Renal replacement therapy was an early pioneer in both extra-corporeal organ replacement and whole organ transplantation. Today, the success of this pioneering work is directly demonstrated in the millions of patients worldwide successfully treated with dialysis and kidney transplantation. However, there remain significant shortcomings to current treatment modalities that limit clinical outcomes and quality of life. To address these problems, researchers have turned to using cell-based therapies for the development of a bioartificial kidney. These approaches aim to recapitulate the numerous functions of the healthy kidney including solute clearance, fluid homeostasis and metabolic and endocrine functions. This review will examine the state-of-the-art in kidney bioengineering by evaluating the various techniques currently being utilized to create a bioartificial kidney. These promising new technologies, however, still need to address key issues that may limit the widespread adoption of cell therapy including cell sourcing, organ scaffolding, and immune response. Additionally, while these new methods have shown success in animal models, it remains to be seen whether these techniques can be successfully adapted for clinical treatment in humans.

Active transport by renal proximal tubules plays a significant role in drug disposition. During drug development, estimates of renal excretion are essential to dose determination. Kidney bioreactors that reproduce physiologic cues in the kidney, such as flow-induced shear stress, may better predict in vivo drug behavior than do current in vitro models. In this study, we investigated the role of shear stress on active transport of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) by Madin-Darby canine kidney cells exogenously expressing the human organic cation transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1). Cells cultured in a parallel plate under continuous media perfusion formed a tight monolayer with a high barrier to inulin. In response to increasing levels of shear stress (0.2-2 dynes/cm²), cells showed a corresponding increase in transport of ASP+, reaching a maximal 4.2-fold increase at 2 dynes/cm² compared with cells cultured under static conditions. This transport was inhibited with imipramine, indicating active transport was present under shear stress conditions. Cells exposed to shear stress of 2 dynes/cm² also showed an increase in RNA expression of both transfected human and endogenous OCT2 (3.7- and 2.0-fold, respectively). Removal of cilia by ammonium sulfate eliminated the effects of shear on ASP+ transport at 0.5 dynes/cm² with no effect on ASP+ transport under static conditions. These results indicate that shear stress affects active transport of organic cations in renal tubular epithelial cells in a cilia-dependent manner.

Introduction

Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na⁺/H⁺ exchange and H₂O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously.

Methods

We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport.

Results

piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport.

Conclusions

Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

Novel biomaterials for medical device applications must be stable throughout all stages of preparation for surgery, including sterilization. There is a paucity of information on the effects of sterilization on sub-10 nm-thick polymeric surface coatings suitable for silicon-based bioartificial organs. This study explores the effect of five standard sterilization methods on three surface coatings applied to silicon: polyethylene glycol (PEG), poly(sulfobetaine methacrylate) (pSBMA), and poly (2-methacryloyloxyethyl phosphorylcholine) (pMPC). Autoclave, dry heat, hydrogen peroxide (H₂ O₂ ) plasma, ethylene oxide gas (EtO), and electron beam (E-beam) treated coatings were analyzed to determine possible polymer degradation with sterilization. Poststerilization, there were significant alterations in contact angle, maximum change resulting from H₂ O₂ (Δ - 14°), autoclave (Δ + 15°), and dry heat (Δ + 23°) treatments for PEG, pSBMA, and pMPC, respectively. Less than 5% coating thickness change was found with autoclave and EtO on PEG-silicon, E-beam on pSBMA-silicon and EtO treatment on pMPC-silicon. H₂ O₂ treatment resulted in at least 30% decrease in thickness for all coatings. Enzyme-linked immunosorbent assays showed significant protein adsorption increase for pMPC-silicon following all sterilization methods. E-beam on PEG-silicon and dry-heat treatment on pSBMA-silicon exhibited maximum protein adsorption in each coating subset. Overall, the data suggest autoclave and EtO treatments are well-suited for PEG-silicon, while E-beam is best suited for pSBMA-silicon. pMPC-silicon was least impacted by EtO treatment. H₂ O₂ treatment had a negative effect on all three coatings. These results can be used to determine which surface modifications and sterilization processes to utilize for devices in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2327-2336, 2018.

Most current microfluidic cell culture systems are integrated single use devices. This can limit throughput and experimental design options, particularly for epithelial cells, which require significant time in culture to obtain a fully differentiated phenotype. In addition, epithelial cells require a porous growth substrate in order to fully polarize their distinct apical and basolateral membranes. We have developed a modular microfluidic system using commercially available porous culture inserts to evaluate polarized epithelial cells under physiologically relevant fluid flow conditions. The cell-support for the bioreactor is a commercially available microporous membrane that is ready to use in a 6-well format, allowing for cells to be seeded in advance in replicates and evaluated for polarization and barrier function prior to experimentation. The reusable modular system can be easily assembled and disassembled using these mature cells, thus improving experimental throughput and minimizing fabrication requirements. The bioreactor consists of an apical microfluidic flow path and a static basolateral chamber that is easily accessible from the outside of the device. The basolateral chamber acts as a reservoir for transport across the cell layer. We evaluated the effect of initiation of apical shear flow on short-term intracellular signaling and mRNA expression using primary human renal epithelial cells (HRECs). Ten min and 5 h after initiation of apical fluid flow over a stable monolayer of HRECs, cells demonstrated increased phosphorylation of extracellular signal-related kinase and increased expression of interleukin 6 (IL-6) mRNA, respectively. This bioreactor design provides a modular platform with rapid experimental turn-around time to study various epithelial cell functions under physiologically meaningful flow conditions.

Primary cells cultured in vitro gradually lose features characteristic of the in vivo phenotype. Culture techniques that help maintain cell-specific phenotype are advantageous for development of tissue engineered and bioartificial organs. Here we evaluated the phenotype of primary human renal tubular epithelial cells subjected to fluid shear stress by culturing the cells on an orbital shaker. Transepithelial electrical resistance (TEER), cell density, and gene and protein expression of proximal tubule-specific functional markers were measured in cells subjected to orbital shear stress. Cells cultured on an orbital shaker had increased TEER, higher cell density, and enhanced tubular epithelial specific gene and protein expression. This is likely due at least in part to the mechanical stress applied to the apical surface of the cells although other factors including increased nutrient and oxygen delivery and improved mixing could also play a role. These results suggest that orbital shaker culture may be a simple approach to augmenting the differentiated phenotype of cultured renal epithelial cells.

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories-coagulation (thrombin-antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems.

Microelectromechanical systems (MEMS) have enabled the fabrication of silicon nanopore membranes (SNM) with uniform non-overlapping "slit shaped" pores. The application of SNM has been suggested for high selectivity of biomolecules in a variety of medical filtration applications. The aim of this study was to rigorously quantify the differences in sieving between slit pore SNM and more commonly modeled cylindrical pore membranes, including effects of the extended Derjaguin, Landau, Verwey, and Overbeek (XDLVO) interactions. Applying equations derived for SNM in previous work, we compare the partition coefficient of slit and cylindrical pore membranes while accounting for both steric and XDLVO interactions. Simple, steric approximations demonstrate that slit pore membranes exhibit significantly lower partition coefficients than cylindrical pore models. Incorporating XDLVO interactions results in an even more marked difference between slit pore and cylindrical pore membranes. These partition coefficients were used to evaluate changes in beta-2-microglobulin (B2M) selectivity. The data demonstrate that XDLVO interactions increase the selectivity advantage that slit pores possess over cylindrical pores, particularly for larger values of the acid-base decay constant. Finally, the bovine serum albumin (BSA) to B2M selectivity ratio was investigated. The selectivity ratio appears larger in slit pores than cylindrical pores for all cases, indicating that slit pores are particularly well suited for hemofiltration applications. The results of this study have significant implications for the application of SNM in membrane processes where highly selective separation of biomolecules is desirable.

Microelectromechanical systems (MEMS), a technology that resulted from significant innovation in semiconductor fabrication, have recently been applied to the development of silicon nanopore membranes (SNM). In contrast to membranes fabricated from polymeric materials, SNM exhibit slit-shaped pores, monodisperse pore size, constant surface porosity, zero pore overlap, and sub-micron thickness. This development in membrane fabrication is applied herein for the validation of the XDLVO (extended Derjaguin, Landau, Verwey, and Overbeek) theory of membrane transport within the context of hemofiltration. In this work, the XDLVO model has been derived for the unique slit pore structure of SNM. Beta-2-microglobulin (B2M), a clinically relevant "middle molecular weight" solute in kidney disease, is highlighted in this study as the solute of interest. In order to determine interaction parameters within the XDLVO model for B2M and SNM, goniometric measurements were conducted, yielding a Hamaker constant of 4.61× 10^-21 J and an acid-base Gibbs free energy at contact of 41 mJ/m². The XDLVO model was combined with existing models for membrane sieving, with predictions of the refined model in good agreement with experimental data. Furthermore, the results show a significant difference between the XDLVO model and the simpler steric predictions typically applied in membrane transport. The refined model can be used as a tool to tailor membrane chemistry and maximize sieving or rejection of different biomolecules.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water. We previously identified transforming growth factor-beta as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active inhabitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5' AMP-activated kinase phosphorylation. The signals that undermine in vitro differentiation are complex, but susceptible to pharmacologic intervention. This achievement overcomes a major hurdle limiting the development of a bioreactor of cultured cells for renal replacement therapy that encompasses not only endocrine and metabolic functions but also transport and excretion. Impact statement Clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this study, we build on our prior work by using small molecules to modulate pathways affected by substrate elasticity. In doing so, we are able to enhance differentiation of these cells on conventional noncompliant substrates and show transport. These results are fundamentally enabling a new generation of cell-based renal therapies.