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Many factors can affect the characterisation of biomaterials during testing. Among them is the type of the testing solution, such as human, animal blood or saline solutions of even different composition. Further influencing factors are drugs that have been administered to the blood donor prior to testing. Experimental details affecting the quality of testing refer to the guidance of blood across the biomaterial leading to different shear stress on blood cells and specific blood donor conditions, such as the age, race and gender. Some of the misconceptions in testing are described here and serve to indicate that a systems approach, and not only individual test parameters, is best when reliably testing for the biocompatibility of biomaterials for further use in medical devices.

Initial results of the clinical use of primary porcine liver cells for extracorporeal liver support are being reviewed as the cell source is controversial. According to Eurotransplant data 20 -25% of explanted donor livers are not transplanted, due to factors such as steatosis or cirrhosis. This number corresponds to the number of patients with acute liver failure who require bridging therapy to transplantation. Primary human liver cells from transplant discards can be isolated, purified and maintained in bioreactors and provide an alternative for cell-based extracorporeal liver support therapy. A four-compartment bioreactor enables recovery from preservation and isolation injury in a three-dimensional network of interwoven capillary membranes with integrated oxygenation, rendering the liver cells from these discarded donor organs viable for clinical utilization. Patient contact with additional animal-derived biomatrix and fetal calf serum can be avoided. The initiation of an in vitro cultivation phase allows cell stabilization, quality control, and immediate availability of a characterized system without cryopreservation. The hypothesis of this paper is that with appropriate logistics and four-compartment bioreactor technology, cells from human liver transplant discards can serve the demand for cell-based therapy, including extracorporeal liver support.

Polymers employed as biomaterials in nephrology serve for different applications: they form membranes for dialysis and plasmapheresis, are used as materials for dialyser housings and as a potting mass for capillary membranes, they make up tubing-systems for extracorporeal circuits and – in the form of beads – act as parts of adsorber columns for hemoperfusion or immunoadsorption.However, generally speaking, many polymers have not yet been designed for their final application. To date, many polymers are still taken from the chemist's shelf according to their alleged performance properties or to their sterilisability.When used in medical application, polymers must show a high purity. Uncontrolled leaching of oligomers from the polymer backbone or of additives from or during the manufacturing process must be avoided. Blood and other body fluids are extremely effective in extracting any loosely bound polymers. During long-term application, e.g. in patients suffering from chronic diseases, these effects may lead to an accumulation of these compounds in circulating blood, tissue, or joints. Consequently, polymers should show an excellent biostability and not degrade during their ageing process. The amount of extractable material should be kept low in order to avoid inflammatory reactions. Polymers must have high blood compatibility in terms of minimized cell-and complement activation.Polymers for medical application should at best be able to stand high temperatures in order to survive steam sterilisation. If this is impossible, their release kinetics for residual quantities of sterilizing agents should be fast.Finally, protein adsorption should appear under controlled conditions, otherwise a reduced performance through protein adsorption will take place. Further, the uncontrolled activation of biochemical cascades, such as the coagulation, complement or contact phase cascade, following blood/material contact must be minimized. A final aspect has been recently made responsible for adverse patients reactions, the interaction between polymers and medicinal drugs. This drug/material interaction must be low, at best zero, apart form those situations, where a controlled drug-release is wanted.The chemical variety of polymers for medical application is large. However, all typical requirements cannot be met by one single polymer. Compromises have to be found between properties and application. Polymer selection for application in nephrology has always to be made under the premise of final application.

Secondary amyloidosis due to beta-2-microglobulin (ß2-m) is a serious long-term complication in patients on regular dialysis therapy. ß2-m can be considered a middle-molecule marker used to facilitate the assessment of dialysis efficacy. For this purpose, a validated model that calculates characteristic efficacy parameters, such as Kt/V, TAC and generation rate, is needed. There is general agreement that ß2-m-kinetics should be described by a two-pool model, but little has been published to validate such an approach.We measured the ß2-m concentration profiles of eight stable patients during hemodialysis (HD) at the start of treatment, after 30 minutes, after 60 minutes, and every hour until the end. Thereafter they were measured at 10-minute intervals for an hour. The dialyser clearances were determined from the plasma concentrations in front of and behind the dialyser twice during each session – after 1 hour, and 4 hours from the start of treatment. The kinetic parameters of a two-pool model (e.g. the compartment volumes V1and V2, the mass transfer coefficient K12and the generation rate G) were determined from the optimal fit of the measured concentration profile.The table below summarises the results by giving the mean and standard deviation for each parameter:V (liters) V1/V2V % TBW K12m(ml/min) G (mg/kg/day)10.0 ± 1.6 4.60 ± 1.8 28.4 ± 3.1 56.3 ± 25.2 2.50 ± 0.66Inter-individual differences in V1/V2and K12were high, ranging from 2.5 to 10.0 for V1/V2and from 26 to 140 for K12. Error analysis suggested that these wide ranges were due to the method and that in reality the probable range of V is 25–36% of TBW, of V1/V23.5–5.3, and of K1230–80 ml/min. With standard values for these three parameters (V = 30% of TBW, V1/V2= 4.4 and K12= 55 ml/m), equal for all patients, and their respective ranges, Kt/V can be calculated with a standard deviation of 13%. Kt/V > 1.2 secures the maximum possible ß2-m removal with three HD treatments a week.Conclusions The parameters of a two-pool model of ß2-m kinetics can be derived from concentration profiles obtained under routine dialysis conditions, but accuracy is not completely satisfactory. Similar to the dialysis dose for urea (Kt/Vurea) the dialysis dose for ß2-m (Kt/Vß2-m) can be calculated from the pre- and post-dialysis concentrations of ß2-m, body weight, ultrafiltration and dialysis time. Kt/Vß2-m> 1.2 secures the maximum possible removal of ß2-m in HD with three sessions per week.

An in vitro set-up has been designed to study the hydraulic permeability of hollow fiber dialysers. Forward and reverse dialysate ultrafiltration were determined using both sterile dialysers and samples with a protein layer settled on the membrane (Fresenius F6, F8, F60 and F80). The ultrafiltration coefficient KUF(ml/h.mmHg) was calculated as the ratio of volumetrical flow (QUF) and transmembrane pressure (TMP) measurements. The protein layer on the membrane was induced either by recirculating human plasma through the dialysers (in vitro) or by a standard hemodialysis session (in vivo).KUFis largely independent of TMP up to 600mmHg (low flux) and 60mmHg (high flux) for forward and reverse flow. In sterile dialysers, backfiltration yields a significantly different KUFexcept for the F80. An in vitro induced protein layer on the membrane decreases KUF15–30% (forward) and 4–12% (backward) in low flux and 45–70% (forward) and 65–73% (backward) in high flux dialysers.

In recent years the percentage of diabetic patients on haemodialysis has increased. Considering the high frequency of intradialytic hypotensive and hypoglycaemic episodes experienced by these patients, it was the aim of the present study to evaluate the influence of different dialysate glucose concentrations (5.5 mmol/L or 11 mmol/L) on blood pressure and glycaemic regulation, using special dialysis equipment - the GENIUS® System.This cross-over, prospective and randomised study, total duration 14 weeks, included 20 diabetic patients on maintenance haemodialysis. Group 1: 9 patients dialysed using dialysate with a glucose concentration of 5.5 mmol/L and after 7 weeks switched to dialysate with a glucose concentration of 11 mmol/L. Group 2: vice versa.Results show a statistically higher number of patients with hypoglycaemic and hypotensive episodes using dialysate with a 5.5 mmol/L glucose concentration. Also, mean serum glucose values were higher during haemodialysis sessions with a glucose dialysate concentration of 11 mmol/L. There were no statistical differences between the groups in laboratory values, HbA1C, insulin doses or in anthropometric parameters.Our results suggest that fewer diabetic patients undergoing haemodialysis using a higher dialysate glucose concentration of 11 mmol/L have hypoglycaemic and hypotensive episodes. Since this dialysate glucose concentration had no influence on lipid or hepatic metabolism, anthropometric parameters and especially HbA1C values in this short-term study, the long term examination of its effects is warranted.

This study investigates genomic damage in peripheral lymphocytes from patients with moderate to severe chronic renal insufficiency and those on long-term maintenance hemodialysis (MHD) and hemodiafiltration therapy. As a measure for genomic damage, the comet assay (single-cell gel electrophoresis) was applied. This test detects single- and double-strand breaks and alkali labile sites through electrophoretic mobility of the resulting fragments. The average damage (percentage of DNA in the tail region of the comet) observed in cells of the control group of 21 healthy subjects was 10.5% [plusmn] 0.8%. There was a significant increase to 14.7% [plusmn] 3.5% in cells of 23 patients with chronic renal failure, and a further increase to 17.1% [plusmn] 3.5% in the subgroup of 12 patients with serum creatinine values greater than 6 mg/dL. Damage was 16.7% [plusmn] 4.2% in cells of the MHD group (26 patients) and 20.1% [plusmn] 3.0% in the subgroup with MHD therapy longer than 10 years (8 patients). Cellular DNA damage in the group of 15 maintenance hemodiafiltration patients was 15.6% [plusmn] 2.1%, ranging between predialysis and MHD patients, and did not seem to increase with treatment time. These results, together with previously observed elevated frequencies of micronuclei, decreased DNA repair, and increased cancer incidence described for these patient groups, emphasize the need to further optimize the current therapy for reducing the degree of genomic damage. [copy ] 2001 by the National Kidney Foundation, Inc.

The accumulation of advanced glycation end-products (AGEs) in end-stage renal disease (ESRD) influenced by dialysis modalities is of current interest. Highly permeable membranes in haemodialysis or haemofiltration should be able to eliminate circulating AGEs as well as their AGE precursors more efficiently.

Summary Protoplasts ofAvena sativa rotate in an alternating electric field provided that at least two cells are located close to each other. An optimum frequency range (20 to 30 kHz) exists where rotation of all cells exposed to the field is observed. Below and above this frequency range, rotation of some cells is only occasionally observed. The angular velocity of rotation depends on the square of the electric field strength. At field strengths above the value leading to electrical breakdown of the cell membrane, rotation is no longer observed due to deterioration of the cells. The absolute value of the angular velocity of rotation at a given field strength depends on the arrangement of the cells in the electric field. A maximum value is obtained if the angle between the field direction and the line connecting the two cells is 45o. With increasing distance between the two cells the rotation speed decreases. Furthermore, if two cells of different radii are positioned close to each other the cell with the smaller radius will rotate with a higher speed than the larger one. Rotation of cells in an alternating electric field is described theoretically by interaction between induced dipoles is adjacent cells. The optimum frequency range for rotation is related to the relaxation of the polarization process in the cell. The quadratic dependence of the angular velocity of rotation on the field strength results from the fact that the torque is the product of the external field and the induced dipole moment which is itself proportional to the external field. The theoretical and experimental results may be relevant for cyclosis (rotational streaming of cytoplasm) in living cells.

An investigation was performed to demonstrate that time-lapse cinematography and computer aided video analysis of cell morphology is suitable to study and compare the characteristics of hepatocytes during the adhesion process to membranes. We chose to compare ordinary cellulose Cuprophan membranes and membranes coated with collagen or fibronectin. Striking differences between uncoated cellulose and fibronectin or collagen coating were seen in the cell count per square millimeter and adhesion behaviour. On the investigated uncoated Cuprophan the hepatocytes were found to attach but not to spread whilst on collagen coated Cuprophan most of the cells spread spherically, and on fibronectin coated membranes most of the cells flattened spherically or polygonally. Time-lapse video microscopy seems to be a valuable technique for assessing the morphologic behaviour of cells in a detailed and quantitative manner in order to improve the hepatocyte culture technique in bioreactors for hybrid systems.

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.

During acute liver failure, hybrid liver support therapy could serve as a bridge to liver transplantation. In this desired temporary use, immune competent cell responses, such as the production of cytokines, might be of limiting relevance. We have investigated the Tumor Necrosis Factor-α (TNF) liberation in two models using pigs, connected with an extracorporeal bioreactor with homologous hepatocytes: TNF liberation was measured in arterial plasma during a 4 day perfusion time in untreated animals, model (i), and during short term perfusion of hepatectomized pigs in model (ii). Animals four days after catheter implantation in model (i) had TNF values of < 5 pg/ml. After connecting the system without hepatocytes, TNF rose to 9.7 ± 2 within 120 min and rose further to 32.6 ± 6 pg/ml within 4 hours after filling the system with the homologous hepatocytes. After 24 hours of continuous perfusion and during four days of perfusion, the TNF levels were lowered to baseline levels. In model (ii), TNF rose to 220 ± 130 pg/ml within 180 min and decreased to 110 ± 10 pg/ml within six hours, whereas controls without hepatocytes showed mean levels with a maximum of 120 ± 20 pg/ml. In both models, there was no correlation between TNF levels and clinical abnormalities such as fever or shock symptoms. There is evidence for an activation of blood cells during experimental extracorporeal hybrid support. No typical side effects were, however, observed. Thus, TNF mediated extracorporeal cell activation does not appear to limit the application of homologous hybrid liver support therapy.

One major goal of dialysis therapy has become the removal of ß2-microglobulin (ß2-m). The interdialytic elimination of ß2-m was studied using a newly developed high-flux cellulose acetate (CA) membrane.The results show that high-flux CA dialyzers offer better biocompatibility than classical Cuprophan or high-flux Cuprophan devices, with regard to leukopenia, C3a desarg generation, and elastase release from polymorphonuclear (PMN) leukocytes. Compared to high-flux CA membranes, high-flux PMMA membranes induce less C3a desarg formation but comparable leukopenia. High-flux PMMA membranes, however cause greater leukocyte stimulation than CA as demonstrated by more PMN elastase release during hemodialysis.Using high-flux CA or high-flux PMMA membranes, serum ß2-m levels decreased 32% during dialysis. Serum ß2-m dropped 10% with high-flux Cuprophan membranes, but remained unchanged with conventional Cuprophan dialyzers.Sieving coefficients for ß2-microglobulin (ß2-m) were virtually zero with classical Cuprophan and 0.66 with high-flux cellulose acetate membranes. High-flux membranes made of Cuprophan and PMMA gave coefficients of 0.25 and 0.45, respectively. This indicates the high removal capacity of the new CA-membrane for substances with high molecular weight.This high-flux CA membrane thus appears to combine a good degree of biocompatibility with a high capacity for ß2-m removal.

Following electro-fusion of plant protoplasts the volume of the fused cell is the sum of the volumes of the parent cells. As shown for mesophyll protoplasts from leaves of Kalanchoë daigremontiana, the excess in membrane material arising from the reduction in membrane area is removed-at least to a larger extent — by the formation of vesicles which are visible in the light microscope. These vesicles, which may have been formed by the fusion of sub-microscopic vesicles, are observed in the contact zone of the fusing cells. The mechanism of the formation of vesicles during electro-fusion is discussed.

Freeze fractures of the plasmalemma membranes of guard-cell and mesophyll protoplasts of Vicia faba demonstrate that the inner monolayer of the plasmalemma is compartmentalized into areas with distinct, highly organized structures. Between areas of intramembranous particles dispersed randomly on a relatively smooth fracture face, membrane domains showing an extremely regular planar, hexagonal array of particles are interspersed. The dimensions of these hexagonal lattices are about 0.5 µm in diameter, the center-to-center spacing is about 22 nm, and the particle size is about 9 nm. The particle in the hexagonal arrays are accompanied by complementary pits in the opposite monolayer fracture of the plasmalemma membrane.

Cells suspended in a low-conducting medium were exposed to an alternating electric field whose frequency was altered between 1 kHz and 2 MHz. A resonance frequency was observed at which all suspended cells rotated about an axis normal to the field lines (when the electric field strength was larger than a threshold value of about 400 V/cm). This resonance frequency varied from species to species of cells (mesophyll protoplasts of Avena sativa = 20-40 kHz, human erythrocytes and ghost cells = 80-100 kHz, yeast cells = 140-180 kHz, Friend cells = 30-40 kHz, at room temperature).

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.

Methods for the detection of positive or negative charges on the surface of biomaterials/membranes and inside a membrane are important for the characterisation of such materials. We tested different dyes and optimized staining procedures. Under standardized conditions negatively charged membranes were stained with cationic triarylmethane compounds such as crystal violet and positively charged membranes with the anionic anthraquinone dye anthralan blue B. There was no staining of uncharged cellulose membranes. The applicability of these methods was demonstrated on membranes coated to varying degrees with charged compounds such as heparin, these changes in charge being detectible quantitatively by photometry. The distribution of charges inside a membrane was detected by optical sectioning across the stained (FITC labelled poly-L-lysine) membrane using confocal laser scanning microscopy (LSM). LSM offers a completely new application possibility in biomaterial and biocompatibility research.

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.

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.

Summary Animal-plant heterokaryons were produced by electrically-induced fusion between mouse erythroleukaemic cells (Friend cells) andPetunia protoplasts. Such heterokaryons exhibited the animalspecific function of DMSO-stimulated haemoglobin synthesis, and the plant-specific function of cell wall regeneration.

An experimental procedure is described for the reproducible production of hybridoma cells using the electrofusion technique. High yields can be obtained when fusion is performed in isotonic inositol solutions containing Ca 2+and Mg 2+in a ratio of 1:5 in the millimolar range. The hybridoma cells are transferred 10 min after the field pulse application into a balanced salt solution for 30 min at 37°C.

A technique is presented which allows electrofusion of single cells under sterile conditions. The electrofusion chamber is placed in a Petri dish. Before a droplet of the fusion medium is pipetted between the electrodes, the chamber is completely covered with vaseline, which prevents the fusion medium evaporating. Additionally, the fusion chamber is treated with solutions containing poly(L)-lysine and pronase which results in a decreased movement of the cells on the glass between the electrodes and which allows electrofusion without any proteolytic pretreatment.

An experimental procedure is described for the reproducible production of hybridoma cells using the electrofusion technique. High yields can be obtained when fusion is performed in isotonic inositol solutions containing Ca2+and Mg2+in a ratio of 1:5 in the millimolar range. The hybridoma cells are transferred 10 min after the field pulse application into a balanced salt solution for 30 min at 37°C.

A technique is presented which allows electrofusion of single cells under sterile conditions. The electrofusion chamber is placed in a Petri dish. Before a droplet of the fusion medium is pipetted between the electrodes, the chamber is completely covered with vaseline, which prevents the fusion medium evaporating. Additionally, the fusion chamber is treated with solutions containing poly(L)‐lysine and pronase which results in a decreased movement of the cells on the glass between the electrodes and which allows electrofusion without any proteolytic pretreatment.

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Today, more than 30 different polymers or polymer blends are used as materials for dialysis membranes. They can be categorized following the scheme of a family tree of haemodialysis membranes. The trunk represents membranes from regenerated cellulose, major branches show either synthetically modified cellulose membranes or membranes manufactured from synthetic polymers. As the latter are standardly hydrophobic, small branches elucidate the technique on how these materials have been rendered partially or completely hydrophilic. Complications may arise, when comparing membranes only following their polymer names, such as polysulfone, polyacrylonitrile or polyamide. Due to varying polymer compositions, membranes with the same polymer names may differ in their haemocompatibility, flux properties and adsorption characteristics. Adsorption of proteins like β2-microglobulin, fibrinogen and coagulation factors, complement proteins, or hormones like parathormon and erythropoietin are differently adsorbed by dialysis membranes and thus adsorption contributes to the removal characteristics. Of central interest for membrane development and application is the question of how these membranes can be sterilized, as a series of patient adverse reactions has been attributed to the dialyser sterilization procedures. Apart from the cellulosic membranes Cuprophan and Hemophan, the majority of membranes cannot be sterilized by steam, as these materials degrade when exposed to above their class-point temperature. Finally, future aspects of modern membrane development should not neglect the needs of patient populations with specific blood properties, such as diabetics.