In vitro study of dual layer mixed matrix hollow fiber membranes for outside-in filtration of human blood plasma.

Hemodialysis mainly removes small water-soluble uremic toxins but cannot effectively remove middle molecules and protein-bound uremic toxins. Besides, the therapy is intermittent leading to fluctuating blood values and fluid status which adversely impacts patients' health. Prolonged hemodialysis (with adequate anticoagulation) could improve the removal of toxins and the development of portable and wearable artificial kidneys could offer more flexibility in the dialysis scheme. This would enhance patients' overall health, autonomy, mobility and flexibility, allowing patients to participate in social and economic life. However, the time that patients' blood is exposed to the dialyzer material is longer during prolonged hemodialysis, and blood clots could obstruct the fiber lumen, resulting in a decrease of the effective membrane surface area available for toxin removal. The outside-in filtration (OIF) mode, wherein blood flows through the inter-fiber space instead of through the fiber lumina, has been applied widely in blood oxygenators to prevent fiber clotting, but not in hemodialysis. In this study, we present for the first time the development of a mixed matrix membrane (MMM) for OIF of human blood plasma. This MMM combines diffusion and adsorption and consists of a polymeric membrane matrix with activated carbon (AC) particles on the inside layer, and a polymeric particle-free layer on the outer fiber layer. Our results show that in vitro MMM fibers for OIF demonstrate superior removal of the protein-bound uremic toxins, indoxyl sulfate and hippuric acid, compared to both earlier MMM fibers designed for inside-out filtration mode and commercial high-flux fibers. STATEMENT OF SIGNIFICANCE: Current hemodialysis therapy cannot effectively remove protein-bound toxins. Prolonged hemodialysis could improve toxin removal. However, during prolonged hemodialysis, blood clots could obstruct the fiber lumen, resulting in decreased effective membrane surface area available for toxin removal. We have prepared, for the first time, dual layer mixed matrix hollow fiber membranes (MMM) for outside-in filtration (OIF). The OIF mode wherein blood would flow through the inter-fiber space instead of through the fiber lumina could prevent fiber clotting. Moreover, the MMMs combine diffusion and adsorption to improve (protein-bound) toxin removal. We believe that the new design of our MMM fibers is an important contribution concerning the development of artificial kidney systems and the improvement of the health and well-being of patients with renal failure.

3D alveolar in vitro model based on epithelialized biomimetically curved culture membranes.

There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, preserving the mainly spherical geometry of the cells' native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future.

Ex vivo evaluation of the blood compatibility of mixed matrix haemodialysis membranes.

The patients with end stage kidney disease need haemodialysis therapies, using an artificial kidney. Nevertheless, the current therapies cannot remove a broad range of uremic toxins compared to the natural kidney. Adsorption therapies, using sorbent-based columns, can improve the clearance of uremic toxins, but the sorbent particles often require polymeric coatings to improve their haemocompatibility leading to mass transfer limitations and to lowering of their performance. Earlier, we have developed a dual layer Mixed Matrix fiber Membrane (MMM) based on polyethersulfone/polyvinylpyrrolidone (PES/PVP) polymer blends. There, the sorbent activated carbon particles are embedded in the outer membrane layer for achieving higher removal whereas the inner blood contacting selective membrane layer should achieve optimal blood compatibility. In this work, we evaluate in detail the haemocompatibility of the MMM following the norm ISO 10993-4. We study two generations of MMM having different dimensions and transport characteristics; one with low flux and no albumin leakage and another with high flux but some albumin leakage. The results are compared to those of home-made PES/PVP single layer hollow fiber and to various control fibers already applied in the clinic. Our results show that the low flux MMM successfully avoids contact of blood with the activated carbon and has good haemocompatibility, comparable to membranes currently used in the clinic. STATEMENT OF SIGNIFICANCE: Haemodialysis is a life-sustaining extracorporeal treatment for renal disease, however a broad range of uremic toxins cannot still be removed. In our previous works we showed that a double layer Mixed Matrix Membrane (MMM) composed of polyethersulfone/polyvinylpyrrolidone and activated carbon can achieve higher removal of uremic toxics compared to commercial haemodialysers. In this work we evaluate the haemocompatibility profile of the MMM in order to facilitate its clinical implementation. The lumen particle-free layer of the MMM successfully avoids the contact of blood with the poorly blood-compatible activated carbon. Moreover, thanks to the high amount of polyvinylpyrrolidone and to the smoothness of the lumen layer, the MMM has very good haemocompatibility, comparable to membranes currently used in the clinic.

Tough combinatorial poly(urethane-isocyanurate) polymer networks and hydrogels synthesized by the trimerization of mixtures of NCO-prepolymers.

The development of tough hydrogels is an essential but challenging topic in biomaterials research that has received much attention over the past years. By the combinatorial synthesis of polymer networks and hydrogels based on prepolymers with different properties, new materials with widely varying characteristics and unexpected properties may be identified. In this paper, we report on the properties of combinatorial poly(urethane-isocyanurate) (PUI) type polymer networks that were synthesized by the trimerization of mixtures of NCO-functionalized poly(ethylene glycol) (PEG), poly(propylene gylcol) (PPG), poly(ε-caprolactone) (PCL) and poly(trimethylene carbonate) (PTMC) prepolymers in solution. The resulting polymer networks showed widely varying material properties. Combinatorial PUI networks containing at least one hydrophilic PEG component showed high water uptakes of >100 wt%. The resulting hydrogels demonstrated elastic moduli of up to 10.1 MPa, ultimate tensile strengths of up to 9.8 MPa, elongation at break values of up to 624.0% and toughness values of up to 53.4 MJ m-3. These values are exceptionally high and show that combinatorial PUI hydrogels are among the toughest hydrogels reported in the literature. Also, the simple two-step synthesis and wide range of suitable starting materials make this synthesis method more versatile and widely applicable than the existing methods for synthesizing tough hydrogels. An important finding of this work is that the presence of a hydrophobic network component significantly enhances the toughness and tensile strength of the combinatorial PUI hydrogels in the hydrated state. This enhancement is the largest when the hydrophobic network component is crystallizable in nature. In fact, the PUI hydrogels containing a crystallizable hydrophobic network component are shown to be semi-crystalline in the water-swollen state. Due to their high toughness values in the water-swollen state together with their water uptake values, elastic moduli and ultimate tensile strengths, the developed hydrogels are expected to be promising materials for biomedical coating- and adhesive applications, as well as for tissue-engineering. STATEMENT OF SIGNIFICANCE: The development of tough hydrogels is a challenging topic that has received much attention over the past years. At present, double network type hydrogels are considered state-of-the-art in the field, demonstrating toughness values of several tens of MJ m-3. However, in terms of ease and versatility of the synthesis method, the possibilities are limited using a double network approach. In this work, we present combinatorial poly(urethane-isocyanurate) type polymer networks and hydrogels, synthesized by the trimerization of mixtures of NCO-functionalized prepolymers. The resulting hydrogels demonstrate exceptionally high toughness values of up to 53 MJ m-3, while the synthesis method is versatile and widely applicable. This new class of hydrogels is therefore considered highly promising in the future development of load-bearing biomaterials.

A bioartificial kidney device with polarized secretion of immune modulators.

The accumulation of protein-bound toxins in dialyzed patients is strongly associated with their high morbidity and mortality. The bioartificial kidney device (BAK), containing proximal tubule epithelial cells (PTECs) seeded on functionalized synthetic hollow fibre membranes, may be a powerful solution for the active removal of those metabolites. In an earlier study, we developed an upscaled BAK containing conditionally immortalized human PTEC with functional organic cationic transporter 2. Here, we first extended this development to a BAK device having cells with the organic anionic transporter 1, capable of removing anionic uraemic wastes. We confirmed the quality of the conditionally immortalized human PTEC monolayer by confocal microscopy and paracellular inulin-fluorescein isothiocyanate leakage, as well as by the active transport of anionic toxin, indoxyl sulphate. Furthermore, we assessed the immune safety of our system by measuring the production of relevant cytokines by the cells after lipopolysaccharide stimulation. Upon lipopolysaccharide treatment, we observed a polarized secretion of proinflammatory cytokines by the cells: 10-fold higher in the extraluminal space, corresponding to the urine compartment, as compared with the intraluminal space, corresponding to the blood compartment. To the best of our knowledge, our work is the first to show this favourable cell polarization in a BAK upscaled device.

Carbon Adsorbents With Dual Porosity for Efficient Removal of Uremic Toxins and Cytokines from Human Plasma.

The number of patients with chronic kidney disease increases while the number of available donor organs stays at approximately the same level. Unavoidable accumulation of the uremic toxins and cytokines for these patients comes as the result of malfunctioning kidneys and their high levels in the blood result in high morbidity and mortality. Unfortunately, the existing methods, like hemodialysis and hemofiltration, provide only partial removal of uremic toxins and/or cytokines from patients' blood. Consequently, there is an increasing need for the development of the extracorporeal treatments which will enable removal of broad spectrum of uremic toxins that are usually removed by healthy kidneys. Therefore, in this work we developed and tested ordered mesoporous carbons as new sorbents with dual porosity (micro/meso) that provide selective and efficient removal of a broad range of uremic toxins from human plasma. The new sorbents, CMK-3 are developed by nanocasting methods and have two distinct pore domains, i.e. micropores and mesopores, therefore show high adsorption capacity towards small water soluble toxins (creatinine), protein-bound molecules (indoxyl sulfate and hippuric acid), middle molecules (ß-2-microglobulin) and cytokines of different size (IL-6 and IL-8). Our results show that small amounts of CMK-3 could provide selective and complete blood purification.

Bioengineered kidney tubules efficiently excrete uremic toxins.

The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

Fabrication of three-dimensional bioplotted hydrogel scaffolds for islets of Langerhans transplantation.

In clinical islet transplantation, allogeneic islets of Langerhans are transplanted into the portal vein of patients with type 1 diabetes, enabling the restoration of normoglycemia. After intra-hepatic transplantation several factors are involved in the decay in islet mass and function mainly caused by an immediate blood mediated inflammatory response, lack of vascularization, and allo- and autoimmunity. Bioengineered scaffolds can potentially provide an alternative extra-hepatic transplantation site for islets by improving nutrient diffusion and blood supply to the scaffold. This would ultimately result in enhanced islet viability and functionality compared to conventional intra portal transplantation. In this regard, the biomaterial choice, the three-dimensional (3D) shape and scaffold porosity are key parameters for an optimal construct design and, ultimately, transplantation outcome. We used 3D bioplotting for the fabrication of a 3D alginate-based porous scaffold as an extra-hepatic islet delivery system. In 3D-plotted alginate scaffolds the surface to volume ratio, and thus oxygen and nutrient transport, is increased compared to conventional bulk hydrogels. Several alginate mixtures have been tested for INS1E ß-cell viability. Alginate/gelatin mixtures resulted in high plotting performances, and satisfactory handling properties. INS1E ß-cells, human and mouse islets were successfully embedded in 3D-plotted constructs without affecting their morphology and viability, while preventing their aggregation. 3D plotted scaffolds could help in creating an alternative extra-hepatic transplantation site. In contrast to microcapsule embedding, in 3D plotted scaffold islets are confined in one location and blood vessels can grow into the pores of the construct, in closer contact to the embedded tissue. Once revascularization has occurred, the functionality is fully restored upon degradation of the scaffold.

Development of multilayer constructs for tissue engineering.

The rapidly developing field of tissue engineering produces living substitutes that restore, maintain or improve the function of tissues or organs. In contrast to standard therapies, the engineered products become integrated within the patient, affording a potentially permanent and specific cure of the disease, injury or impairment. Despite the great progress in the field, development of clinically relevantly sized tissues with complex architecture remains a great challenge. This is mostly due to limitations of nutrient and oxygen delivery to the cells and limited availability of scaffolds that can mimic the complex tissue architecture. This study presents the development of a multilayer tissue construct by rolling pre-seeded electrospun sheets [(prepared from poly (l-lactic acid) (PLLA) seeded with C2C12 pre-myoblast cells)] around a porous multibore hollow fibre (HF) membrane and its testing using a bioreactor. Important elements of this study are: 1) the medium permeating through the porous walls of multibore HF acts as an additional source of nutrients and oxygen to the cells, which exerts low shear stress (controllable by trans membrane pressure); 2) application of dynamic perfusion through the HF lumen and around the 3D construct to achieve high cell proliferation and homogenous cell distribution across the layers, and 3) cell migration occurs within the multilayer construct (shown using pre-labeled C2C12 cells), illustrating the potential of using this concept for developing thick and more complex tissues.

Corrugated round fibers to improve cell adhesion and proliferation in tissue engineering scaffolds.

Optimal cell interaction with biomaterial scaffolds is one of the important requirements for the development of successful in vitro tissue-engineered tissues. Fast, efficient and spatially uniform cell adhesion can improve the clinical potential of engineered tissue. Three-dimensional (3-D) solid free form fabrication is one widely used scaffold fabrication technique today. By means of deposition of polymer fibers, scaffolds with various porosity, 3-D architecture and mechanical properties can be prepared. These scaffolds consist mostly of solid round fibers. In this study, it was hypothesized that a corrugated fiber morphology enhances cell adhesion and proliferation and therefore leads to the development of successful in vitro tissue-engineered constructs. Corrugated round fibers were prepared and characterized by extruding poly(ethylene oxide terephthalate)-co-poly(butylene terephthalate) (300PEOT55PBT45) block co-polymer through specially designed silicon wafer inserts. Corrugated round fibers with 6 and 10 grooves on the fiber surface were compared with solid round fibers of various diameters. The culture of mouse pre-myoblast (C2C12) cells on all fibers was studied under static and dynamic conditions by means of scanning electron microscopy, cell staining and DNA quantification. After 7days of culturing under static conditions, the DNA content on the corrugated round fibers was approximately twice as high as that on the solid round fibers. Moreover, under dynamic culture conditions, the cells on the corrugated round fibers seemed to experience lower mechanical forces and therefore adhered better than on the solid round fibers. The results of this study show that the surface architecture of fibers in a tissue engineering scaffold can be used as a tool to improve the performance of the scaffold in terms of cell adhesion and proliferation.

A modular versatile chip carrier for high-throughput screening of cell-biomaterial interactions.

The field of biomaterials research is witnessing a steady rise in high-throughput screening approaches, comprising arrays of materials of different physico-chemical composition in a chip format. Even though the cell arrays provide many benefits in terms of throughput, they also bring new challenges. One of them is the establishment of robust homogeneous cell seeding techniques and strong control over cell culture, especially for long time periods. To meet these demands, seeding cells with low variation per tester area is required, in addition to robust cell culture parameters. In this study, we describe the development of a modular chip carrier which represents an important step in standardizing cell seeding and cell culture conditions in array formats. Our carrier allows flexible and controlled cell seeding and subsequent cell culture using dynamic perfusion. To demonstrate the application of our device, we successfully cultured and evaluated C2C12 premyoblast cell viability under dynamic conditions for a period of 5 days using an automated pipeline for image acquisition and analysis. In addition, using computational fluid dynamics, lactate and BMP-2 as model molecules, we estimated that there is good exchange of nutrients and metabolites with the flowing medium, whereas no cross-talk between adjacent TestUnits should be expected. Moreover, the shear stresses to the cells can be tailored uniformly over the entire chip area. Based on these findings, we believe our chip carrier may be a versatile tool for high-throughput cell experiments in biomaterials sciences.

Development of novel membranes for blood purification therapies based on copolymers of N-vinylpyrrolidone and n-butylmethacrylate.

Developments in membrane based blood purification therapies often come with longer treatment times and therefore longer blood-material contact, which requires long-term membrane biocompatibility. In this study, we develop for the first time membranes for blood purification using the material SlipSkin™, which is a copolymer, made from N-vinylpyrrolidone (NVP) and butylmethacrylate (BMA). Specific attention is focused on understanding the mechanism of pore formation and the tailoring of the membrane mechanical and transport properties to obtain the optimal membrane for blood purification therapies. Polymer composition, solvent type and solvent evaporation time influence membrane morphology and membranes with sieving properties of cascade filters in plasma fractionation applications are developed. The new membranes have very good blood compatibility properties; in fact compared to benchmark flat membranes currently used in the clinic, they have lower platelet adhesion while all other properties (contact activation, thrombogenicity, leukocyte adhesion, hemolysis and complement activation) are also very good and comparable to the benchmarks.

Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs.

Sufficient nutrient and oxygen transport is a potent modulator of cell proliferation in in vitro tissue-engineered constructs. The lack of oxygen and culture medium can create a potentially lethal environment and limit cellular metabolic activity and growth. Diffusion through scaffold and multi-cellular tissue typically limits transport in vitro, leading to potential hypoxic regions and reduction in the viable tissue thickness. For the in vitro generation of clinically relevant tissue-engineered grafts, current nutrient diffusion limitations should be addressed. Major approaches to overcoming these include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers and pre-vascularization of the engineered tissues. This study focuses on the development and utilization of a new perfusion culture system to provide adequate nutrient delivery to cells within large three-dimensional (3D) scaffolds. Perfusion of oxygenated culture medium through porous hollow fiber (HF) integrated within 3D free form fabricated (FFF) scaffolds is proposed. Mouse pre-myoblast (C2C12) cells cultured on scaffolds of poly(ethylene-oxide-terephthalate)-poly(butylene-terephthalate) block copolymer (300PEOT55PBT45) integrated with porous HF membranes of modified poly(ether-sulfone) (mPES, Gambro GmbH) is used as a model system. Various parameters such as fiber transport properties, fiber spacing within a scaffold and medium flow conditions are optimized. The results show that four HF membranes integrated with the scaffold significantly improve the cell density and cell distribution. This study provides a basis for the development of a new HF perfusion culture methodology to overcome the limitations of nutrient diffusion in the culture of large 3D tissue constructs.

Morphology and microtopology of cation-exchange polymers and the origin of the overlimiting current.

In electrodialysis desalination processes, the operating current density is limited by concentration polarization. In contrast to other membrane processes such as ultrafiltration, in electrodialysis, current transport above the limiting current is possible. In this work, the origin of the overlimiting current at cation-exchange polymers is investigated. We show that, under certain experimental conditions, electroconvection is the origin of the overlimiting conductance. The theory concerning electroconvection predicts a shortening of the plateau length of membranes with increased conductive or geometrical heterogeneity. We investigate the influence of these two parameters and show that the creation of line undulations on the membrane surface normal to the flow direction, having distances in the range of approximately 50-200% of the boundary-layer thickness, lead to an earlier onset of the overlimiting current. The plateau length of the undulated membranes is reduced by up to 60% compared to that of a flat membrane. These results verify the existence of electroconvection as a mechanism destabilizing the laminar boundary layer at the liquid-membrane interface and causing ionic transport above the limiting current density.

Passive and iontophoretic controlled delivery of salmon calcitonin through artificial membranes.

The development of a transdermal delivery system for drug molecules of high molecular weight (peptides or proteins) is nowadays a great scientific and commercial challenge. For these molecules, the passive transport through the skin is generally very low and should be enhanced by the application of the electrical current (a method called iontophoresis). A very important component of a transdermal iontophoretic system is the artificial membrane, which acts as the interface between the drug reservoir and the skin. The optimum membrane should (i) provide an effective drug delivery; (ii) have low electrical resistance and (ii) have low drug adsorption. In this work, the selection of membrane(s) for a transdermal iontophoretic salmon calcitonin (sCT, MW approximately 3500) system is performed. The passive and iontophoretic transport of sCT through porous artificial membranes, the sCT adsorption to them and the electrical resistance of all porous membranes in iontophoretic experiments is studied. The sCT transport through the membranes is compared with that through human skin, and based on the above three criteria the optimum membranes are selected for the sCT transdermal system.

In vitro evaluation of a hydroxypropyl cellulose gel system for transdermal delivery of timolol.

In this work, the development of a gel reservoir for a timolol (TM) transdermal iontophoretic delivery system is investigated. TM gel is prepared using hydroxypropyl cellulose (HPC) and the permeability of TM from the gel through an artificial membrane (Polyflux) and pig stratum corneum (SC) is studied. For a constant TM donor concentration, the TM transport across the Polyflux membrane alone decreases when the concentration of the gel increases due to increase of the gel viscosity. For constant gel concentration, however, the TM permeation across the membrane increases when the TM donor concentration increases. In addition, no effect of the electrical current (iontophoresis, current density 0.5 mA cm-2) on the TM permeation is found. For the combination of the Polyflux membrane with pig SC, the TM transport is much lower than for the membrane alone and the SC fully controls the TM delivery. In this case, the application of electrical current enhances the TM delivery 13-15 times in comparison to passive (no current) transport. According to our estimation, the daily TM dose (10-60 mg) can be delivered by an iontophoretic patch with Polyflux membrane area of 6-36 cm2 containing 20% (w/w) HPC gel and 15 mg cm-3 of TM.