenia. However, by modification of the cellobiosic unit it is possible to produce a membrane whose biocompatibility is similar to that of a membrane manufactured from a synthetic co-polymer such as polysulphone.

Determination of contact phase activation by the measurement of the activity of supernatant and membrane surface‐adsorbed factor XII (FXII): Its relevance as a useful parameter for the in vitro assessment of haemodialysis membranes

We investigated hemodialysis membrane biocompatibility with respect to contact phase activation by determination of FXII-like activity (FXIIA) on the membrane surface and in the supernatant phase, during plasma contact with various hemodialysis membranes using an in vitro incubation test cell. The results were compared to the influence of these membranes on the activation of purified FXII. A time course for the generation of activated FXII using purified FXII solution at physiologic concentrations on two similar negatively charged polymers was performed. The membranes assessed were regenerated cellulose (Cuprophan; Akzo Faser AG, Germany), modified cellulosic (Hemophan; Akzo Faser AG), acrylonitrile-sodium methallyl copolymer-based membrane AN69S (Hospal, France), and SPAN, a new polyacrylonitrile-based copolymer (akzo Nobel AG). The plasma FXIIA at the membranes surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, activation of purified FXII in a plasma-free system with respect to supernatant activity indicated significant differences between the materials. A similar finding for the membrane-bound factor XIIA was also observed when purified factor XII was used. The membrane-bound FXIIA values observed in the plasma system containing heparin were significantly greater than in citrated plasma. This demonstrated the strong influence of heparin and the interaction of other plasma components to the membrane surface on the activation of contact phase of coagulation.

Cellulose-Ester as Membrane Materials for Hemodialysis

The majority of dialysis membranes are fabricated from regenerated unmodified cellulose. This standard type of cellulosic membrane is frequently under attack because of its alleged lack of biocompatibility. Recent developments, however, have proven that a chemical modification of the reactive surface groups of regenerated cellulose, the hydroxylgroups, limits the complement-activating potential of these materials and thus improves its blood-compatibility. We extended the idea of modifying cellulose for improved blood-compatibility to a series of different cellulose esters. Special focus was directed towards the question whether a variation of the type of substituent and degree of substitution could influence the blood-compatibility pattern of these materials: the analysis of blood-compatibility profiles showed a direct dependency on the type of substituent and the degree of substitution (DS). As an example, it was found that the DS, necessary for a complete reduction of complement activation, decreases with increasing chain lengths of aliphatic substituents. Optimal degrees of substitution are characteristic of the type of substituents and enable us to tailor materials specifically for optimized blood compatibility.

In vitro contact phase activation with haemodialysis membranes : role of pharmaceutical agents

Contact phase activation was investigated in vitro using flat sheet type of haemodialysis membranes, Cuprophan (Akzo, Faser, Germany) and AN69S (Hospal, France), and a negatively charged polyamide Ultipor NR 14225 membrane as a control. The investigation focussed on the determination of factor XII-like activity (FXIIA) as an indicator of contact phase activation in the supernatant phase and at the membrane surface after plasma-membrane contact using an incubation test cell. The findings were compared with the observations from a plasma-free system utilizing purified unactivated factor XII. The plasma FXIIA bound to the membrane surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, the plasma-free system exhibited significant differences in the supernatant FXIIA and membrane-bound FXIIA for all the materials used and the magnitude of the activity was significantly greater for negatively charged materials. This finding demonstrated the strong influence of the interaction of other plasma constituents on the membrane surface and as such the binding and subsequent activation of factor XII may be altered possibly due to competitive binding and steric hindrance. On the addition of anticoagulants such as heparin, low-molecular-weight heparin, citrate and hirudin, no significant differences were observed in plasma supernatant phase FXIIA. However, each anticoagulant appears to have a distinct influence on the magnitude of plasma membrane-bound FXIIA. On the addition of aprotinin (a kallikrein inhibitor), no significant differences were observed in the plasma supernatant FXIIA. In contrast, aprotinin appears to significantly reduce membrane-bound FXIIA on Cuprophan and polyamide NR, but significantly increase the magnitude of the membrane-bound FXIIA on AN69S.

Ex vivo complement protein adsorption on positively and negatively charged cellulose dialyser membranes

An ex vivo test system was used to measure complement protein C3 and factor B adsorption onto small dialyser modules made from regenerated and modified cellulosic hollow fibre membranes in which positive diethylaminoethyl (DEAE) or negative carboxymethyl (CM) groups were introduced into the cellulose matrix. The extracorporeal system, which included test-dialysers and the dialysis environment, allowed the use of labelled proteins without contaminating the blood donors which were connected in an open-loop fashion to the extracorporeal test system. The modules were removed at selected time points from the extracorporeal system for radioactivity counting. The results were used to evaluate the mechanisms involved in complement reactions to foreign surfaces. The system therefore allowed the analysis of complement protein adsorption occurring in the dialyser modules and its relationship to the complement generation rate in the extracorporeal system to be evaluated. It was possible to demonstrate that significant complement C3 and factor B adsorption occurred in the test modules made of cellulosic membranes. Complement adsorption as a function of the pH and the release reaction of the adsorbed C3 and factor B after membrane blood perfusion were therefore found to be variable according to the cellulosic membrane type and the presence of positive or negative charged groups within the cellulose matrix. The data obtained from the ex vivo model therefore provided additional evidence on the discussion of the mechanisms involved in the increased complement activation by regenerated cellulose and in its attenuation by DEAE- or CM-modified cellulose.

Membranes and polymer structures—Biocompatibility aspects with respect to production limits☆

Plasmapheresis can be performed by centrifugation and by use of membrane technology. With the latter technique we receive a plasma which is absolutely free from platelets. This is why membranes are gaining market shares in this particular field of medical application. Today plasmapheresis membranes are mostly fabricated from synthetic polymers, such as polypropylene (e.g. PLASMAPHAN), polysulfone, polyacrylonitrile, polymethylmethacrylate, polyvinylalcohol and others, the only exception being cellulose acetate. Parameters determining the biocompatibility of plasmapheresis membranes are generation of complement C3a or C5a, hemolysis and possible thrombus formation. These parameters depend on various properties of the membrane polymer: e.g. the nature of the molecular end/side-groups, the distribution of electrical charges on the polymer surface and the different chemical structures and conformation of the polymer. In addition, membrane properties like pore distribution and geometry or the flow characteristics of a particular device-design may trigger cell activation or influence biocompatibility through the adsorption of various plasmacomponents. Most of the polymers which are used today for manufacturing plasmapheresis membranes have not been developed for this purpose. They were originally selected to be used as textile fibers. Further, no present membrane polymer has been specifically developed to achieve high biocompatibility. The membrane profile was designed in such a way that pheresis properties were met rather than optimizing biochemical blood/polymer interactions. One reason for this decision may be that the market volume of plasmapheresis technology is too small in order to justify specific and high-cost developments of polymers for this purpose. Polymer selection to achieve excellent biocompatibility profiles is determined by polymer-availability, costs, membrane-forming processes and environmental aspects related to possible pollution during the manufacturing process. The production of PLASMAPHAN by the unique Accurel-process combines several of these parameters. The main membrane production processes and especially the Accurel-process are described here. The influence of polymer-surface properties, membrane structure and module-design on the biocompatibility of plasmapheresis treatments are discussed and explained by appropriate examples.

Variables associated with the assessment of systemic tumor necrosis factor alpha levels during hemodialysis.

Conflicting results have been published concerning the systemic induction of the cytokine tumor necrosis factor alpha (TNFα) during hemodialysis (HD). We therefore evaluated in vitro TNFα production in whole blood as well as in vivo variability of TNFα levels in patients on long-term HD. Whole blood was incubated at room temperature (RT) with or without exogenously added endotoxin (ET), and plasma-TNFα was measured after 5, 30, 120, 240, and 960 min by specific enzyme immunoassay. Additionally, plasma-TNFα before and after 120 and 240 min HD was studied longitudinally once a week over a period of 4 weeks in 36 patients on Cuprophan® (CU, n=23) or polysulfone-F60 (PSu, n=13) HD. Mean plasma TNFα levels in vitro rose from (mean) 8 pg/ml after 5 min to 12 pg/ml (120′) and 32 pg/ml (960′) even without ET addition, and to 18 pg/ml (after 120′) and 88 pg/ml (after 960′) when 0.1 μg/ml ET were added. Pre-dialytic as well as intradialytic TNFα levels in patients showed high intra-individual variability. A substantial (> 100%) increase in plasma TNFα was observed during only 14 out of 84 treatments with CU and 20 out of 47 with PSu, however, the increase in TNFα was not statistically significant in either group. We conclude that the sampling procedure, if not carefully standardized, is a potential source of artifacts with regard to “systemic” TNFα levels. The high intra and inter-individual variability of plasma TNFα suggests that results of cross-sectional studies are questionable.