Bioengineered Kidney Tubules Efficiently Clear Uremic Toxins in Experimental Dialysis Conditions.

Patients with end-stage kidney disease (ESKD) suffer from high levels of protein-bound uremic toxins (PBUTs) that contribute to various comorbidities. Conventional dialysis methods are ineffective in removing these PBUTs. A potential solution could be offered by a bioartificial kidney (BAK) composed of porous membranes covered by proximal tubule epithelial cells (PTECs) that actively secrete PBUTs. However, BAK development is currently being hampered by a lack of knowledge regarding the cytocompatibility of the dialysis fluid (DF) that comes in contact with the PTECs. Here, we conducted a comprehensive functional assessment of the DF on human conditionally immortalized PTECs (ciPTECs) cultured as monolayers in well plates, on Transwell® inserts, or on hollow fiber membranes (HFMs) that form functional units of a BAK. We evaluated cell viability markers, monolayer integrity, and PBUT clearance. Our results show that exposure to DF did not affect ciPTECs' viability, membrane integrity, or function. Seven anionic PBUTs were efficiently cleared from the perfusion fluid containing a PBUTs cocktail or uremic plasma, an effect which was enhanced in the presence of albumin. Overall, our findings support that the DF is cytocompatible and does not compromise ciPTECs function, paving the way for further advancements in BAK development and its potential clinical application.

Portable, wearable and implantable artificial kidney systems: needs, opportunities and challenges.

Haemodialysis is life sustaining but expensive, provides limited removal of uraemic solutes, is associated with poor patient quality of life and has a large carbon footprint. Innovative dialysis technologies such as portable, wearable and implantable artificial kidney systems are being developed with the aim of addressing these issues and improving patient care. An important challenge for these technologies is the need for continuous regeneration of a small volume of dialysate. Dialysate recycling systems based on sorbents have great potential for such regeneration. Novel dialysis membranes composed of polymeric or inorganic materials are being developed to improve the removal of a broad range of uraemic toxins, with low levels of membrane fouling compared with currently available synthetic membranes. To achieve more complete therapy and provide important biological functions, these novel membranes could be combined with bioartificial kidneys, which consist of artificial membranes combined with kidney cells. Implementation of these systems will require robust cell sourcing; cell culture facilities annexed to dialysis centres; large-scale, low-cost production; and quality control measures. These challenges are not trivial, and global initiatives involving all relevant stakeholders, including academics, industrialists, medical professionals and patients with kidney disease, are required to achieve important technological breakthroughs.

The European Green Deal and nephrology: a call for action by the European Kidney Health Alliance.

The world faces a dramatic man-made ecologic disaster and healthcare is a crucial part of this problem. Compared with other therapeutic areas, nephrology care, and especially dialysis, creates an excessive burden via water consumption, greenhouse gas emission and waste production. In this advocacy article from the European Kidney Health Alliance we describe the mutual impact of climate change on kidney health and kidney care on ecology. We propose an array of measures as potential solutions related to the prevention of kidney disease, kidney transplantation and green dialysis. For dialysis, several proactive suggestions are made, especially by lowering water consumption, implementing energy-neutral policies, waste triage and recycling of materials. These include original proposals such as dialysate regeneration, dialysate flow reduction, water distillation systems for dialysate production, heat pumps for unit climatization, heat exchangers for dialysate warming, biodegradable and bio-based polymers, alternative power sources, repurposing of plastic waste (e.g. incorporation in concrete), registration systems of ecologic burden and platforms to exchange ecologic best practices. We also discuss how the European Green Deal offers real potential for supporting and galvanizing these urgent environmental changes. Finally, we formulate recommendations to professionals, manufacturers, providers and policymakers on how this correction can be achieved.

One-Step Fabrication of Porous Membrane-Based Scaffolds by Air-Water Interfacial Phase Separation: Opportunities for Engineered Tissues.

Common methods for fabricating membrane-based scaffolds for tissue engineering with (hydrophobic) polymers include thermal or liquid-phase inversion, sintering, particle leaching, electrospinning and stereolithography. However, these methods have limitations, such as low resolution and pore interconnectivity and may often require the application of high temperatures and/or toxic porogens, additives or solvents. In this work, we aim to overcome some of these limitations and propose a one-step method to produce large porous membrane-based scaffolds formed by air-water interfacial phase separation using water as a pore-forming agent and casting substrate. Here, we provide proof of concept using poly (trimethylene carbonate), a flexible and biocompatible hydrophobic polymer. Membrane-based scaffolds were prepared by dropwise addition of the polymer solution to water. Upon contact, rapid solvent-non-solvent phase separation took place on the air-water interface, after which the scaffold was cured by UV irradiation. We can tune and control the morphology of these scaffolds, including pore size and porosity, by changing various parameters, including polymer concentration, solvent type and temperature. Importantly, human hepatic stellate cells cultured on these membrane-based scaffolds remained viable and showed no signs of pro-inflammatory stress. These results indicate that the proposed air-water interfacial phase separation represents a versatile method for creating porous membrane-based scaffolds for tissue engineering applications.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells.

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial-endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air-liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial-endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial-endothelial and alveolar-endothelial models with primary lung epithelial cells.

In vivovascularization and islet function in a microwell device for pancreatic islet transplantation.

Islet encapsulation in membrane-based devices could allow for transplantation of donor islet tissue in the absence of immunosuppression. To achieve long-term survival of islets, the device should allow rapid exchange of essential nutrients and be vascularized to guarantee continued support of islet function. Recently, we have proposed a membrane-based macroencapsulation device consisting of a microwell membrane for islet separation covered by a micropatterned membrane lid. The device can prevent islet aggregation and support functional islet survivalin vitro. Here, based on previous modeling studies, we develop an improved device with smaller microwell dimensions, decreased spacing between the microwells and reduced membrane thickness and investigate its performancein vitroandin vivo. This improved device allows for encapsulating higher islet numbers without islet aggregation and by applying anin vivoimaging system we demonstrate very good perfusion of the device when implanted intraperitoneally in mice. Besides, when it is implanted subcutaneously in mice, islet viability is maintained and a vascular network in close proximity to the device is developed. All these important findings demonstrate the potential of this device for islet transplantation.

Data Sharing Under the General Data Protection Regulation: Time to Harmonize Law and Research Ethics?

The General Data Protection Regulation (GDPR) became binding law in the European Union Member States in 2018, as a step toward harmonizing personal data protection legislation in the European Union. The Regulation governs almost all types of personal data processing, hence, also, those pertaining to biomedical research. The purpose of this article is to highlight the main practical issues related to data and biological sample sharing that biomedical researchers face regularly, and to specify how these are addressed in the context of GDPR, after consulting with ethics/legal experts. We identify areas in which clarifications of the GDPR are needed, particularly those related to consent requirements by study participants. Amendments should target the following: (1) restricting exceptions based on national laws and increasing harmonization, (2) confirming the concept of broad consent, and (3) defining a roadmap for secondary use of data. These changes will be achieved by acknowledged learned societies in the field taking the lead in preparing a document giving guidance for the optimal interpretation of the GDPR, which will be finalized following a period of commenting by a broad multistakeholder audience. In parallel, promoting engagement and education of the public in the relevant issues (such as different consent types or residual risk for re-identification), on both local/national and international levels, is considered critical for advancement. We hope that this article will open this broad discussion involving all major stakeholders, toward optimizing the GDPR and allowing a harmonized transnational research approach.

Frontiers in hemodialysis: Innovations and technological advances.

As increasing demand for hemodialysis (HD) treatment incurs significant financial burden to healthcare systems and ecological burden as well, novel therapeutic approaches as well as innovations and technological advances are being sought that could lead to the development of purification devices such as dialyzers with improved characteristics and wearable technology. Novel knowledge such as the development of more accurate kinetic models, the development of novel HD membranes with the use of nanotechnology, novel manufacturing processes, and the latest technology in the science of materials have enabled novel solutions already marketed or on the verge of becoming commercially available. This collaborative article reviews the latest advances in HD as they were presented by the authors in a recent symposium titled "Frontiers in Haemodialysis," held on 12th December 2019 at the Royal Society of Medicine in London.

Development of Porous and Flexible PTMC Membranes for In Vitro Organ Models Fabricated by Evaporation-Induced Phase Separation.

Polymeric membranes are widely applied in biomedical applications, including in vitro organ models. In such models, they are mostly used as supports on which cells are cultured to create functional tissue units of the desired organ. To this end, the membrane properties, e.g., morphology and porosity, should match the tissue properties. Organ models of dynamic (barrier) tissues, e.g., lung, require flexible, elastic and porous membranes. Thus, membranes based on poly (dimethyl siloxane) (PDMS) are often applied, which are flexible and elastic. However, PDMS has low cell adhesive properties and displays small molecule ad- and absorption. Furthermore, the introduction of porosity in these membranes requires elaborate methods. In this work, we aim to develop porous membranes for organ models based on poly(trimethylene carbonate) (PTMC): a flexible polymer with good cell adhesive properties which has been used for tissue engineering scaffolds, but not in in vitro organ models. For developing these membranes, we applied evaporation-induced phase separation (EIPS), a new method in this field based on solvent evaporation initiating phase separation, followed by membrane photo-crosslinking. We optimised various processing variables for obtaining form-stable PTMC membranes with average pore sizes between 5 to 8 µm and water permeance in the microfiltration range (17,000-41,000 L/m2/h/bar). Importantly, the membranes are flexible and are suitable for implementation in in vitro organ models.

Membranes for Modelling Cardiac Tissue Stiffness In Vitro Based on Poly(trimethylene carbonate) and Poly(ethylene glycol) Polymers.

Despite the increased expenditure of the pharmaceutical industry on research and development, the number of drugs for cardiovascular diseases that reaches the market is decreasing. A major issue is the limited ability of the current in vitro and experimental animal models to accurately mimic human heart disease, which hampers testing of the efficacy of potential cardiac drugs. Moreover, many non-heart-related drugs have severe adverse cardiac effects, which is a major cause of drugs' retraction after approval. A main hurdle of current in vitro models is their inability to mimic the stiffness of in vivo cardiac tissue. For instance, poly(styrene) petri dishes, which are often used in these models, have a Young's modulus in the order of GPa, while the stiffness of healthy human heart tissue is <50 kPa. In pathological conditions, such as scarring and fibrosis, the stiffness of heart tissue is in the >100 kPa range. In this study, we focus on developing new membranes, with a set of properties for mimicry of cardiac tissue stiffness in vitro, based on methacrylate-functionalized macromers and triblock-copolymers of poly(trimethylene carbonate) and poly(ethylene glycol). The new membranes have Young's moduli in the hydrated state ranging from 18 kPa (healthy tissue) to 2.5 MPa (pathological tissue), and are suitable for cell contraction studies using human pluripotent stem-cell-derived cardiomyocytes. The membranes with higher hydrophilicity have low drug adsorption and low Young's moduli and could be suitable for drug screening applications.

A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans.

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas.

Drugs Commonly Applied to Kidney Patients May Compromise Renal Tubular Uremic Toxins Excretion.

In chronic kidney disease (CKD), the secretion of uremic toxins is compromised leading to their accumulation in blood, which contributes to uremic complications, in particular cardiovascular disease. Organic anion transporters (OATs) are involved in the tubular secretion of protein-bound uremic toxins (PBUTs). However, OATs also handle a wide range of drugs, including those used for treatment of cardiovascular complications and their interaction with PBUTs is unknown. The aim of this study was to investigate the interaction between commonly prescribed drugs in CKD and endogenous PBUTs with respect to OAT1-mediated uptake. We exposed a unique conditionally immortalized proximal tubule cell line (ciPTEC) equipped with OAT1 to a panel of selected drugs, including angiotensin-converting enzyme inhibitors (ACEIs: captopril, enalaprilate, lisinopril), angiotensin receptor blockers (ARBs: losartan and valsartan), furosemide and statins (pravastatin and simvastatin), and evaluated the drug-interactions using an OAT1-mediated fluorescein assay. We show that selected ARBs and furosemide significantly reduced fluorescein uptake, with the highest potency for ARBs. This was exaggerated in presence of some PBUTs. Selected ACEIs and statins had either no or a slight effect at supratherapeutic concentrations on OAT1-mediated fluorescein uptake. In conclusion, we demonstrate that PBUTs may compete with co-administrated drugs commonly used in CKD management for renal OAT1 mediated secretion, thus potentially compromising the residual renal function.

Development of a villi-like micropatterned porous membrane for intestinal magnesium and calcium uptake studies.

Intestinal enterocytes are key players in the absorption of magnesium (Mg2+) and calcium (Ca2+). Understanding the exact molecular mechanisms by which their absorption behavior is regulated could greatly improve treatment strategies for stimulating intestinal absorption in diseases with Mg2+ and/or Ca2+ deficiency. However, such studies are hampered by the lack of in vitro intestinal cell models mimicking the mechanical and physiological properties of the gut. In this study we develop an in vitro gut model based on porous micropatterned membranes with villi-like surface topography and mechanical properties closely mimicking that of intestinal tissue. These membranes are prepared via phase separation micromolding using poly-ε-caprolactone/poly-lactic-glycolic acid (PCL/PLGA) polymer blend and can facilitate cellular differentiation of Caco-2 cells similar to native enterocytes. In fact, cells cultured on these micropatterned membranes form a brush border of microvilli with spatial differences in morphology and tight junction formation along the villous-base axis. Moreover, cells cultured on our membranes show a 2-fold increased alkaline phosphatase activity at the end of differentiation. Finally, we demonstrate that cells cultured on our micropatterned membranes have a 4- and 1.5-fold increased uptake of 25Mg and 45Ca, respectively, compared to non-patterned membranes. These results indicate that the new membranes can mimic the intestinal environment and therefore can have a great impact on mineral uptake in vitro. STATEMENT OF SIGNIFICANCE: This study presents the development of an in vitro gut model consisting of villi-like PCL/PLGA micropatterned membranes. These membranes are prepared via phase separation micromolding (PSµM), a technique which allows tailoring of the membrane surface topography combined with membrane porosity and interconnectivity which are important parameters for membranes used for in vitro transport studies. The culture of Caco-2 cells on these micropatterned membranes shows that they facilitate cellular differentiation similar to gut enterocytes. Our data indicate that mimicking the 3D geometry of the gut is very important for improving the physiological relevance of in vitro gut models. In the future, our micropatterned membranes with segment-specific geometries, in combination with isotopic measurements, would be applied to perform detailed ion uptake and transport studies.

Application of microstructured membranes for increasing retention, selectivity and resolution in asymmetrical flow field-flow fractionation.

In the present proof-of-concept study, we demonstrate that retention time, selectivity and resolution can be increased in asymmetrical flow field-flow fractionation (AF4) by introducing microstructured ultrafiltration membranes. Evenly spaced micron-sized grooves, that are placed perpendicular to the channel flow on the accumulation wall of a field-flow fractionation system, cause a decrease in the zone velocity which is stronger for larger solutes. This has been demonstrated in thermal field-flow fractionation, and we prove that this is also the case in AF4. We examine the hypothesis theoretically and experimentally, by both computational and physical experiments. By means of moment analysis, we derive theoretically a set of equations which, under certain conditions, describe the mass transport and relate retention time, selectivity and plate height to the dimensions of the grooves. Physical experiments are carried out using microstructured polyethersulfone membranes fabricated by hot embossing, and the experimental results are compared with computational fluid dynamics experiments.

In Vitro Evaluation of Small Molecule Delivery into Articular Cartilage: Effect of Synovial Clearance and Compressive Load.

Intra-articular injection of drug depots is considered as a therapeutic strategy for the treatment of osteoarthritis. In this study, we designed an in vitro assay in a previously described bioreactor system to evaluate the uptake of a small molecule drug mimic as a function of drug clearance by the synovium and compressive load. Bromophenol blue (BPB) loaded hydrogels were placed on top of bovine articular cartilage explants and were compressed in a dual flow bioreactor. As a control, BPB was directly injected in the bioreactor compartment mimicking the synovial fluid. Subsequently, diffusion coefficients of the dye were estimated based on Fick's law. Mimicking synovial clearance revealed that dye penetration of BPB when released from a drug delivery system placed on top of a cartilage explant was enhanced compared to direct injection of BPB into a simulated synovial fluid. Furthermore, we show the synergistic effect of the amount of load and the frequency on drug uptake by the cartilage. In the described model, we have shown that, under compressive load, drug delivery from a depot was beneficial over conventional intra-articular drug administration. The assay mimics the complexity of the knee joint in several key aspects, which results in a more close representation of the expected drug outcome. In this study, we have evaluated the penetration of a model small molecule drug into articular cartilage under compressive conditions, and future development will focus on incorporating synovial(-like) fluid, synovium, and bone to increase the predictive potential of the assay further.

In vitro assessment of mixed matrix hemodialysis membrane for achieving endotoxin-free dialysate combined with high removal of uremic toxins from human plasma.

For a single hemodialysis session nearly 500 L of water are consumed for obtaining pyrogen-free dialysis fluid. However, many efforts are required to avoid biofilm formation in the system and risk of contamination can persist. Water scarcity and inadequate water purification facilities worsen contamination risk in developing countries. Here, we investigated the application of an activated carbon (AC)/polyethersulfone/polyvinylpyrrolidone mixed matrix membrane (MMM) for achieving for the first time endotoxin-free dialysate and high removal of uremic toxins from human plasma with a single membrane. The MMM, thanks to sorbent AC, can remove approximately 10 times more endotoxins from dialysis fluid compared to commercial fibers. Pyrogens transport through the MMM was investigated analyzing inflammation in THP-1 monocytes incubated with samples from the dialysis circuit, revealing safety-barrier properties of the MMM. Importantly, endotoxins from dialysate and protein-bound toxins from human plasma can be removed simultaneously without compromising AC adsorption capacity. We estimated that only 0.15 m2 of MMM is needed to totally remove the daily production of the protein-bound toxins indoxyl sulfate and hippuric acid and to completely remove endotoxins in a wearable artificial kidney (WAK) device. Our results could open up new possibilities for dialysis therapy with low water consumption including WAK and where purity and scarcity of water are limiting factors for hemodialysis treatment. STATEMENT OF SIGNIFICANCE: Hemodialysis is a life-sustaining extracorporeal treatment for renal disease, however the production of pyrogen-free dialysate is very costly and water demanding. Biofilm formation in the system worsens bacteria contamination risk. Pyrogens could be transferred into the patients' blood and trigger inflammation. Here, we show for the first time that a mixed matrix membrane composed of polyethersulfone/polyvinylpyrrolidone and activated carbon can achieve simultaneous complete removal of endotoxins from dialysate and high removal of uremic toxins from human plasma without compromising activated carbon adsorption capacity. The mixed matrix membrane could find future applications for simultaneous blood purification and dialysate depyrogenation thus lowering water consumption as for wearable artificial kidney devices and where purity and scarcity of water hamper hemodialysis treatment.

The Trimerization of Isocyanate-Functionalized Prepolymers: An Effective Method for Synthesizing Well-Defined Polymer Networks.

For the study of polymer networks, having access to polymer networks with a controlled and well-defined microscopic network structure is of great importance. However, typically, such networks are difficult to synthesize. In this work, a simple, effective, and widely applicable method is presented for synthesizing polymer networks with a well-defined network structure. This is done by the functionalization of polymeric diols using a diisocyanate, and their subsequent trimerization. Using hexamethylene diisocyanate and hydroxyl-group-terminated poly(ε-caprolactone) and poly(ethylene glycol), it is shown that both hydrophobic and hydrophilic poly(urethane-isocyanurate) networks with a well-defined network structure can readily be synthesized. By using in situ infrared spectroscopy, it is shown that the trimerization of isocyanate endgroups is clearly the predominant reaction pathway of network formation, supporting the proposed mechanism and network structure. The resulting networks possess excellent mechanical properties in both the dry and in the wet state.